Cystatin C, a secreted cysteine protease inhibitor, is abundantly expressed in retinal pigment epithelium (RPE) cells. A mutation in the protein’s leader sequence, corresponding to formation of an alternate variant B protein, has been linked with an increased risk for both age-related macular degeneration (AMD) and Alzheimer’s disease (AD). Variant B cystatin C displays intracellular mistrafficking with partial mitochondrial association. We hypothesized that variant B cystatin C interacts with mitochondrial proteins and impacts mitochondrial function. We sought to determine how the interactome of the disease-related variant B cystatin C differs from that of the wild-type (WT) form. For this purpose, we expressed cystatin C Halo-tag fusion constructs in RPE cells to pull down proteins interacting with either the WT or variant B form, followed by identification and quantification by mass spectrometry. We identified a total of 28 interacting proteins, of which 8 were exclusively pulled down by variant B cystatin C. These included 18 kDa translocator protein (TSPO) and cytochrome B5 type B, both of which are localized to the mitochondrial outer membrane. Variant B cystatin C expression also affected RPE mitochondrial function with increased membrane potential and susceptibility to damage-induced ROS production. The findings help us to understand how variant B cystatin C differs functionally from the WT form and provide leads to RPE processes adversely affected by the variant B genotype. CST3, encoding the protein cystatin C.Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world , and is The 14 kDa cysteine protease inhibitor cystatin C is an abundantly expressed and directionally secreted protein from the RPE ,7,8,9,10An increasing number of studies have highlighted the maintenance of mitochondrial homeostasis as an essential process in promoting RPE cellular health with age. In general, cells undergo a decline in mitochondrial activity with aging . HoweverGiven its unusual intracellular behavior, marked by an apparent mitochondrial association identified using visual analysis techniques, we hypothesized that variant B cystatin C may interact with one or more mitochondrial proteins that could have an impact on downstream mitochondrial function. Here we performed affinity purification mass spectrometry to elucidate and compare interactomes of wild type and risk-associated variant B cystatin C in RPE cells. Using Halo-tag technology, cystatin C fusion proteins were used as baits to capture interacting proteins on Halo-link resin, with eluates subjected to mass spectrometry analysis. We identified several proteins that exclusively bound to variant B cystatin C, including outer mitochondrial membrane (OMM) proteins, translocator protein (TSPO), and cytochrome B5 type B (CYB5B). In addition, analyses of mitochondrial functions revealed that the expression of variant B cystatin C in RPE cells resulted in alterations in susceptibility to mitochondrial ROS production and mitochondrial membrane potential (Δψm). This data provides for the first time information regarding the protein-protein interactions formed by the disease-linked variant B of cystatin C, and further demonstrates its impact on mitochondrial function.2. Upon reaching 80% confluency, cells were routinely passaged at a 1:3 ratio by dissociation with trypsin-EDTA solution .ARPE-19 cells  were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham  supplemented with 10% fetal bovine serum  at 37 °C, 5% CO® CMV-neo plasmid  between the EcoRI and XhoI restriction sites, using standard molecular biology techniques. Sequences were confirmed by Sanger sequencing . Preparation of plasmids encoding EGFP-tagged WT or variant B cystatin C has previously been described [cDNA encoding WT or variant B cystatin C (encoding 1–146 aa full length protein) were subcloned into the pHTC HaloTagescribed . For mam7 ARPE-19 cells were washed in DPBS and resuspended in 500 µL Neon resuspension buffer R. A total of 5 µg of plasmid was added to the mixture and electroporation was performed using 100 µL tips with the settings 1350 V, 20 ms, 2 pulses, after which the cells were immediately added to the culture medium. Cells were incubated in a T75 flask for 24 h, followed by washing once in ice-cold DPBS and harvesting by scraping.Halo-tagged cystatin C was transfected in ARPE-19 cells by electroporation using the Neon system . For preparation of cell lysates for pull down assay, 1 × 10Fluorescent visualization of Halo-tagged cystatin C was performed with HaloTag TMRDirect ligand  added to the culture medium of transfected cells following the manufacturer’s instructions.g for 5 min, and the supernatant was diluted to 1 mL using TBS and added to 50 µL Halo-link resin  prewashed three times with wash buffer (TBS + 0.05% IGEPAL-630). Protein complexes were allowed to bind to the resin overnight at 4 °C using end-over-end mixing. The resin was washed three times in wash buffer and once in TBS, after which bound proteins were eluted in 50 µL 50 mM ammonium bicarbonate supplemented with 0.1% Rapigest SF  at 80 °C for 10 min. Eluted samples were stored at −80 °C until analyzed.ARPE-19 cells expressing Halo-tagged proteins were resuspended in 300 µL mammalian lysis buffer supplemented with protease inhibitor . Following 5 min of incubation on ice, the lysis mixture was homogenized by passing through a 25G needle 10 times. The sample was clarified by centrifugation at 14,000× 10 µL of eluted samples were mixed 1:1 in 2× SDS-PAGE sample buffer, boiled at 95 °C for 5 min, and resolved by SDS-PAGE on a 12% gel, followed by visualization by silver staining using a Pierce silver stain kit . Gels were imaged using a ChemiDoc XRS+ imaging system .−1, after which peptides were eluted onto an Acclaim PepMap RSLC 75 µm × 25 cm 2 µm 100 Å  at 300 nL min−1 by increasing the mobile phase B concentration from 4–35% over 72 min, then to 80% over 3 min, followed by a 15 min re-equilibration at 4%.Mass spectrometry experiments and analysis were performed by the Warwick proteomics research technology platform using similar methods as described previously ,33,34. Fm/z were performed at 120K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 1.2 Th using the quadrupole, HCD fragmentation with a normalized collision energy of 33, and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 1 × 104 and the max injection time was 200 ms. Precursors with a charge state of 2–6 were selected and sampled for MS2. The dynamic exclusion duration was set to 40 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 2 s cycles.Eluting peptides were converted to gas-phase ions by means of electrospray ionization and analyzed on a Thermo Orbitrap Fusion . Survey scans of peptide precursors from 375 to 1575 https://www.biochem.mpg.de/6304115/maxquant (accessed on 1 November 2022)) against Homo sapiens database (http://www.uniprot.org/ (accessed on 1 November 2022)) and the common contaminant database from MaxQuant. Peptides were generated from a tryptic digestion with up to two missed cleavages, carbamidomethylation of cysteines as fixed modifications, and the oxidation of methionines as variable modifications. Precursor mass tolerance was 10 ppm, and product ions were searched at 0.8 Da tolerances. The raw data was processed using MaxQuant engine  was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at >95.0% probability and contained ≥2 identified peptides. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Protein groups with significant total spectra intensity were determined according to the T-Test, using Benjamini-Hochberg multiple test correction and Crude mitochondrial fractions from ARPE-19 cells expressing Halo-tagged WT or variant B cystatin C were isolated by differential centrifugation using a mitochondrial isolation kit , following the manufacturer’s instructions. The mitochondrial pellet was solubilized in 2 × SDS-PAGE sample buffer, and the mitochondrial and cytosolic fractions were analyzed by immunoblotting.Following SDS-PAGE using 12% gels, proteins were transferred to a nitrocellulose membrane using standard molecular biology procedures. Primary antibodies used were against cystatin C , GAPDH , CYB5B , TOM20 , TSPO , IRAP , and α-tubulin . After blocking in 5% milk in TBS supplemented with 0.1% Tween-20 (TBST) and probing with primary antibody, membranes were washed three times with TBST and incubated with secondary antibody conjugated to HRP , and then washed for an additional three times with TBST and developed using either Radiance Plus chemiluminescent substrate  or SuperSignal™ West Pico PLUS Chemiluminescent Substrate . Blots were imaged using a ChemiDoc XRS+ imaging system . Band intensity was assessed via densitometry and normalized against indicated housekeeping protein, and was used as a semi-quantitative estimate of protein concentrations. 5 per well) were transfected with 0.5 µg of either EGFP only, WT, or variant B cystatin C endotoxin free plasmid DNA using the Neon electroporation system . Mitochondrial ROS detection experiments were performed using a commercially available kit  following the manufacturer’s protocol. Briefly, 24 h post-transfection, ARPE-19 cells were detached from wells using trypsin, resuspended in media, and centrifuged at 400× g for 5 min. Cell pellets were then resuspended in 100 µL of cell-based assay buffer  and centrifuged again at 400× g for 2 min. Cell pellets were resuspended in 500 µL of 20 µM Mitochondrial ROS detection reagent  and incubated for 30 min at 37 °C. Cell suspensions were centrifuged again and pellets were washed with Hank’s Balanced Salt Solution (HBBS) and centrifuged at 400× g for 5 min (step repeated twice). The final cell pellet was resuspended in 500 µL of warm HBBS +/− 10 µM antimycin A reagent  and incubated for a further 1 h at 37 °C. Cell suspensions were processed for flow cytometry analysis for mitochondrial ROS detection using the BD Accuri C6TM instrument . A minimum of 4000 events were collected in the P1 gate for each condition. Samples were analyzed using the BD Accuri C6 program and were utilized to detect fluorescent intensity that correlated to ROS levels (FL2 channel) from transfected cells only (FL1 channel). Flow cytometry analysis, using the BD Accuri C6 SoftwareTM , provided transfection efficiency measurements, but more importantly allowed for the selection of only transfected green cells (FL1 channel) from mixed non-transfected and transfected populations. Mitotracker Red FM staining was measured on the FL2 channel with fluorescent intensity numbers used for quantification.ARPE-19 cells seeded into 6-well plate . 24 h post-transfection, ARPE-19 cells were stained with 100 nM Mitotracker Red FM , a dye whose uptake is dependent on mitochondrial membrane potential (Δψm) for 30 min after which flow cytometry analysis was performed. A minimum of 10,000 events were collected in the P1 gate for each condition. Samples were analyzed on the BD Accuri C6 Flow Cytometry System  in the same manner as for mitochondrial ROS detection in order to select transfected green cells (FL1 channel) from the mixed population. Mitotracker Red FM staining was measured on the FL3 channel with fluorescent intensity numbers used for quantification.1.5 × 10To evaluate the different interaction partners of WT and variant B cystatin C, the two proteins were first expressed as Halo-tagged fusion proteins in ARPE-19 cells. The Halo-tag is a versatile fusion protein that allows irreversible attachment to a wide range of ligands while being biologically inert. In addition, successful expression can be confirmed directly by linking the tag to a fluorescent ligand. Expression constructs were generated by subcloning the full-length cystatin C protein coding sequence between the EcoRI and XhoI sites in the pHTC plasmid, resulting in a sequence-encoding cystatin C with a C-terminal Halo-tag attached. Positioning of the tag at the C-terminus allowed normal processing of precursor cystatin C, which involves cleavage of the N terminal signal sequence to form the mature protein. A high degree of expression of the respective proteins was achieved in transfected ARPE-19 cells a. WesterProteins from ARPE-19 whole cell lysates interacting with either WT or variant B cystatin C fused to a C-terminal Halo-tag were isolated by immobilizing the bait proteins via their Halo-tag on Halo-link resin, followed by extensive washing to remove contaminants/non-interacting proteins and elution of interacting proteins in Rapigest buffer c. As RapAs expected, the majority of interacting proteins, including most of the high abundance proteins, were similar across WT and variant B pull downs. These included cathepsins C, L, K, H, B, O, F, and V, which are known targets of cystatin C, providing confidence that the assay and analysis had worked as expected and that the Halo-fusion forms of cystatin C were active. However, a number of different interacting proteins were also identified. Of particular interest, among the eight proteins that were exclusively pulled down by variant B cystatin C, three  are known to be abundantly distributed in mitochondria. Previous studies have indicated that a fraction of variant B cystatin C molecules mislocalize to the mitochondria , which mTo test whether the Halo-tagged WT and variant B cystatin C differ in their subcellular localization when expressed in ARPE-19 cells, crude mitochondrial fractions were isolated from cytoplasm using differential centrifugation. Western blot analysis of the fractions confirmed the findings of previous studies showing association of a portion of variant B cystatin C pool and mitochondria by showing that the protein was enriched in the mitochondrial fraction in comparison to the WT protein a. FurtheAfter observing that variant B cystatin is significantly enriched in mitochondrial fractions compared to WT cystatin C, we next sought to see if the presence of variant B had an effect on mitochondrial function in relation to mitochondrial ROS production and Δψm. In relation to ROS production, a significant decrease in ROS production was observed in variant B-transfected RPE cells compared to WT cells c. IntereNext, in order to quantitatively evaluate the effects of variant B cystatin C on Δψm in RPE cells, the optimization of Mitotracker Red FM concentration was performed in order to use the dye in its sensitive range of concentration where differences can be detected. This showed that a concentration of 100 nM was sufficient to stain most cells whilst also allowing changes to be detected at a fluorescent and cell number level when CCCP, an uncoupling agent that disrupts oxidative phosphorylation, was added a,b. The In the present study, we have identified, for the first time to our knowledge, the interactome of WT cystatin C and AMD/AD-associated variant B cystatin C. The findings highlighted specific variant B cystatin C interacting mitochondrial proteins while also confirming the enrichment of variant B in mitochondrial fractions isolated from RPE cells expressing this form of the protein. Furthermore, we showed that the presence of variant B cystatin C in RPE cells leads to alterations in mitochondrial ROS production and Δψm. Thus, this study delineates possible routes on which variant B cystatin C interacts with mitochondrial processes and proteins, impacting mitochondrial function and contributing to mitochondrial dysfunction, which is a characteristic feature described for both AMD and AD.Our mass spectrometry and pull down data showed that variant B cystatin C interacts with CYB5B and TSPO. This is of interest due to the fact that both CYB5B and TSPO are localized to the outer mitochondrial membrane, thus comprising plausible anchor points for mistrafficked cystatin C. TSPO has been previously highlighted for its potential role in neurodegeneration seen in AD . While iRecent studies have demonstrated other roles in a wide range of processes for TSPO, such as oxidative stress, calcium transport, mitochondrial function, apoptosis, inflammation, and perhaps most intriguing, autophagy, all of which have been linked to RPE dysfunction and AMD development ,47,48,49TNFR1), a change which causes an autoinflammatory disorder called tumor necrosis factor receptor-associated periodic syndrome (TRAPs) [The functional effects of variant B cystatin C enrichment at the mitochondria were investigated through measurement of mitochondrial ROS and Δψm levels. Expression of variant B cystatin C caused a decrease in basal mitochondrial ROS levels in RPE cells, but an overall increase in fold change of mitochondrial ROS levels upon the addition of antimycin A compared to cells expressing WT cystatin C. ROS are generally produced from the mitochondria and play an important role, either physiological or pathophysiological, directly dependent of their level, in a wide range of cellular processes such as cell survival/death and inflammation ,52,53. E (TRAPs) . Further (TRAPs)  that incJurkat cells resulted in increased gene expression of mitochondrial ETC molecules and ATP production [The concept of protection is supported by findings in hypoxic cells, specifically that an adaptive response mediated by hypoxia-inducible factor 1 (HIF-1) reduces mitochondrial ROS production levels through multiple mechanisms, including increasing efficiency of electron transport chain (ETC) components . It has oduction , thus evoduction .Here we also identified an interaction between variant B cystatin C and OMM CYB5B, comprising an additional plausible site for ETC modulation. An overall increase in fold change mitochondrial ROS levels (upon the addition of antimycin A) in variant B-expressing cells compared to WT-expressing RPE cells suggests that the presence of variant B renders RPE cells more susceptible and responsive to ETC inhibitors such as antimycin A. In relation to RPE cells, one such stress known to accumulate with age in cells and in the Bruch’s membrane (BrM) is represented by advanced glycation end products (AGEs), a group of heterogeneous molecules that impact RPE function. AGEs have been shown to increase mitochondrial ROS , impair In addition to altered ROS levels, an increase in ΔΨm was determined, indicating that variant B cystatin C-expressing RPE cells contain functionally altered hyperpolarized mitochondria. Hyperpolarized mitochondria have been observed in iPSC-derived neurons isolated from patients carrying a mutation in the Tau gene that causes the neurodegenerative disease frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) . This hy3 with antioxidant effects, displayed lowered mitochondrial ROS and increased ΔΨm as well as, intriguingly, extended cell life span [Generally, a positive correlation exists between ΔΨm and mitochondrial ROS production . Howeverife span . This exife span . It was ife span .Opening and closing of mPTP formed on the inner mitochondrial membrane is involved in regulating calcium levels that are driven by ΔΨm . OpeningLimitations of this study include the use of the ARPE19 cell line as opposed to using primary or iPSC-derived RPE tissue, as well as allowing cells to go into suspension for electroporation instead of allowing them to form confluent and polarized monolayers. Although such alternative systems may have provided a higher level of physiological relevance, the choice of ARPE19 cells was founded on the critical need to achieve high levels of transient expression of fusion protein in a well-characterized model system. It is therefore important that future studies focus on validating results in primary tissue to understand the mechanistic relationship between variant B cystatin C and AMD development. Furthermore, although our experiments indicate a functional effect of variant B cystatin C on mitochondria, the exact mechanism and conditions involved need to be assessed in future studies to fully understand their impact on mitochondria and link with age-related diseases.In conclusion, we identified for the first time mitochondrial proteins that interact with variant B cystatin C. In addition, our data shows that the presence of variant B cystatin C impacts mitochondrial function, as observed through decreased mitochondrial ROS production and increased ΔΨm. Notably, other models that show similar results suggest these mechanisms are indicative of protection mechanisms for the cells. However, fold change mitochondrial ROS data (upon the addition of antimycin A) suggests that variant B cystatin C-expressing cells are more susceptible to detrimental ETC modulating stresses such as AGE, which accumulate with age.


This is especially relevant because the treatment response of tics can be variable across patients and often necessitates a combination of interventions.3 early behavioural model of tic control theorized that tics are voluntary motor responses to undesired somatosensory experiences known as premonitory urges, perpetuated because of negative reinforcement. Other theoretical frameworks conceptualized tics as prepotent actions that are not perceived as voluntary and are modulated by a voluntary inhibitory tic control that becomes increasingly efficient throughout development. For the generation of tics and premonitory urges, these later models called into play neural hubs that are primarily involved in the generation of voluntary actions and urges to act, i.e. medial frontal cortical areas—like the supplementary motor area (SMA) and the pre-SMA—the insular cortex and the striatum. Although experimental data have not yet provided a comprehensive mechanistic model of tic emergence, both clinical observations and behavioural data indicate that the interaction between the generation of actions, tics and volitional motor control lies at the core of tic pathophysiology.3Historically, there have been many models to explain the occurrence of tics. Bliss’5 thus suggesting potential overlap in neuroanatomical substrates between tic inhibition and action control. The right IFG and pre-SMA are engaged when preparing to stop an action and while stopping an action outright.6 Interestingly, this motor inhibition network is recruited across action-stopping tasks of variable complexity, suggesting that its involvement might be comparable during the suppression of both simple and complex tics.7 However, the neuroanatomical overlap between tic inhibition and action control poses an interesting neuroscientific question. How does the brain discern between voluntary actions and tics in order to achieve selective control of one type of motor output over the other?Voluntary tic inhibition may be mediated by a functional network that involves frontal cortical regions [inferior frontal gyrus (IFG) and pre-SMA] and the basal ganglia,et al.8 chose an experimental paradigm that allowed them to compare action and inhibition processes between adults with Tourette syndrome and age-matched healthy volunteers independent of tics. Their study exploited the computational granularity of MRI-based representational similarity analysis (RSA), which discerns the similarity and dissimilarity of neural responses within a brain region based on the stimuli received or task conditions. They performed representational similarity analysis in relation to externally cued and internally chosen action and inhibition motor outcomes (right index button presses), in six pre-selected regions that, as summarized above, are putatively involved in tic-related mechanisms .To explore this discriminatory ability, Rae et al.8 suggest that this indicates reduced accuracy in differentiating between these different action outcomes. Even with its limitations in statistical power, this study provides initial support to the authors’ hypothesis that the representation of action and inhibition within a network putatively controlling generation and inhibition of voluntary actions is more blurred in Tourette syndrome patients compared to healthy subjects.The study did not report any difference in behavioural performance on the externally cued selection of action and inhibition motor outcomes between the two groups of participants. Despite this equiperformance, the authors detected a greater representational similarity in Tourette syndrome subjects between externally cued action and inhibition, internally chosen action and inhibition, externally cued and internally chosen action and externally cued and chosen inhibition, all at the level of the right pre-SMA. Although statistical significance was reached only for the last of these contrasts, this finding suggests potentially similar neural activations between both action and inhibition and externally and internally cued motor programmes at the level of the pre-SMA. In turn, Rae At the same time, this observation stirs reflection and highlights several questions that are worthy of further investigation. The most obvious one, which the cross-sectional nature and the limited statistical power of this study do not allow to answer, is whether this blurring of contrast between motor outcomes is a primary, developmental ‘trait’ associated with Tourette syndrome or, rather, the ‘long-term outcome’ of exposure to tic behaviours. Interestingly, the representational similarity in pre-SMA did not correlate with tic severity or urge intensity, which were instead correlated with greater representational similarity in other regions , although only at a level of statistical trend.9 Finally, voluntary motor action/inhibition discrimination in this study was referred to a single body region (hand), therefore not exploring somatotopic differences that are important in tic disorders, given the inverse somatotopic gradient in tic expression and tic suppression.10Following up on the previous consideration, the design of this study does not allow to clarify whether a direct mechanistic link exists between the blurred neural representation of action and inhibition in pre-SMA and the voluntary control of tics. All the actions or action inhibitions processed by the participants of this experimental protocol were voluntary. By instructing Tourette syndrome participants not to intentionally suppress their tics, the voluntary motor outcomes used in the study were kept, to the best of the research team’s abilities, unconditioned by tic expression or tic suppression. However, it is possible that Tourette syndrome participants may have implicitly suppressed their tics during the scanning sessions, although this was not directly measured. Representational similarity of action and inhibition was not associated with tic expression during the experiment, indicating that the former is not a state marker of the propensity to tic. Furthermore, the presence of concurrent urges may have confounded the discrimination between externally and internally cued voluntary motor actions, but real-time urge intensity was not measured in the study. It is also striking that some of the pre-selected brain regions for this analysis, including pre-SMA, were not detected by recent lesion network maps identified from tic-inducing lesions.et al.8 may not directly expand our understanding of the mechanisms of tics and their suppression, they offer important insights with respect to the mechanisms of behavioural and neuromodulatory treatment strategies for tics. Both the habit reversal and the exposure–response prevention behavioural treatment approaches for tics instruct patients to treat internal cues (urges) as external commands to inhibit, ultimately teaching them to discern internal signals more accurately.3 Comparing representational similarity at the level of pre-SMA and of the IFG before and after these behavioural treatments might help elucidating the key mechanisms of behavioural therapy for tics or even assess whether this imaging marker is able to predict responsiveness to behavioural therapy. In a similar fashion, the change in representational similarity at the level of the pre-SMA in Tourette syndrome patients might support a rationale to use (facilitatory) non-invasive neuromodulation, for example targeting the IFG, to potentiate its inhibitory drive on motor output for treatment purposes. Ultimately, scoping the variability of prefronto–pre-SMA–basal ganglia connections across different subgroups and age groups of patients with tic disorders, and in relation to response to pharmacological therapies, will be necessary to confirm if the pattern of neural activation along this pathway is a useful physiomarker of tic disorders.While the findings from Rae


Proton exchange membrane (PEM) fuel cell faces the inevitable challenge of the cold start at a sub‐freezing temperature. Understanding the underlying degradation mechanisms in the cold start and developing a better starting strategy to achieve a quick startup with no degradation are essential for the wide application of PEM fuel cells. In this study, the comprehensive in situ non‐accelerated segmented techniques are developed to analyze the icing processes and obtain the degradation mechanisms under the conditions of freeze–thaw cycle, voltage reversal, and ice formation in different components of PEM fuel cells for different freezing time. A detailed degradation mechanism map in the cold start of PEM fuel cells is proposed to demonstrate how much degradation occurs under different conditions, whether the ice formation is acceptable under the actual operating conditions, and how to suppress the ice formation. Moreover, an ideal starting strategy is developed to achieve the cold start of PEM fuel cells without degradation. This map is highly valuable and useful for researchers to understand the underlying degradation mechanisms and develop the cold start strategy, thereby promoting the commercialization of PEM fuel cells. The degradation mechanisms under the conditions of freeze–thaw cycle, voltage reversal, and ice formation in different components of PEM fuel cells for different freezing time are revealed. The different degradation degrees and mechanisms under conditions are summarized into a degradation mechanism map, which can help researchers understand the underlying degradation mechanisms in various processes and develop the cold start strategy. As same as the other power sources used in transportation, such as internal combustion engines and lithium batteries, PEM fuel cell also faces the challenge of starting at a sub‐freezing temperature. Specially, the water as the only product will freeze during the cold start and thus highly affects the performance and durability of PEM fuel cell, which is the great limit of PEM fuel cell commercialization.During the transformation of global energy structure, emission‐free or nearly emission‐free electrification is essential in many fields, especially in transportation field.2, 3 Under a sub‐freezing temperature, the ice formation might block the reactant gas paths and cover the catalyst sites, thereby leading to the failure of cold start and the degradation of PEM fuel cell. In addition, the inevitable freeze–thaw cycle and the possible voltage reversal during the cold start will also affect the durability of PEM fuel cell. Nowadays, many countries and regions have put forward the specific standards on the durability of PEM fuel cell, such as the United States, Japan, and European Union. Therefore, the experimental research on the degradation mechanisms of PEM fuel cell during the cold start, especially caused by ice formation, is essential to develop the cold start strategies to suppress degradation, thereby improving the durability of PEM fuel cell.During the cold start, the water has several states, including vapor, liquid, super‐cooled water, ice, as well as frozen/non‐frozen membrane water, of which the phase change determines whether the cold start of PEM fuel cell can success.13 Und During the cold start, the ice formation, freeze–thaw cycle, and voltage reversal mainly cause the irreversible degradation of PEM fuel cell. Many experiments have been carried out to investigate the degradation phenomena during the cold start. Park et al. summarized the GDL degradation under the freeze‐thaw condition, including mechanical degradation and chemical degradation. Tabe et al. used a cryo‐scanning electron microscope to observe the cross‐sectional CL after the cold start and found that the region with ice formation moves from the membrane to the GDL during the freezing period. A simplified accelerated process with a PEM single‐cell was used to reveal the irreversible changes of the cathode during the repeated freeze cycles by Sabawa et al. Ozden et al. studied the degradation caused by the ice formation using the electrode specimens immersed in the deionized water under the continuous freeze–thaw cycles and found that the structural degradation of electrodes mainly depends on the ionomer type instead of the Pt loading. Zhong et al. studied the low temperature durability of a stack through segmented fuel cell and confirmed that the structure of electrodes is damaged due to the ice stress, resulting in the detachment and agglomeration of catalysts and even the delamination of CLs. However, these experiments adopted the ex‐situ test technique or in‐situ accelerated test technique, which is different from the real operating conditions. Moreover, the understandings of the underlying degradation mechanisms are scarce, due to the lack of comprehensive experimental techniques.The degradation of PEM fuel cell includes the reversible degradation, which can be mitigated by recovery procedures, and the irreversible degradation, which should be avoided. The irreversible degradation is usually caused by the irreversible damage to the structures of PEM fuel cell components, including membrane, catalyst layer (CL) and gas diffusion layer (GDL).25 DurTherefore, it is still necessary to develop the comprehensive experimental techniques to reveal the underlying degradation mechanisms and summarize which degradation during the cold start is acceptable. In this study, the comprehensive in situ non‐accelerated techniques, including electrochemical performance test, electrochemical surface area (ECSA) degradation test, current and temperature distribution measurements, and pore characterization, are developed to analyze the degradation mechanisms under the conditions of freeze‐thaw cycle, voltage reversal, and ice formation. By analyzing the degradation caused by different factors, we summarize the degradation mechanisms into the degradation mechanism map in cold start of PEM fuel cell. This map demonstrates whether the ice formation is acceptable under the actual operating conditions and how to suppress the ice formation, and helps researchers understand the degradation mechanisms and develop the starting strategy to migrate the degradation.2FigureTable Therefore, during the test of voltage reversal, the fuel cell first starts with the CC strategy and then artificially shuts down when the voltage reversal is observed (≈6 s). It is noteworthy that the water is not produced in the PEM fuel cell via the electrochemical reaction in this process.The icing‐independence processes, including the freeze–thaw cycle and voltage reversal, and the icing‐dependence processes, including the ice formation in catalyst coated membrane (CCM) and GDL, are studied using the in situ non‐accelerated segmented fuel cell techniques in this study Figure1. The  The constant voltage (CV) strategy is employed to avoid the voltage reversal in these processes. The first strategy is to make the cold start quickly fail to ensure that the produced water only freezes in CCM and cannot flow into GDL. Then the PEM fuel cell is directly heated up above 0 °C using the auxiliary heating device, or continuously freezes for 9900 s and then is heated up, to investigate the degradation caused by the icing in CCM for different freezing time. To achieve the ice formation in GDL, the second strategy is to make the PEM fuel cell work for a period to ensure that the produced water can flow into GDL. It can be judged whether the water flows into GDL based on the cathode pressure drop (CPD). Then the PEM fuel cell is frozen for 180 or 9900 s, followed by the heating up, to investigate the degradation caused by the icing in GDL for different freezing time. Based on our previous work, the PEM fuel cell is frozen for 180 s to ensure that ice forms in the GDL. At this time, the order of magnitude of the 180 s is at 102 s. In order for the ice stress to be fully effective, we set the PEM fuel cell to freeze for 104 s. Considering the convenience of timing, we choose 9900 s as the freezing time, i.e., 2 h and 45 min. Finally, the strategy to suppress the ice formation and achieve the cold start without degradation is discussed, and the degradation mechanisms are summarized to guide the starting strategy development.Furthermore, the degradation caused by ice formation is explored when the electrochemical reaction occurs in the PEM fuel cell. Based on our previous work, two cold start strategies are designed to form the severe icing in CCM and GDL, respectively, to investigate the degradation.32 TheMore experimental details are provided in the Supporting Information, including Figures 2.1FigureFreeze–thaw cycle is an inevitable process in the cold start of PEM fuel cell. Therefore, the degradation caused by freeze–thaw cycle is first investigated. The black line in 2.22 outlet, where the current density is high and a lot of heat is generated in a short time due to the voltage reversal. The highest current density and temperature raise to 2.13 A cm−2 and −6.81 °C, respectively, as shown in the black boxes in Figure 2 outlet.Generally, the cold start with the CC strategy is better than that with the CV strategy. However, the voltage reversal easily occurs in the first few seconds when starting with the CC strategy. Therefore, the degradation caused by voltage reversal is investigated in this subsection. As shown in Figure −2 and −7.94 °C, respectively, as shown in the black boxes in Figure 2 outlet, despite the same operating condition. It means that the gas starvation can possibly occur at the side of PEM fuel cell besides at the H2 outlet. Although the voltage reversal can provide a lot of heat for the cold start, it should be avoided due to the severe ECSA degradation. In future, developing the novel catalysts with a high tolerance of voltage reversal can help reduce the degradation.Interestingly, the voltage reversal is also observed at the side of PEM fuel cell in few experiments, as shown in Figure radation.342.3FigureA lot of ice formation will not only lead to the failure of cold start but also cause the serious degradation of PEM fuel cell. The water is generated in the CCM when the electrochemical reaction occurs. Therefore, the icing in CCM is explored to know whether it is acceptable during the cold start. We also consider the long‐time icing in CCM when the PEM fuel cell is frozen at a sub‐freezing temperature environment for a long time. The PEM fuel cell starts with a CV strategy at 0.45 V from −15 °C, which leads to a quick failure of cold start due to the severe icing in CCM. One PEM fuel cell is directly heated up after failure, while another one continues to be frozen for 9900 s before heating up. Figure  The HFR is appropriately 1.9 mΩ when the electrolyte and membrane are sufficiently wetted, while it is higher than 1.9 mΩ when the fuel cell is frozen, which means that the ice formation will affect the proton transport in CCM. The HFR values under the short‐time icing and long‐time icing condition are nearly the same, which indicates that long‐time icing does not lead to more ice formation than short‐time icing. Therefore, the more severe degradation under the long‐time icing condition in CCM is ascribed to the long presence of ice instead of more ice.The hydration of electrolyte and membrane in CCM can be represented by the high frequency resistance (HFR) value as shown in Figure 36 TheIn addition, the heating time under the long‐time icing condition in CCM is longer than that under the short‐time icing condition in CCM, as shown in Figure Furthermore, the amount of charge produced by electrochemical reaction can represent the amount of produced water. We collect the amount of accumulated charge in the 27 segments of fuel cell after every cycle, as shown in Figure 2.4Figure Then the PEM fuel cells are frozen for 180 and 9900 s, respectively, to investigate the effects of short‐time and long‐time icing in GDL. As shown in Figure In order to investigate the icing in GDL, the PEM fuel cell starts with a CV of 0.3 V at −10 °C, to ensure that the produced water can flow from CCM into GDL. As shown in The HFR values of different conditions are measured to further understand the underlying mechanism. The comparison of HFR values under the short‐time icing conditions in CCM (blue line) and GDL (light green line) is shown in Figure  Therefore, the porosity in segment 14 is the largest, while that in segment 20 is least. To furthermore analyze the underlying mechanism, the accumulated charges of segment 5, that is located above the segment 14, and segments 3, 14, and 20 are compared as shown in Figure The accumulated charges in each segment of the fuel cell with icing in GDL are counted and their distributions are shown in Figure 25 TheThe degradation mechanism caused by long‐time icing in GDL is summarized and shown in Figure 2.5−2, are investigated. Figure−2), a failure process caused by flooding (0.5 A cm−2), and a success process with local ice formation (0.8 A cm−2), respectively. Figure Based on the above analyses, the ice formation will significantly lead to the degradation of PEM fuel cells. Therefore, suppressing the ice formation through developing the starting strategy is necessary during the cold start of PEM fuel cells. In this subsection, the ice formation phenomena under the different starting current densities, including 0.2, 0.5, and 0.8 A cm2.6FigureThe effects of freeze–thaw cycle, voltage reversal, and ice formation on the degradation of PEM fuel cells are investigated in this study. The degradation mechanism map considering different processes is shown in 2 outlet. A higher staring current density is preferred to quickly warm the PEM fuel cells without ice formation and achieve the ideal cold start without degradation.The degradation mechanism map proposed in this study is highly valuable. First, this map can help researchers understand the underlying degradation mechanisms in various processes. Furthermore, this map can also help develop the starting strategy with the low experiment and time cost. For example, the linear degradation, i.e., degree I shown in Figure 3In this study, we investigated the degradation mechanisms under the freeze–thaw cycle, voltage reversal, and ice formation in CCM and GDL using in situ non‐accelerated test techniques, including electrochemical performance test, ECSA degradation test, pore characterization, and current density/temperature distribution measurement. Based on the experimental results, the degradation under the freeze–thaw cycle is slight and acceptable, while that under the voltage reversal is avoidable using the novel catalysts with a high voltage reversal tolerance. Moreover, the ice formation in different components of PEM fuel cells for different freezing time will lead to different degradation degrees. The long‐time icing in CCM will lead to a more severe degradation than the short‐time icing due to the influence of long‐time ice stress, although the amount of ice is the same. The water flows into GDL when more water is generated in CCM, which tends to slow down the icing process in CCM and decrease the degradation to some degree. However, long‐time freezing of water in GDL damages the GDL structure and increases the porosity of GDL, which leads to more water stored in GDL and thus more severe degradation due to the more and quicker icing in the next cold start. Therefore, the ice formation is the major factor that causes the degradation during the cold start of PEM fuel cells. A higher starting current density is preferred to avoid the ice formation and achieve the ideal cold start without degradation. Degradation mechanism map in the cold start of PEM fuel cells is summarized in this paper, which can help researchers understand the underlying degradation mechanisms in various processes and develop the cold start strategy.The authors declare no conflict of interest.J.L. and K.J. conceptualized the study; J.L., L.F., Q.D., Y.Y., and K.J. contributed to methodology; J.L. performed investigations; L.F. and K.J. acquired resources; J.L. and L.F. wrote the original draft; J.L., L.F., and K.J. reviewed and edited the final manuscript; K.J. supervised the study; L.F., Q.D., and Y.Y. acquired funding.Supporting InformationClick here for additional data file.


To study their role(s) in cyanobacteria, we investigated a collection of 11 completely segregated spk mutants among the 12 annotated Spks in the model cyanobacterium Synechocystis sp. PCC 6803. Screening of the mutant collection revealed that especially the mutant defective in SpkB encoded by slr1697 showed clear deviations regarding carbon metabolism, that is, reduced growth rates at low CO2 or in the presence of glucose, and different glycogen accumulation patterns compared to WT. Alterations in the proteome of ΔspkB indicated changes of the cell surface but also metabolic functions. A phospho-proteome analysis revealed the absence of any phosphorylation in two proteins, while decreased phosphorylation of the carboxysome-associated protein CcmM and increased phosphorylation of the allophycocyanin alpha subunit ApcA was detected in ΔspkB. Furthermore, the regulatory PII protein appeared less phosphorylated in the mutant compared to WT, which was verified in Western blot experiments, indicating a clearly delayed PII phosphorylation in cells shifted from nitrate-containing to nitrate-free medium. Our results indicate that SpkB is an important regulator in Synechocystis that is involved in phosphorylation of the PII protein and additional proteins.Protein phosphorylation   •via serine/threonine protein kinases (Spk) is a widespread mechanism.Protein phosphorylation •spk mutants was investigated in Synechocystis PCC 6803.A collection of 11 completely segregated •slr1697 showed clear deviations regarding carbon metabolism.Especially the mutant defective in SpkB encoded by •A phospho-proteome analysis revealed the absence of any phosphorylation in two proteins.•II protein appeared less phosphorylated in the mutant compared to WT.Furthermore, the regulatory P Synechocystis sp. PCC 6803. A subsequent phospho-proteome analysis revealed the absence of any phosphorylation in two proteins, while decreased phosphorylation of the carboxysome-associated protein CcmM and the regulatory PII protein was observed. Our results indicate that SpkB is an important regulator of the carbon/nitrogen metabolism in Synechocystis.Mutation of the serine/threonine protein kinase SpkB resulted in many phenotypical alterations in the cyanobacterium  Then e.g., , 9). The. The2-con , 12 different Spks have been annotated in its genome (http://genome.microbedb.jp/cyanobase/GCA_000009725.1) , but the.g., ). Among 5.1) see , 17. In st years , 19, 20,spk mutants in Synechocystis revealed that SpkE is associated with the response to cold stress, while the eukaryotic-like kinases SpkC and SpkF are involved in phosphorylation of GroES  of Spks in Synechocystis sp. strain PCC 6803M (WT M)   or straiM (WT M) . The kinM (WT M) . The ideM (WT M) . All str2  at 30 °C and 120 μmol photons m−2 s−1 for 3 days. Eventually, Synechocystis suspension was adjusted to an optical density at 720 nm (OD720) of 0.2 with BG 11 TES pH 7.0 and grown under ambient air . The growth was monitored with the Multi-Cultivator MC-1000-OD system (Photon Systems Instruments), in which the OD720 increase was continuously recorded at 30 °C and 100 μmol photons m−2 s−1 for 4 days. Samples from HC- and LC-acclimated cells were used to record absorption spectra from 400 to 750 nm. Spectra data were concomitantly utilized for pigment quantification. Raw growth data were normalized to their start OD720 (OD720N = OD720n/OD7200) and subsequently normalized to the WT from 0 to 1 (OD720NMut = (OD720Mut − OD720NWTmin)/(OD720NWTmax − OD720NWTmin)).WT and mutant cells were precultivated in glass tubes filled with BG11  aerated−2 s−1 and 30 °C for 7 days. Ahead of the growth experiment, cultures were adjusted to OD750 of 0.5 with BG11 (TES pH 8.0) and subsequently split into two separate cultures with one supplemented with 10 mM glucose whereas the remaining served as a control. The strains were grown shaking at ambient air with 35 μmol photons m−2 s−1 and 30 °C. Growth was monitored by measuring manually the OD750 using a photometer every 24 h until the cultures reached the stationary growth phase. Raw growth data were normalized to their start OD750 and subsequently normalized to the WT from 0 to 1 (OD750NMut = (OD750Mut − OD750WTmin)/(OD750WTmax − OD750WTmin)).Cells of WT and mutants deficient in Spks were inoculated from material maintained on solid medium into Erlenmeyer flasks filled with 100 ml BG11. The strains were precultivated shaking (140 rpm) at ambient air (LC) with 35 μmol photons m−2 s−1 in an LC environment until they reached the desired OD750. Upon drop dilution assay, the suspension was adjusted to the OD750 of 0.2 with BG11 (TES pH 8.0). Then, the suspension was serial diluted to 1:10, 1:100, or 1:1000. Two microliters of the dilution series were spotted on solid BG11 plates  containing different supplements. For mixotrophic conditions, 10 mM glucose and for a salinity environment 500 mM NaCl were added to the medium. Plates were either incubated at 30 °C and 100 μmol photons m−2 s−1 at continuous light  or at diurnal light conditions (12 h light/12 h darkness) at 30 °C and 75 μmol photons m−2 s−1 for 4 days. Pictures were taken together with their respective control plate.Kinase-deficient mutants together with their respective WT were precultivated in shaking flask with BG11 (TES pH 8.0) at 30 °C and 100 μmol photons m2O2. Strains were precultivated in shaking flasks in BG11 (TES pH 8.0) at 30 °C and light of 100 μmol photons m−2 s−1 in an LC environment until they reached the desired OD750. Cell suspensions were then adjusted to OD750 of 0.4 and probed with either 3 mM or 4 mM of H2O2 followed by an incubation under grow light conditions for 1 h. After H2O2 incubation, the suspension was serial diluted to 1:10, 1:100, or 1:1000. Two microliters of the dilution series were spotted on solid BG11 plates . Surviving cells were recovered for 4 days at 30 °C and 100 μmol photons m−2 s−1 at constant light. Pictures were taken together with their respective control plate.To test the sensitivity of the mutant strains towards externally supplied reactive oxygen species (ROS), cells were probed with increasing amount of HSynechocystis was precultivated in a batch culture under HC (BG11 TES pH 8.0) conditions at 30 °C and 100 μmol photons m−2 s−1 continuous light until desired OD750 of 1 was reached. Upon shift to LC, cells were spun down  and resuspended in BG11 TES pH 7.0. Synechocystis was further cultivated under LC condition for 24 h. Samples for glycogen quantification were taken at an HC-acclimated state and 3 h and 24 h of LC acclimation. Cellular glycogen content was determined by applying the method described  until desired OD750 was reached. The night before the initial shift, cells were adjusted to OD750 of 1 in BG11 (TES 7.0) and transiently acclimated to LC conditions. Before the NO3 deprivation, cells were harvested  and immediately snap frozen in liquid nitrogen until further processing. Remaining cells were spun down  and resuspended in BG11 TES pH 7.0 without the addition NaNO3. Samples were taken 20, 40, 60, or 120 min after the initial nitrogen shift and snap frozen in liquid nitrogen.Cells of the II-antibody  and subsequently broken by sonicating . The disrupted cell suspension was then spun down for 20 min at 3000 rpm and 4 °C. The crude extract was used for quantifying total protein content (ROTI Nanoquant). Crude extracts were probed with native sample buffer  glycerol, 0.0015% (v/v) bromophenol blue) in a 3:1 ratio. Ten micrograms of crude protein extract were loaded on a native-PAGE  and separated for about 3 h at 170 V. Proteins were transferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific) using a semidry blot system (Thermo Fisher Scientific) applying a constant current of 6 V for 90 min. The immunoblot was probed with a Pantibody  that was3−-dependent photosynthetic O2-evolution rates were quantified using an S1 Oxygraph (Hansatech Instruments). Suspensions of HC- or LC-acclimated cells were adjusted to Chla 10 μg ml−1 with CO2-free BG11. Three milliliters of the adjusted suspension were used to quantify O2 evolution rates at 30 °C and at a saturating light intensity of 300 μmol photons m−2 s−1 in the presence of increasing HCO3− concentrations . Photosynthetic rates at each HCO3− concentration were recorded at 30 s intervals.HCO−2 s−1 until desired OD750 of 1 was reached. Upon shift, cells were spun down  and resuspended in either BG11 TES pH 7.0 or BG11 TES 8.0. Cells were further cultivated under either LC or HC condition for 24 h. A 5 ml sample was taken at a long-term HC- and LC-acclimated state and after 1, 3, 6, or 24 h of acclimating to LC and HC environment, respectively. Cells were harvested by quick filtration on nitrocellulose filters . Metabolites were quantified as described  or HC (BG11 TES pH 8.0) conditions at 30 °C and continuous light of 100 μmol photons mpkB were precultivated in glass tube batch cultures under HC conditions (5% CO2) in buffered BG11 medium (TES pH 8.0) at 30 °C and 120 μmol photons m−2 s−1 until the cell suspension reached OD750 of ∼1. The cultures were transferred daily into fresh BG11 medium to avoid nutrient limitation. Two days before the shift experiment, the precultures were adjusted to OD750 of 1.0 and split into three individual cultures that were kept under HC conditions. After growth for 24 h, the cell suspensions were again readjusted to OD750 of 1.0. Cells were harvested by centrifugation and resuspended in fresh BG11 medium to inoculate six individual cultures of 120 ml with an OD750 of 1.0. To allow recovery and acclimation to the new conditions, cells were aerated with 5% CO2 in the light for 2 h before HC samples were taken. For harvesting, 40 ml cell suspension were separated from each individual culture and split into two equal portions and transferred into 50 ml tubes filled with ice for rapid inhibition. The cells were immediately pelleted by centrifugation . Cell pellets were washed with 7 ml ice-cold PBS-buffer, snap-frozen in liquid nitrogen, and stored at −80 °C until further processing. For the shift to LC conditions, the medium of the remaining culture was removed by centrifugation and the cells were resuspended in the same volume of BG11 (TES pH 7.0) and further cultivated with ambient air (0.04% CO2) aeration. Samples from LC-shifted cultures were harvested after 3 h and 24 h. Three independent cultivations were conducted for replicated phospho-proteome analyses.Cells of the WT and mutant Δsg, 15 min, 20 °C) and the supernatant was subjected to protein precipitation with acetone/methanol (8 + 1 sample volume) at −20 °C overnight. Protein pellets were washed using ice cold 80% v/v acetone until the supernatant was colorless and air dried, followed by resuspension in 2 ml urea buffer . Protein concentrations were measured by Bradford assay and 3 mg protein per sample were digested to peptides at room temperature using proteomics grade endoproteinases Lys-C  and trypsin (30 μg) diluted in four sample volumes of 1 mM Tris–HCl, pH 8. After overnight incubation, peptide solutions were acidified with TFA to pH 2.5. One third (1 mg) of the material was separated from each sample and added to a standard mixture, containing equal fractions from all conditions and replicates. Subsequently, samples and standard mixture were loaded on C18 columns  and dimethyl labeled as described previously (spkB (intermediate label) from the same condition as well as the standard mixture (heavy label) for quantitative comparison between different conditions, as outlined in the experimental design (2 chromatography using Sachtopore-NP 5 μm titania spheres (ZirChrom Separations) in a 10:1 peptide:TiO2 ratio in 6% v/v TFA/80% v/v acetonitrile solution for 10 min. For each mix, eight consecutive enrichment rounds were performed, of which rounds 1 + 2, 3 to 5, and 6 to 8 were pooled, since our previous studies using this method  supplemented with phosphatase inhibitors  to 95 °C for 10 min, followed by sonification for 30 s using a micro-tip. Sample extracts were then reduced in presence of 10 mM DTT and alkylated in the presence of 5.5 mM iodoacetamide in the dark, each for 1 h at room temperature. Samples were centrifuged  revealed  in the positive-ion mode. During data-dependent acquisition, MS spectra were acquired with an m/z range of 300 to 1750 at resolution 60,000. The 20 most intense multiple charged ions were selected for fragmentation by higher-energy collisional dissociation and fragment ions were recorded at MS2 resolution 30,000. Automatic gain control targets and maximum injection times were set for MS and MS2 scans to instrument setting “standard” and “auto”, respectively. Dynamic exclusion of previously sequenced precursor ions was set to 30 s in all measurements.For MS measurements, 250 ng material from proteome fractions and all available material from pooled phosphopeptide enrichment fractions were loaded onto a C18 nanoHPLC column , in-house packed with ReproSil-Pur 1.9 μm C18 material (Dr Maisch), and separated by RP-chromatography on an EASY-nLC 1200 system using 60 min segmented linear gradients at a constant flow rate of 200 nl minhttps://www.maxquant.org/) using the following search parameters: Trypsin was defined as a cleaving enzyme and up to two missed cleavages were allowed. Dimethylation on peptide N-termini and lysine residues was defined as light (+28.03 Da), intermediate (+32.06 Da), and heavy (+36.08 Da) labels. Carbamidomethylation of cysteines was set as fixed modification, and methionine oxidation, protein N-terminal acetylation and phosphorylation of serine, threonine and tyrosine residues were set as variable modifications. “Requantification,” “match between runs,” and “iBAQ” were enabled. The initial mass tolerance of precursor ions was limited to 20 ppm and 0.5 ppm for fragment ions. False discovery rates of (modified)peptides and proteins were limited to 1% each. Protein groups identified based on single peptides were only accepted with comprehensive fragment ion series , and the standard mixture  were present. Nine proteome fractions and three pooled phosphopeptide fractions per replicate resulted in 36 samples (27 proteome and nine phospho-proteome measurements) per condition. The total of 108 samples from all experimental conditions was processed in MaxQuant with an experimental design defining condition and replicate of each sample (https://maxquant.net/perseus/). For the direct comparison of ΔspkB to WT at each condition , normalized M/L label ratios were analyzed. For the strain-specific comparison of the proteome between different conditions, normalized ratios relative to the H-labeled standard mixture were calculated as 1/[(H/X LC condition)/(H/X HC condition)], with X = L for WT and X = M for ΔspkB, respectively. Student’s t test was used to determine proteins and p-events with significantly changed abundances compared to the overall distribution between independent replicates. A p-value ≤0.05 was defined as cutoff in all analyses.MS-based phospho-proteome analyses were performed for each experimental condition  separately in three independent biological replicates. In each replicate, triplex dimethyl labeled peptides of the WT , strain Δh sample . Statistspk genes were interrupted by an antibiotic gene cartridge using appropriate restriction sites as done in a previous study to analyze the role of Spks in Synechocystis (sll0005 encoding SpkH could never be completely removed from the mutant genome. These results indicated that SpkH or other protein(s) encoded in the sll0005-containing operon is essential for viability of Synechocystis under our laboratory conditions, whereas all other Spks are dispensable.The coding sequences of all annotated hocystis . Subsequhocystis . Only onspk mutants was then screened under different growth conditions. Initially, the ability of all mutants to acclimate to LC conditions was analyzed. To this end, mutant strains were precultured under HC conditions and then transferred to LC conditions for 4 days in continuous light. In addition to the previously characterized mutant ΔspkC .The collection of 11 completely segregated nt ΔspkC , the mutnding WT . Especia culture . The slod pVZ322 . Growth spkE showed slower growth than WT under diurnal conditions, whereas all other strains grew similar as WT . Only mutant Δar as WT . Next, tor ΔspkI . Another than WT .Synechocystis is not only able to grow photoautotrophic but can also use external glucose  in cells of the mutant ΔspkB compared to WT , the latter also comprises an SLH domain and might be functionally connected to the S-layer (slr2015-17) that are essential for cell motility are found at higher abundances in mutant ΔspkB, whereas the amount of the type 4 pilin-like protein Slr1929 is strongly diminished  and the alkaline phosphatase Sll0654 accumulated in ΔspkB, which point at a phosphate limitation in the mutant because these three proteins belong to the phosphate regulon in Synechocystis  were found among the proteins with lowered abundance in mutant ΔspkB  were reliably identified on 115 different proteins , whereas other multiply phosphorylated peptides reveal one constant and several occasional p-events   . Due to or T387) .spkB  subunit PsaE was significantly lower at LC conditions in ΔspkB, while the corresponding protein levels were only slightly reduced. PsaE is situated at the donor site of PSI and plays an important role in the ferredoxin docking and cyclic electron transport  protein at S49 was strongly reduced in ΔspkB compared to the WT when grown at HC and especially when shifted for 3 h to LC  at T31. Two further p-sites showed similar trends at HC and partially at LC . Intere h to LC . The PIIiewed in ). Contraly at LC . ApcA isly at LC .Fig. 5Que.g., . Subseqynthesis .II phosphorylation states between WT and ΔspkB by LC-MS/MS, we aimed to verify this observation by Western-blotting after protein separation under native conditions. This technique permitted the distinct visualization of the differentially phosphorylated PII trimers, which can be separated in four bands . Thus, ed to WT . These rSynechocystis and are summarized in the database CyanoEXpress (http://cyanoexpress.sysbiolab.eu/). In several cases, a good correlation between the transcriptome and proteome has been reported for Synechocystis, for example, after high salt or low CO2 stress . Th. ThSynec2 supply . One oftbacteria . TherefoSynechocystis under our test conditions, whereas other spk mutations resulted in clear phenotypic alterations  are more abundant in cells with limited Ci availability . Such cy . Howeve  and Slr0483 (T34 or T35) were exclusively found at different Ci conditions in WT samples but never in ΔspkB, which highlights them as potential substrates of SpkB. Slr1545 is the glutathione S-transferase, Gst1, which plays important role in the redox regulation of proteins among cyanobacteria . Previously, the GlyS (glycyl-tRNA synthetase subunit beta), a protein from the same functional group as Slr0483 that, however, was found to be nonphosphorylated in the present study, has been reported as SpkB substrate  and coubacteria . Phycobibacteria . ApcA isbacteria . Changedbacteria . HoweverII protein was detected in the phospho-proteome analysis and supported by Western blots detecting the different PII phosphorylated trimers (II protein in cyanobacteria (II has been identified as PphA (II-specific Spk. It has been shown that cells grown under LC conditions in N-rich BG11 medium show the least PII phosphorylation, which is then triggered in response to N-limitation (II phosphorylation in the mutant ΔspkB and WT. Corresponding to the putative role of SpkB as PII-specific kinase, the PII phosphorylation level was always lower than in WT. Especially after shift into N-free BG11 medium, a substantial amount of PII remained nonphosphorylated (see II became still phosphorylated, which points to the presence of a substituting kinase that can phosphorylate PII with lower efficiency. It should be noted that similar observations were made in a mutant of S. elongatus PCC 7942, in which the spkB ortholog synpcc7942_1294 was inactivated (II in the mutant ΔspkB could also explain the observed changes in the PII phosphorylation level. Clearly, more experiments are needed in the future, especially in vitro phosphorylation assays of pure SpkB and PII protein with and without PphA, to reveal whether or not SpkB is indeed a specific PII kinase or if the observed phosphorylation changes are indirectly related to the mutation of spkB.Finally, significant less phosphorylation at S49 of the P trimers  and 6. Dbacteria , the inv as PphA . Hence, mitation , 45. Theated see . Neverthctivated . HoweverII phosphorylation was recently observed in a mutant defective in the histidine kinase Hik8 (Synechocystis (II in the hik8 mutant was accompanied by a significantly reduced glycogen accumulation, which was reversed to WT levels by expression a PII variant with mutated S49 phosphorylation site (spkB with decreased PII phosphorylation at S49 contains more glycogen under HC conditions. However, PII is not directly acting on glycogen synthesis or carbon metabolism. Recently, the PII-interacting protein PirC has been identified as key regulator to switch carbon flow between lower glycolysis and glycogen accumulation because PirC can inhibit phosphoglycerate mutase under N-limiting conditions when the accumulation of the metabolic regulator 2-oxoglutarate releases it from its PII-bound state (2 FC −0.6, below our significance threshold, spkB than WT under HC conditions, while the PirC and the glycogen amounts are similar 24 h after LC shift. Hence, in addition to the changed PII phosphorylation, the changes of PirC amounts in mutant ΔspkB could also contribute to the observed alterations in glycogen contents.A strongly increased Pase Hik8 , which hhocystis , 53. Intion site . These fnd state . Our provia Spks play an important role in stress acclimation of cyanobacteria. Among the annotated Spks, SpkB seems to be of particular importance because its absence directly or indirectly results in many phenotypic alterations and changes in the phospho-proteome. However, it seems to act in a kinase network as it is exemplified in the PII phosphorylation. The action of multiple kinases on specific target proteins in Synechocystis has also been shown in the phosphorylation of GroES (via acetyl phosphate (Synechocystis proteins (Synechocystis, which can regulate protein activities such as RubisCO but also the PII protein in a Ci-dependent manner (Collectively, our results strongly support the notion that protein phosphorylation of GroES . This coof GroES . Furtherhosphate , is muchproteins . Finallyt manner . Summarivia the PRIDE (The original contributions presented in the study are included in the article and the associated he PRIDE  partner This article contains The authors declare no competing interests.


Ubiquitin–proteasome-system-mediated clearance of misfolded proteins is essential for cells to maintain proteostasis and reduce the proteotoxicity caused by these aberrant proteins. When proteasome activity is inadequate, ubiquitinated proteins are sorted into perinuclear aggresomes, which is a significant defense mechanism employed by cells to combat insufficient proteasome activity, hence mitigating the proteotoxic crisis. It has been demonstrated that phosphorylation of SQSTM1 is crucial in regulating misfolded protein aggregation and autophagic degradation. Although SQSTM1 S403 phosphorylation is essential for the autophagic degradation of ubiquitinated proteins, its significance in proteasome inhibition-induced aggresome formation is yet unknown. Herein, we investigated the influence of SQSTM1 S403 phosphorylation on the aggresome production of ubiquitinated proteins during proteasome suppression.We examined the phosphorylation levels of SQSTM1 S403 or T269/S272 in cells after treated with proteasome inhibitors or/and autophagy inhibitors, by western blot and immunofluorescence. We detected the accumulation and aggresome formation of ubiquitinated misfolded proteins in cells treated with proteasome inhibition by western blot and immunofluorescence. Furthermore, we used SQSTM1 phosphorylation-associated kinase inhibitors and mutant constructs to confirm the regulation of different SQSTM1 phosphorylation in aggresome formation. We examined the cell viability using CCK-8 assay.Herein, we ascertained that phosphorylation of SQSTM1 S403 did not enhance the autophagic degradation of ubiquitinated proteins during proteasome inhibition. Proteasome inhibition suppresses the phosphorylation of SQSTM1 S403, which facilitated the aggresome production of polyubiquitinated proteins. Interestingly, we found proteasome inhibition-induced SQSTM1 T269/S272 phosphorylation inhibits the S403 phosphorylation. Suppressing S403 phosphorylation rescues the defective aggresome formation and protects cells from cell death caused by unphosphorylated SQSTM1 (T269/S272).This study shows that inhibition of SQSTM1 S403 phosphorylation facilitates the aggresome formation of ubiquitinated proteins during proteasome dysfunction. SQSTM1 T269/S272 phosphorylation inhibits the S403 phosphorylation, boosting the aggresome formation of ubiquitinated protein and shielding cells from proteotoxic crisis.The online version contains supplementary material available at 10.1186/s11658-023-00500-6. Removing misfolded proteins by the ubiquitin–proteasome-system (UPS) is essential for cells to maintain proteostasis and reduce the proteotoxicity caused by these aberrant proteins , 2. WhenAggresome formation is a complex biological process governed by numerous functional factors . AlthougAs multifunctional scaffold proteins, SQSTM1 is a key regulator of aggrephagy and aggresome formation , 22. SQSIn this report, we examined the influence of SQSTM1 S403 phosphorylation on the aggresome production of ubiquitinated proteins during proteasome suppression. We demonstrated that phosphorylation of S403 inhibits the proteasome inhibitor-induced aggresome production of polyubiquitinated proteins. T269/S272 phosphorylation in SQSTM1 enhances aggresome formation by inhibiting S403 phosphorylation. Our findings indicate a crosstalk mechanism between SQSTM1 S403 and T269/S272 phosphorylation during ubiquitinated protein-associated aggrephagy and aggresome formation.MG132 ; Bafilomycin A1 ; Bortezomib ; CX-4945 ; BX795 ; SBI-0206965 ; Doramapimod ; Wortmannin .pcDNA3.1-SQSTM1 , pcDNA3.1-FLAG-p38δ  were constructed as described previously [l-glutamine  in a 5% CO2 incubator. All the cell lines were authenticated and tested for contamination. Plasmid transfection was carried out with Megatran  following the manufacturer's instructions.Wild-type AD293, SQSTM1 knockout AD293, and SQSTM1 re-expressing cell lines were obtained and constructed as described in previous studies , 20. HelTo generate stably re-expressed wildtype or mutated SQSTM1 cell lines, the DNA fragments corresponding to the ORFs of SQSTM1 were cloned into lentiviral vectors, then transfected into HEK293FT cells with helper plasmids to package lentiviral. Stable cell selection was carried out as described previously .The total protein extract was prepared by homogenizing the cells in 1 × SDS sample buffer. All the protein samples were boiled for 5 min and then resolved in SDS-PAGE. The protein samples separated by SDS-PAGE were transferred to the Immunobilon-FL PVDF membrane . After blocking with 5% non-fat milk in TBST , the membrane was incubated with primary antibody at 4 ℃ overnight. The membrane was washed and incubated with secondary antibodies in the dark for 2 h. The image was acquired by Li-Cor Odyssey Clx Infrared Imaging System . The following primary and secondary antibodies were used for western blotting assay: SQSTM1 , ubiquitin ; Phospho-SQSTM1 (Thr269/Ser272)-specific antibody ; Phospho-SQSTM1 (S403)-specific antibody ; FLAG tag ; GAPDH ; β-actin . Dylight 680, or Dylight 800-conjugated secondary antibodies . See Additional file Immunostaining was performed as described previously . In brieCells were seeded into 96-well plates. After treated with the indicated reagent, the cell viability was determined by Cell Counting-8 Kit  as recommended by the manufacturers. The efficacy of drugs on cell growth was normalized to untreated control.t test (2 groups) or one-way ANOVA analysis (> 2 groups). Differences were considered statistically significant when P < 0.05. All statistical analyses were performed using Graph Prim 7.0.Each experiment was performed at least three times. Western blot was measured by ImageJ. Fluorescence images from aggresome formation results were quantified manually. Data were represented as mean ± SEM. Comparisons between individual data points were made using a two-tailed Student Since SQSTM1 S403 phosphorylation is essential for the aggrephagy of ubiquitinated proteins , we studNext, we examined whether the phosphorylation of SQSTM1 S403 influences the autophagic degradation of ubiquitinated proteins during proteasome suppression. As depicted in Fig. Next, we explored whether SQSTM1 S403 phosphorylation influences the proteasome inhibition-induced aggresome generation of ubiquitinated proteins. According to previous research, the rate of aggresome production in Hela cells was slower than AD293 and A375 cells , 20. IntAccording to previous work, autophagic sequestration of SQSTM1 prevented the formation of ubiquitinated protein aggresomes . We assuOur previous studies have revealed that compared to AD293 cells and A375 cells, the SQSTM1 T269/S272 phosphorylation was limited in Hela cells due to the much lower expression of p38γ/δ that can phosphorylate SQSTM1 at T269/S272 under proteasome inhibition . TherefoWe predicted that SQSTM1 S403 phosphorylation would limit the aggresome formation of ubiquitinated proteins and suppress T269/S272 phosphorylation-enhanced aggresome formation. Next, we examined the impact of S403 phosphorylation on T269/S272 phosphorylation-regulated aggresome production. First, we discovered that a mutation of S403A can repair the impairment in the aggresome formation of ubiquitinated proteins caused by the T269A/S272A mutant Fig. A, B. We Since aggresome formation protects against cell death caused by proteasome inhibition, we wondered if blocking S403 phosphorylation might protect cells against proteasome inhibitor-induced cell death. To investigate this, we first studied the effect of SQSTM1 mutants on the cell viability of SQSTM1 knockout cells after being treated with proteasome inhibitors. Figure Phosphorylation alters SQSTM1's biological function. The S403 phosphorylation can enhance the affinity of SQSTM1 to ubiquitinated proteins and promote their degradation by aggrephagy . HoweverProteasome inhibition eliminates misfolded protein-related proteotoxic crises in two steps. One is that when proteasome activity is inhibited, misfolded proteins are sequestrated into aggresome, considered the critical protective mechanism in cells at the early stage of proteasome inhibition , 28. AggSQSTM1 S403 phosphorylation is an important regulatory point in the aggrephagy of ubiquitinated proteins. However, S403 phosphorylation enhances the autophagic breakdown of ubiquitinated proteins, Shinrye Lee et al. reported that in neuronal cells, S403A mutants can decrease cell damage by enhancing the autophagic clearance of insoluble ubiquitinated proteins during UPS dysfunction . MoreoveWe previously found that SQSTM1 T269/S272 phosphorylation during proteasome inhibition prevents autophagic sequestration and promotes the aggresome formation of ubiquitinated protein by weakening its binding to LC3B . Here, wIn conclusion, SQSTM1 T269/S272 phosphorylation inhibits S403 phosphorylation during proteasome inhibition, boosting the aggresome formation of ubiquitinated protein and shielding cells from proteotoxic crisis. Our research showed that SQSTM1 phosphorylation links autophagic degradation and aggresome production of ubiquitinated proteins.Additional file 1: Table S1. Sequences of the primers used for ORF amplification. Table S2 Antibody information.


Colon cancer is a major health problem, and its occurrence is rising particularly among young adults. Preventing this cancer is urgently needed. Earlier studies in animal models as well as on human have shown promising preventive effect of agents like Sulindac and DFMO. Unfortunately, the long-term use of these agents at high doses is associated with some side effects, thus alternative strategies are being explored to employ these drugs for preventing CRC. In the present study, we used approaches such as (i) Combination of low dose of the two agents, (ii) testing the efficacy of sulindac derivatives. For this colon cancer was chemically induced in rats, later the test agents, Sulindac, NO-Sulindac and DFMO were administered to the rats, either individually or in combination. At the end of the study, we observed that these treatment regimens inhibited the tumor progression to advanced stage with no evidence of toxicity. Thus our study demonstrate that low dose combination of these agents may be a promising approach to use them for colon cancer prevention without causing any toxicity.p < 0.0001) and 51% (p < 0.0011), respectively, whereas NO-Sulindac had a modest effect . Combinations of DFMO plus Sulindac or NO-Sulindac suppressed adenocarcinoma incidence , and multiplicity . Rats that were fed the combination of DFMO plus Sulindac showed significant inhibition of tumor cell proliferation and induction of apoptosis. In addition, enhancement of p21, Bax, and caspases; downregulation of Ki-67, VEGF, and β-catenin; and modulation of iNOS, COX-2, and ODC activities in colonic tumors were observed. These observations show that a lower-dose of DFMO and Sulindac significantly enhanced CRC chemopreventive efficacy when compared to NO-Sulindac alone, and the combination of DFMO and NO-Sulindac was modestly efficacious as compared to DFMO alone.Non-steroidal anti-inflammatory drugs (NSAIDs) are promising colorectal cancer (CRC) chemopreventive drugs; however, to overcome NSAIDs’ associated side effects, there is a need to develop safer and efficacious approaches. The present study was designed to evaluate (i) the efficacy of nitric-oxide releasing (NO)-Sulindac as compared to Sulindac; (ii) whether NO-Sulindac is superior to Sulindac in enhancing low-dose difluoromethylornithine (DFMO)-induced chemopreventive efficacy, and (iii) assessing the key biomarkers associated with colon tumor inhibition by these combinations. In F344 rats, colonic tumors were induced by azoxymethane (AOM). At the adenoma stage (13 weeks post AOM), groups of rats were fed the experimental diets containing 0 ppm, 500 ppm DFMO, 150 ppm Sulindac, and 200 ppm NO-Sulindac, individually or in combinations, for 36 weeks. Colon tumors were evaluated histopathologically and assayed for expression levels of proliferative, apoptotic, and inflammatory markers. Results suggest that , DFMO, Sulindac individually, and DFMO combined with Sulindac or NO-Sulindac significantly suppressed AOM-induced adenocarcinoma incidence and multiplicities. DFMO and Sulindac suppressed adenocarcinoma multiplicity by 63% ( Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths in the US and worldwide . Over thMin mice -ornithine, L-[3H]-arginine, and 14C-arachidonic acid as substrates [Polyamine analysis: Each colonic mucosal and tumor sample was suspended in ice-cold PBS and homogenized on ice using a Polytron Homogenizer . Protein-free supernatant (500 µL) was mixed with 350 µL saturated sodium carbonate and 400 µL of 37 mmol/L dansyl chloride, then the mixture was incubated at 60 °C for 1 h. Dansylated polyamines were extracted in toluene, dried, then redissolved in 100 µL of acetonitrile, and finally quantified by reverse-phase HPLC method. Briefly, a chromatography procedure using a Shimadzu C18, 33 × 4.6 mm i.d. cartridge column with 10 mmol/L heptane sulfonate buffer, pH 3.4, in acetonitrile gradient at a flow rate of 1.5 mL/min were applied, and dansylated polyamines were detected using fluorescence detection set for excitation at 330 nm and emission at 470 nm in Shimazu fluorescence detector 510.Effect of Chemopreventive Agents on Colonic Tumor ODC, iNOS, and COX-2 Enzyme Activities and Polyamines. Measurement of ODC, iNOS, and COX-enzymes activities were carried out by previously published procedures using [bstrates . Polyamiv/v) Tween 20, and 5% BSA, and incubated overnight with either anti-p21 , anti-Bax , anti-NRF2 , anti-Caspase6 , anti-Caspase8 , or anti-β-actin (1:1000). After washing the blots in TBST, the blots were incubated with horseradish peroxidase-conjugated secondary antibody corresponding to primary antibody followed by washing with TBST. Washed blots were incubated with Super Signal West Pico Chemiluminescence Substrate  and images were captured using a Gbox instrument  [Western Blot. Polyclonal antibodies for p21, Bax, NRF2, and Caspases 6 and 8 were procured from Abcam  or Cell Signal Technology (CST) . Tumors harvested from different groups were lysed in ice-cold RIPA buffer , and Halt™ Protease and Phosphatase Inhibitor Cocktail  and the protein content was determined by using the Bio-Rad Protein Assay reagent . Separation of proteins (50 μg) was resolved on an SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with a solution containing 10 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.01%  among different groups were compared by the Fisher’s Exact test. Tumor multiplicity (number of tumors per rat), organ weights, serum parameters, and protein expression and activities were analyzed by unpaired General Observations. DFMO, Sulindac, and NO-Sulindac administered alone or in combination to male F344 rats, for the experimental period, showed no significant effect on their body weights. During 36 weeks of administration of the above compounds in the diet, there were no apparent adverse effects on the rats. The body weights of all rats that were fed diets of different groups were comparable with those of the corresponding control groups throughout the study A. Chronip < 0.001) and multiplicity by 63% (p < 0.0001); Sulindac at 150 ppm inhibited colon adenocarcinoma incidence by 35.7% (p < 0.005) and multiplicity by 51% (p < 0.0011), whereas NO-Sulindac at 200 ppm inhibited colon adenocarcinoma incidence by 14.25% and multiplicity by 22.8% . In the combination groups, the adenocarcinoma incidence inhibition in DFMO plus Sulindac was 57.1% (p < 0.0001) and multiplicity inhibition was 81% (p < 0.0001), whereas in the DFMO plus NO-Sulindac group, the total adenocarcinoma incidence inhibition was 46.4% (p < 0.0004) and multiplicity inhibition was 62.1% (p < 0.0001). Importantly, as compared to DFMO or Sulindac alone, the combination of DFMO + Sulindac had a significant synergistic inhibitory effect  on colon adenocarcinoma multiplicity. However, the colon adenoma incidence and multiplicities were not significantly impacted by various treatments (The combination of DFMO and Sulindac significantly inhibited the incidence and multiplicity of colon adenocarcinomas. Colonic tumors were classified by histopathology as adenomas (benign tumors with dysplastic glandular epithelium) or adenocarcinomas  . Most of the colonic tumors in this experiment were adenocarcinomas (~80%), predominately tubular adenocarcinomas, and fewer tumors were signet-ring mucinous adenocarcinomas. The majority of the colonic tumors (~80%) originated in the distal region . As showeatments .p < 0.001–0.005), and their combination showed greater inhibitory effect (p < 0.0001) as compared to individual administration alone, whereas NO-Sulindac failed to show any significant effect (p = 0.2) on iNOS activity in colonic tumors, nor provided additional inhibitory effect with DFMO combination. DFMO, Sulindac, NO-Sulindac, DFMO + Sulindac, and DFMO + NO-Sulindac inhibited colon adenocarcinoma COX-2 activities by 27.6% (p = 0.051), 45.4% (p < 0.002), 24.3% (p = 0.07), 55% (p < 0.0001), and 30.2% (p < 0.05), respectively. Sulindac alone and its combination with DFMO have a strong inhibitory effect on the PGE2 formation. With regard to colonic tumor ODC activities, as anticipated, only DFMO or its combinations showed a significant inhibitory effect. However, Sulindac and NO-Sulindac had modest but not significant inhibitory effects. Polyamines analyzed for putrescine, spermidine, and spermine  in cells. ODC is highly expressed in colorectal cancer compared to adjacent normal mucosal tissue [Polyamine levels are tightly regulated by enzyme ODC and the catabolic enzyme spermidine/spermine Nl tissue , and conl tissue . High lel tissue ,35,36. Al tissue ,36, whicl tissue . Althougl tissue . This isl tissue . Thus, Sl tissue , thereby2 levels in the colonic mucosa of patients treated with Sulindac/DFMO combination [The combined chemopreventive efficacy of Sulindac plus DFMO is higher than that of either of the compounds alone or NO-Sulindac plus DFMO. This enhanced efficacy is consistent with the notion that these compounds modulate polyamine biosynthesis synergistically. In addition to modulation of polyamine synthesis, Sulindac was associated with a significant decrease in the expression of inflammatory molecules and increased apoptosis. The role of COX-2 and iNOS in colon carcinogenesis is well established ,39,40,41bination . Earlierbination . In contWnt signaling. There is not much change in β-catenin expression levels in the case of NO-Sulindac, which may be a reason for not showing much tumor inhibitory effect. Although we have no explanation for this discrepancy, the validity of our finding is strongly supported by the observation that the decrease in β-catenin expression levels may contribute, at least in part, to the mechanism by which Sulindac alone and in combination with DFMO suppresses colon tumorigenesis in rats. The chemopreventive efficacy of DFMO/Sulindac was stronger than that of either individual compound, and tumor inhibition was associated with significantly decreased cell proliferation and increased apoptosis.In addition to COX-2 and iNOS, several markers of anti-proliferation and apoptosis have been evaluated in colonic tumors that affect the pathways which play a role in the anti-tumor effects of these drugs. The role of β-catenin in colon carcinogenesis is well established, as hyperactivation of β-catenin signaling by mutations in either APC or β-catenin, leads to excessive colonocyte proliferation that leads to colon cancer development ,46,47 ancip1/wif1 transcription, resulting in decreased p21cip1/wif1 protein [cip1/wif1 in the DFMO treated group individually and in combination with Sulindac and NO-Sulindac. This explains the ODC activity inhibition due to the depletion of polyamine levels in the tumors, thereby decreasing c-myc expression and increasing p21cip1/wif1 expression, leading to tumor growth arrest. Effects of the combination of Sulindac and DFMO on tumor inhibition are mediated by stimulating apoptotic pathways by inhibiting cellular proliferation. From our study, Sulindac or NO-Sulindac alone and their combination with DFMO were able to increase the caspases 6/8, but only the combination of DFMO plus Sulindac or DFMO plus NO-Sulindac was able to increase the pro-apoptotic protein Bax in colonic tumors. However, the enhancement of apoptosis, rather than the reduction of cell proliferation, has been more consistently associated with the ability of Sulindac and NO-Sulindac to inhibit tumor growth in AOM-induced colonic tumors. The above anti-apoptotic effect coupled with downregulation of β-catenin, induction of SSAT gene, and anti-inflammatory effects of Sulindac are proven to be more potent and more effective when combined with DFMO.We evaluated p21 and caspase markers in rat colonic tumors from different experimental groups. We observed a modest induction of p21 in colonic tumors of rats fed Sulindac and NO-Sulindac, but there was a significant induction of p21 in colonic tumors of rats fed DFMO alone or in combination with Sulindac and NO-Sulindac. It is known that ODC gene overexpression increases cellular polyamines, induces c-Myc expression, and inhibits p21 protein . ResultsIn conclusion, our results suggest that, at the adenoma stage, administration of DFMO plus Sulindac bestowed significant adenocarcinoma inhibition with anti-inflammatory, proapoptotic, and anti-proliferative actions more efficiently than a combination of NO-Sulindac plus DFMO. Thus, the combination regimen applied in this study supports our previous low-dose combination approaches to suppress colon cancers in a synergistic and/or additive manner.https://seer.cancer.gov/ (accessed on 1 August 2023)) (https://seer.cancer.gov/tools/solidtumor/Colon_STM.pdf (accessed on 1 August 2023)) by a clinical pathologist, rather than by a rodent histopathologist. ALT levels for all rats, including the untreated, are above the normal range, whichmay be related to the age of the rats or due to AOM treatment.The guidelines used here to classify the tumors are based on the WHO Classification of Tumors of the Digestive System 2010 edition (