PDGF activates Ca2+ elevation by activating the phospholipase C gamma (PLCγ)-signaling pathway, resulting in ER Ca2+ release. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx following depletion of ER Ca2+ stores and stromal interaction molecule 1 (STIM1) is a key molecule in the regulation of SOCE. In this study, wild-type and STIM1 knockout mouse embryonic fibroblasts (MEF) cells were used to investigate the role of STIM1 in PDGF-induced Ca2+ oscillation and its functions in MEF cells. The unexpected findings suggest that STIM1 knockout enhances PDGFR–PLCγ–STIM2 signaling, which in turn increases PDGF-BB-induced Ca2+ elevation. Enhanced expressions of PDGFRs and PLCγ in STIM1 knockout cells induce Ca2+ release from the ER store through PLCγ–IP3 signaling. Moreover, STIM2 replaces STIM1 to act as the major ER Ca2+ sensor in activating SOCE. However, activation of PDGFRs also activate Akt, ERK, and JNK to regulate cellular functions, such as cell migration. These results suggest that alternative switchable pathways can be observed in cells, which act downstream of the growth factors that regulate Ca2+ signaling.Platelet-derived growth factor (PDGF) has mitogenic and chemotactic effects on fibroblasts. An increase in intracellular Ca Moreover, there is a common signal transduction pathway to increase cytosolic Ca2+ concentration. After the ligand binds to receptor tyrosine kinase (RTK), phospholipase C gamma (PLCγ) is phosphorylated and activated, which in turn hydrolyzes membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,4,5-inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 can induce the diffusion of Ca2+ from the ER into the cytoplasm through the IP3 receptor (IP3R) on the ER membrane. Subsequent exhaustion of ER Ca2+ activates a refill mechanism called the store-operated Ca2+ entry (SOCE), which also increases cytosolic Ca2+ concentration.Capoptosis ,2. To coduration ,4. In ce2+ concentration in most non-excitable and a few excitable cells and is triggered following the depletion of ER Ca2+ concentration via the store-operated channels on the plasma membrane [2+ store sensors. Following the depletion of ER Ca2+ concentration, the activated STIM proteins aggregate near the plasma membrane and interact with two Ca2+-permeable components of store-operated channels (SOCs), viz., Ca2+ release-activated calcium modulator 1  and transient receptor potential canonical 1 (TRPC1), which contribute to SOCE [2+ can be passively transported into the cytoplasm through the STIM–Orai1–TRPC1 complex to regulate Ca2+-sensitive enzymes and Ca2+-dependent transcription factors, such as cAMP response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT) [SOCE is the major mechanism that increases and replenishes intracellular Camembrane ,6,7. Str to SOCE ,9,10. Exs (NFAT) ,12,13,142+ and activates store-operated channels (SOCs) to regulate cell proliferation [2+ store depletes through interacting with Ca2+ release-activated calcium modulator 1 (Orai1) in mouse embryonic fibroblasts (MEF) cells [2+ in the ER lumen than STIM1. Thus, it has been suggested that STIM2 responds to minor elevation in ER luminal Ca2+ concentration [2+ regulation is still unclear. There are three homologues of Orais, viz., Orai1, Orai2, and Orai3, and all three Orai proteins are expressed in plasma membranes and mediate SOCE [2+ influx via the STIM1–Orai1–TRPC1 complex [2+ hemostasis [The STIM family senses ER Caferation ,16 and mferation ,18. Therferation ,20,21. SF) cells ,23. Prevntration ,20,24. Hate SOCE ,26,27,28ate SOCE ,29. Of t complex ,31. Prevmostasis ,33.2+ release [2+ store and Ca2+ re-entering from the extracellular solution through SOCs [Platelet-derived growth factor (PDGF) is a mitogen that is involved in cellular migration, proliferation, angiogenesis, and extracellular matrix generation ,35,36,37 release ,43. Prevugh SOCs ,45. Receugh SOCs ,47.2+ elevation including ER Ca2+ release and SOCE, for which STIM1 and Orai1, but not STIM2, are essential in PDGF-BB-induced Ca2+ elevation and cell migration [2+ concentration and replenished Ca2+ concentration through the STIM–Orai puncta-like complex [2+ depletion through the PDGFR–PLCγ–IP3 signaling pathway. However, the role between STIM1 and PDGF-BB signaling is still unclear. Thus, in this study, we employed a particular cell line, mouse embryonic fibroblast-STIM1 knockout cells (MEF-STIM1−/−), to study the effects of STIM1 knockout on PDGF-BB-stimulated Ca2+ response.Mitogens, such as PDGF and EGF, are well recognized as inducing STIM1-mediated Caigration ,47,48. H complex ,24. In m−/−) cell lines were used and intracellular concentration of Ca2+ was monitored by using a ratiometric fluorescent dye, Fura-2/AM. Thapsigargin (TG), a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor and SOCE activator, is usually used to deplete ER Ca2+ store to activate SOCE. The cells were exposed to 2 μM TG in the absence of extracellular Ca2+. An initial transient intracellular Ca2+ elevation was observed due to Ca2+ released from the ER. Next, 2 mM Ca2+ was reintroduced into the extracellular solution, leading to Ca2+ influx, called SOCE  and STIM1 knockout MEF  in the absence of extracellular Ca2+, after which 2 mM Ca2+ was added to the extracellular solution. There was a transient increase in intracellular Ca2+ concentration in MEF-STIM1−/− cells due to the release of ER Ca2+, which then reverted to the baseline concentration. Following the addition of 2 mM Ca2+, Ca2+ influx appeared to replenish the cytosol  and that PDGFR phosphorylation activates PLCγ to hydrolyze PIP2 into DAG and IP3, which leads to a depletion of the ER CaWT cells A, indicaWT cells B and phoWT cells C also coWT cells D. STIM2 WT cells E. We thend STAT3 A. Upon P quickly B. AlthouWT cells C. In addWT cells D. Activaignaling . Our resignaling  resulted/− cells . Moreoverylation . These rrylation  but inhirylation .−/− cells but not in MEF-WT cells. To understand how PDGF-BB induced Ca2+ elevation in MEF-STIM1−/− cells, proteins that contribute to SOCE were examined by immunoblotting analysis. Although STIM1 knockout did not affect STIM2; Orai1, Orai2, Orai3, and TRPC1 were decreased in MEF-STIM1−/− cells  and PLC (U73122 and D609) inhibitors were employed to verify whether the PDGFR–PLCγ axis could be activated by PDGF-BB stimulation in MEF-STIM1/− cells ,50,51. I2+ elevation in a dose-response manner  mouse embryonic fibroblasts were maintained in high-glucose Dulbecco’s modified Eagle’s medium  supplemented with 5% fetal bovine serum , 100 IU/mL penicillin, 100 μg/mL streptomycin, non-essential amino acid , and 1 mM sodium pyruvate  under 5% CO2 at 37 °C. Recombinant mouse PDGF-BB and EGF were purchased from R&D Systems . Wild-type (MEF-WT) and STIM1 knockout . Fura-2 were excited alternatively between 340 nm and 380 nm using the Polychrome IV monochromator (Till Photonics), which is highly sensitive and can detect rapid changes in Ca2+ concentration with high temporal and spatial resolution and images were captured using an Olympus IX71 inverted microscope equipped with a xenon illumination system and an IMAGO CCD camera (Till Photonics). Emission fluorescence intensity of 510 nm was used to calculate intracellular Ca2+ levels using the TILLvisION 4.0 software (Till Photonics).Intracellular CaCell lysates were harvested in radioimmunoprecipitation assay (RIPA) buffer , and the lysates were analyzed by Western blotting using antibodies against STIM1 , Orai1, Orai3 , Orai2 , TRPC1 , PDGFRα, JNK, phospho-EGFR, EGFR , phospho-PDGFRβ, PDGRβ , STIM2, phospho-PLCγ, PLCγ, phospho-Akt, Akt, phospho-JNK, phospho-ERK, ERK, phospho-STAT3, STAT3, phospho-CREB, CREB , and β-actin . The immune complexes were then detected with horseradish peroxidase-conjugated IgG , and the reaction was developed using an enhanced chemiluminescence (ECL) detection kit under an ImageQuant LAS 4000 system .4 cells) were seeded in a 3-cm glass-bottom dish . After adhesion, cells were starved in a serum-free medium for 12 h and then treated with PDGF-BB (100 ng/mL) for 3, 5, and 10 min. Then, cells were fixed with 4% paraformaldehyde  for 10 min; incubated in PBS washing buffer containing 0.1% Triton X-100 for 10 min; and then in CAS-Block solution  for 1 h with gentle shaking. Subsequently, the cells were loaded with primary antibody, anti-STIM1  and anti-STIM2  diluted to 1:100 in filtered PBS. Finally, cells were stored overnight at 4°C. Alexa Fluor-conjugated secondary antibodies (Invitrogen) were used to counteract primary antibodies, and Hoechst 33342 was loaded as the nuclear marker. The fluorophores were excited by laser at 405 and 594 nm and detected by a laser scanning confocal microscope . The FV10-ASW software  was used to access puncta formation and translocation of the STIM proteins.MEF cells  with 0.5% FBS medium and incubated at 37 °C with 5% CO2 overnight. The insert was removed after cells were well-attached and formed a monolayer. The cells were then incubated in DMEM containing 0.1% FBS with 100 ng/mL PDGF-BB. Cell migrating into the gap  was recorded every 6 h via phase-contrast microscopy. The data were collected from three independent experiments and analyzed as wound closure (%) by the ImageJ software .Culture inserts (ibidi) were applied to assess MEF cell migration. The insert consisted of two wells separated by a 500 μm-thick silicon wall. The culture insert was placed into a 3-cm culture dish and slightly pressed on the top of the insert to ensure adhesion on the dish. MEF-WT and MEF-STIM1−/−) were seeded into a 3-cm culture dish with 0.5% FBS medium for cell starvation and incubated at 37 °C and 5% CO2 overnight. After cells had attached properly to the dish, the medium was changed (0.1% FBS with 100 ng/mL PDGF-BB). The cells were collected and fixed with 4% paraformaldehyde at 0, 24, 48, and 72 h. Next, the cells were stained with Hoechst 33342 for 30 min. Fluorescence images were captured randomly and counted using fluorescence microscopy and ImageJ software, respectively.To assess MEF cell proliferation, cells were stained using Hoechst 33342. MEF cells . For statistical analysis, Student’s


Salvia miltiorrhiza (SM) extracts induce tonic contraction of rat LES ex vivo and elucidate the underlying mechanisms. To investigate the mechanism underlying the SM extract-induced contractile effects, rats were pretreated with atropine (a muscarinic receptor antagonist), tetrodotoxin (a sodium channel blocker), nifedipine , and Ca2+-free Krebs-Henseleit solution with ethylene glycol tetraacetic acid (EGTA), followed by administration of cumulative dosages of SM extracts. SM extracts induced dose-related tonic contraction of the LES, which was unaffected by tetrodotoxin, atropine, or nifedipine. However, the SM extract-induced LES contraction was significantly inhibited by Ca2+-free Krebs-Henseleit solution with EGTA. Next, SM extracts significantly induce extracellular Ca2+ entry into primary LES cells in addition to intracellular Ca2+ release and in a dose-response manner. Confocal fluorescence microscopy showed that the SM extracts consistently induced significant extracellular Ca2+influx into primary LES cells in a time-dependent manner. In conclusion, SM extracts could induce tonic contraction of LES mainly through the extracellular Ca2+ influx pathway.Up to 40% of patients with gastroesophageal reflux disease (GERD) suffer from proton pump inhibitor refractory GERD but clinically the medications to strengthen the lower esophageal sphincter (LES) to avoid irritating reflux are few in number. This study aimed to examine whether  Gastroesophageal reflux disease (GERD) is caused by reflux of acid or other irritants from the stomach into the esophagus. The main cause of GERD is incompetence of the lower esophageal sphincter (LES) which normally closes at the esophagogastric junction to avoid the back-up of food and acid injury and transiently relaxes to allow food from the esophagus into the stomach when swallowing . Acid reSalvia miltiorrhiza Bunge (SM), also known as red sage or Danshen in Chinese, is clinically used in Traditional Chinese Medicines and officially listed by the Pharmacopoeia Commission of the People’s Republic of China i flow from extracellular entry or intracellular release were investigated in primary LES smooth muscle cells. The mean concentration-response plots of SM-induced increased [Ca2+]i signals in Ca2+-free medium was shown in 2+]i: 47.03 ± 13.43 nM at the 60th s). After addition of SM extracts, a short decay of [Ca2+]i was noted and rapid rise of [Ca2+]i followed. A sustained [Ca2+]i was noted after [Ca2+]i reached the top. The changes of [Ca2+]i(61–360) were that mean [Ca2+]i during 0–60 s as a background was subtracted from [Ca2+]i at each time point during 61–360 s As showed in 2+]i(61–360) − mean [Ca2+]i(0–60)) was calculated and the results in the groups of high (0.5 μL/mL) and low (0.1 μL/mL) concentration of SM extracts were 37.53 ± 8.83 nM and 18.95 ± 7.20 nM, respectively . This result indicated SM extracts induced intracellular Ca2+ release from intracellular stores (sarcoplasmic reticulum) in a dose-response manner.The effects of SM extracts on [Ca2+]i signals in Ca2+-containing medium are shown in 2+]i: 99.11 ± 4.61 nM at the 60th s). After addition of SM extract, [Ca2+]i had a similar presentation as that in Ca2+-free medium. As shown in 2+]i(61–360) − mean [Ca2+]i(0–60)) was calculated and the results in the groups of high (0.5 μL/mL) and low (0.1 μL/mL) concentration of SM extracts were 76.15 ± 15.65 nM and 20.91 ± 10.26 nM, respectively . Comparing 2+]i(61–360) − mean [Ca2+]i(0–60)) of these groups stimulated by high and low concentration of SM extract in the Ca2+-containing medium were significantly higher than those in the Ca2+-free medium, respectively . Compared to low concentration SM extract, high concentrations of SM extract induce 3.64- and 1.98-fold increases (76.15 ± 15.65/20.91 ± 10.26 vs. 37.53 ± 8.83/18.95 ± 7.20) in Ca2+-containing (2+-free (p < 0.05). These results indicate SM extracts induce extracellular Ca2+ entry into smooth muscle cells in addition to intracellular Ca2+ release in a dose-response manner.The mean concentration-response plots of SM-induced increased  increased gradually from the 105th s (green) to the 300th s (red and bright). On the contrary, the fluorescence images of primary LES cells after SM treatment in Ca2+-free BSS showed a mild increase at the 105th s and had only a little yellow particle on central part at the 300th s 0, where F0 = the first image of the sequence and F = subsequent images) in the Ca2+-containing BSS. The relative fluorescence intensity (423.82 ± 133.68) of intracellular [Ca2+] in primary LES cells at the 300th s was significantly higher than that at 0 s . 0) in Ca2+-free BSS. The change of relative fluorescence intensity from 0 s, and 105th s to 300th s was at a relatively low level. These results indicate SM extracts induce extracellular Ca2+ entry into smooth muscle cells and increase fluorescence intensity. These in vitro findings are in good agreement with our results obtained in the ex vivo study, which shows SM-induced contraction of LES is mainly associated with extracellular Ca2+ influx.2+-free BSS solution (control group) added to 1 mL Ca2+-free BSS at the 10th min. The [Ca2+]i in the SM extract stimulation group was more than in the control group after addition of 2 mM CaCl2 at the 13th min (n = 3). These results indicate SM extracts increase [Ca2+]i in primary LES cells by inducing extracellular Ca2+ entry into smooth muscle cells. N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) , and Ca2+-containing BSS (control group) to 1 mL Ca2+-containing BSS and 1 μM TG was applied to induce Ca2+ influx at the 30th s by depleting the endoplasmic reticulum calcium stores. The [Ca2+]i in the group of only SM extracts pretreatment was more than the other groups (n = 3). These results indicate Ca2+ influx is the main pathway of increased [Ca2+]i induced by SM extracts.2 and all efforts were made to minimize suffering. Male Sprague-Dawley rats were obtained from the BioLASCO Taiwan . Carbachol, atropine, nifedipine, papain, collagenase II, dispase II and buffer reagents were obtained from Sigma-Aldrich . Fura-2-acetoxymethyl ester (Fura-2 AM) and fluo-4 AM were purchased from Invitrogen . Tetrodotoxin was purchased from Tocris Cookson Inc. . TA was obtained from the National Institute for Control of Biological and Pharmaceutical Products of China  and its purity was more than 98% by HPLC analysis.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Council of Agriculture, Executive Yuan, Taiwan. The protocol was approved by the Animal Care and Use Committee at E-Da Hospital (Permit Number: IACUC-103025). All rats were directly sacrificed by CODried roots of SM were obtained from Winpower Biotech. Co., Ltd. . The authentication of SM roots was done on the basis of standards and methods provided as listed in Zhonghua Bencao . A herba® STAR RP-18 endcapped   was applied for the separation and temperature was controlled at 25 °C. The mobile phase was made up of 75% acetonitrile, 12.5% methanol and 12.5% water at volumetric ratios and pH was titrated to 3.0 with glacial acetic acid. The detection was monitored at 270 nm. The mobile phase was delivered at a rate of 1.0 mL/min and the volume of injection loop was 10 μL. Diazepam was used as the internal standard. The sample calibration curve for TA was linear (r = 0.9999) within the range 0–50 mg/mL. Intra- and inter-day coefficients of variation of the assays were less than 5% (n = 6) i and measurement of [Ca2+]i was performed according to the procedure previously described in detail  in primary LES smooth muscle cells were detected using a confocal microscope . Briefly, 105/mL primary LES smooth muscle cells were seeded onto the glass coverslips in six well plastic plates to grow. Two days later, culture medium was removed and cells were washed with BSS and incubated with 1 μM fluo-4 AM solution for 20 min at 37 °C. Fluo-4 AM solution was removed and cells was washed out again with BSS. The coverslip was nipped on special holder and the confocal microscope was used to detect calcium imaging at 3 s intervals for 6 min. The 0.5 μL extracts of SM were sequentially added into 1 mL Ca2+-containing or Ca2+-free BSS on the holder at 1st min. BSS consisted of 5.4 mM KCl, 5.5 mM D-glucose, 1 mM MgSO4, 130 mM NaCl, 20 mM HEPES, 2 mM CaCl2 and pH was adjusted to 7.4. Ca2+-free BSS had a similar composition except Ca2+. Similarly, CellˆR IX81 fluorescence microscope  with fluo-4 AM staining was also applied to detect the intracellular calcium responses to stimulation, such as by CaCl2 or TG. Calcium concentration was calculated using the following formula: [Ca2+]i = KD × [(F − Fmin)/(Fmax − F)]. Plotting the fluorescence intensity versus [Ca2+]i yielded the calibration curve with the formula of: [Ca2+]i = KD × [(F − Fmin)/(Fmax − F)], where KD = 150.5 nM, F = Fluo-4 intensity, Fmax = 640, and Fmin = 21.7 for Fluo-4 [Fluo-4 AM was applied as Car Fluo-4 ,35.t test. In all cases, differences were considered significant when p < 0.05.These data were expressed as means ± standard deviation, and statistical analysis of the results was performed by using Student 2+ influx, especially through store operated calcium channels. Hence, SM has the potential to treat the patients with proton pump inhibitor refractory GERD.The present study demonstrates that SM causes tonic contraction of LES. The SM-induced contraction may not relate to neuronal conduction, muscarinic receptors, and L-type voltage-dependent calcium channels. This study provides the evidence that the SM-induced contraction of LES is mainly associated with extracellular Ca


Idelalisib, a selective PI3Kδ inhibitor, has been approved by the FDA for chronic lymphocytic leukemia/small lymphocytic lymphoma treatment and for follicular lymphoma treatment when combined with rituximab. However, the mechanisms of effective action of idelalisib in hepatocellular carcinoma (HCC) remain unclear. In the current study, we aimed to investigate how idelalisib inhibits the growth of HCC cells and enhances the effects of other chemotherapeutic drugs. Our results show that idelalisib treatment promotes Bim induction in HCC via the FoxO3a pathway following PI3K/AKT inactivation. Moreover, our results show that Bim is required for idelalisib-mediated apoptosis in HCC. Idelalisib also synergizes with sorafenib or doxorubicin to induce significant apoptosis in HCC, and Bim is also necessary for the induction of apoptosis by cotreatment. Furthermore, a xenograft experiment reveals that the Bim deficiency abolishes apoptosis and antitumor effects of idelalisib in vivo. In summary, our results indicate a key role of Bim in mediating the antitumor effects of idelalisib in HCC. Our results also support the clinical significance of the drug. The incidence of HCC in developed countries is on the rise due to the progress of hepatitis C viral (HCV) infection in the elderly, as well as to alcohol consumption, the prevalence of obesity and the nonalcoholic fatty liver disease related to the metabolic syndrome3. Over the past few decades, the treatment of HCC has undergone significant changes, such as an introduction of liver transplantation, liver resection, systemic therapy and hepatic artery-targeted chemotherapy5.Hepatocellular carcinoma (HCC) represents the second leading cause of cancer-related deaths worldwide, especially in developing countries7. Idelalisib is approved by FDA as monotherapy, or in combination with rituximab  and follicular lymphoma8. However, idelalisib has side effects, such as colitis, diarrhea, severe hepatotoxicity, and intestinal perforation10. Idelalisib has also been found to be effective in p53-mutated CLL patients with high-risk genetic traits, which suggests the treatment of p53 deletion/mutant patients should involve screening for idelalisib12. However, the mechanism of autonomous effects of idelalisib, such as cell killing in solid tumors, is unclear.Idelalisib (CAL-101 or GS-1101) belongs to a class of inhibitors of phosphatidylinositol 3 kinase delta (PI3Kδ), a cytosolic tyrosine kinase participating in various signaling pathways in B-cells14. The proapoptotic protein Bim (Bcl2L11) is a Bcl-2 family member and has three major isoforms , which can be produced by alternative mRNA splicing15. Together with other proapoptotic proteins, such as Bad, Bax, PUMA, and anti-apoptotic proteins, such as Bcl-2, Bcl-XL, and Mcl-1, Bim regulates the homeostasis that is essential for normal cell death and survival16. Precise regulation of Bim expression has been shown to be essential for normal development17. Decreasing Bim expression can disrupt tumor suppression, accelerate tumorigenesis, and induce autoimmunity19.Apoptosis, a process of programmed cell death, occurs in multicellular organisms and is regulated by both proapoptotic and anti-apoptotic proteinsIn the current study, our results indicate that idelalisib induces Bim activation via the AKT/FoxO3a pathway, which plays a key role in therapeutic response to idelalisib in HCC.The HCC cell lines, Hep G2, Hep3B, SNU-398, PLHC-1 and embryonic kidney 293T cells were obtained from American Type Culture Collection . HuH-7 cell line was got from Japanese Collection of Research Bioresources (JCRB), normal hepatocyte cell line HL-7702 was got from Institute of Biochemistry and Cell Biology . All HCC cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin and 100 units/mL penicillin . The drugs, including idelalisib, sorafenib, doxorubicin , were diluted with DMSO. Constitutively active AKT was obtained from Addgene .6) were seeded on tissue culture plates (60 mm) 24 h prior to transfection. Subsequently, 1 µg of lentiviral products was mixed with pMD2G and psPAX plasmids, as well as the PolyJet agent in serum-free media. After 15-min incubation at room temperature, the mixture was slowly added to the cells. Medium containing lentiviral particles was obtained after 2 days of transfection. For lentivirus infection, 6-well plates were seeded with HepG2 cells (4–5 × 104 per well). The infected cells were selected with puromycin at a concentration of 2 µg/mL after 1 day of infection and then incubated at 37 °C and 5% CO2. For selecting a single clone, the surviving cells were seeded on a 96-well plate. Western blotting was used to confirm the knockout cells.To generate Bim knockout cells, two gRNA sequences targeting Bim were selected . Single-stranded complementary oligos with BsmBI overhangs were generated. The LentiCRISPR v2 lentiviral vector  was digested using FastDigest BsmBI obtained from Fermentas. The digested product was purified using the QIAquick Gel Extraction Kit , followed by elution in EB buffer. Phosphorylation as well as annealing of the oligos was carried out using T4 polynucleotide kinase in T4 ligation Buffer . The reaction was incubated at 37 °C for 30 min, followed by 90 °C for 5 min, and finally cooled to 25 °C at a rate of 5 °C/min. The ligation reaction was carried out by mixing the oligos to be annealed, the digested LentiCRISPR v2 vector, and the Quick Ligase enzyme included in the Quick Ligase Buffer, before transformation into Stbl3 bacteria. Human 293T cells -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed using the MTS assay kit  according to the manufacturer’s instructions. Luminescence was measured with a Wallac Victor 1420 Multilabel Counter . Each assay was conducted in triplicate and repeated three times.Indicated cell lines were seeded in 96-well plates at a density of 1 × 10TM Probes Supermix (Bio-Rad) in a final reaction volume of 20 μL with gene-specific primer/probe sets, and a standard thermal cycling procedure (35 cycles) in a Bio-Rad CFX96TM real-time PCR system. Bim and β-actin levels were assessed using TaqMan Gene Expression Real-Time PCR assays. The results were expressed as the threshold cycles (Ct). The relative quantification of the target transcripts was determined by the comparative Ct method (ΔΔCt) according to the manufacturer’s protocol. The 2−ΔΔCt method was used to analyze the relative changes in gene expression. Control experiments were conducted without reverse transcription to confirm that the total RNA was not contaminated with genomic DNA. β-actin was used as an internal control gene for normalization.Total RNA was extracted using the TRIzol RNA Kit  according to the manufacturer’s protocol. One µg of total RNA was used to generate cDNA using SuperScript II reverse transcriptase . PCR was performed in triplicate using SsoFasr20, with antibodies against AKT , phospho-AKT , Bid , cleaved-caspase 3 , cleaved-caspase 9 , phospho-FoxO3a , Bak , FoxO3a , cytochrome oxidase subunit IV (Cox IV)  , E2F1 , Egr-1 , cytochrome c , β-actin , Bim  , Mcl-1 , Bcl-2  and Bcl-XL  .Proteins were extracted and western blotting was performed as described previouslyc release, cytosolic fractions were isolated by differential centrifugation and probed by western blotting for cytochrome c. For colony formation assays, the treated cells were plated in 12-well plates at appropriate dilutions and allowed to grow for 2 weeks, followed by crystal violet staining of cell colonies.Apoptosis was analyzed by nuclear staining with Hoechst 33258 . Apoptosis was assessed through microscopic visualization of condensed chromatin and micronucleation. At least three independent experiments were carried out for each condition, and a minimum of 300 cells were counted in each measurement. Annexin V/propidium iodide (PI) staining was performed using annexin-Alexa 488  and PI. For analysis of cytochrome FoxO3a (sc-37887) were from Santa Cruz Biotechnology .Cells were transfected with Lipofectamine 2000  according to the manufacturer’s instructions. The knockdown experiment was performed 24 h prior to the treatment with idelalisib with 200 pmol siRNA. The scrambled siRNA and siRNA for human ChIP with FoxO3a antibody was performed using the Chromatin Immunoprecipitation Assay Kit  according to the manufacturer’s instructions. The precipitates were analyzed by PCR using primers 5′-TGCCACCAAAGATCTCTACC-3′ and 5′-GCATTTCCTCACAGAGTTGG-3′ to amplify a Bim promoter fragment containing putative FoxO3a sites.Bim-KO HepG2 cells were collected, and 4 × 106 cells in 0.2 mL of medium were subcutaneously implanted into the back of athymic nude female mice. After 7 days of tumor growth, mice were treated with idelalisib 30 mg/kg daily for 10 consecutive days through the oral gavage. Tumor growth was monitored with a caliper and tumor volumes were calculated according to the formula ½ × length × width2. Mice were killed when tumors reached ~1.0 cm3 in size. Tumors were dissected and fixed in 10% formalin and embedded in paraffin. TUNEL and active caspase 3 staining were performed on 5-µM paraffin-embedded tumor sections, by using an AlexaFluor 488-conjugated secondary antibody  for signal detection. TUNEL and active caspase 3-positive cells were counted and plotted.All animal experiments were conducted according to the relevant ethical regulations of China Medical University. WT and t test was used to identify the significance of difference of two groups, and one-way analysis of variance (ANOVA) was used for multiple groups. *p < 0.05 was considered to be statistically significant. The means ± SD are shown in the figures.GraphPad Prism IV software was carried out for statistical analyses. Student’s To investigate the effects of idelalisib on HCC, HCC cell lines were treated with increasing concentrations of idelalisib for 72 h, and cell growth was examined by the MTS assay. As shown in Fig. To investigate the role of Bcl-2 family proteins in idelalisib-induced apoptosis, we treated HCC cells with idelalisib and found that it did not promote Bid, Bax and Noxa protein upregulation but did reduce the level of the anti-apoptotic proteins, such as Bcl-2 and Mcl-1 Fig. . Treatin22. In addition, idelalisib inhibited AKT, which phosphorylates FoxO3a and blocks its nuclear localization. Therefore, we examined whether FoxO3a and AKT are involved in idelalisib-induced Bim expression. As shown in Fig. Next, we identified the Bim induction mechanism in response to idelalisib. Transcription factors, such as E2F1, Egr-1 and myc, were not involved as seen from their unchanged expression following idelalisib treatment Fig. . PreviouNext, we examined whether the PI3K/AKT signaling pathway is involved in the action of idelalisib on Bim expression. Idelalisib treatment markedly inhibited AKT phosphorylation at S473 while reducing FoxO3a phosphorylation Fig. , which iBim-KO HepG2 cells  proteins with the ability to bind all anti-apoptotic Bcl-2 proteins with high affinity to trigger cell death30. Our results suggest that Bim expression can be used as a biomarker for the prediction of the antitumor effect of idelalisib in HCC.In the current study, we found that idelalisib promotes Bim induction via AKT/FoxO3a pathway and initiates apoptosis of the HCC via the intrinsic apoptosis pathway. Bim induction plays a key role in apoptosis in response to a variety of chemotherapeutic agents and may be a useful indicator of chemosensitivity. In addition, recent studies showed that the response of isolated mitochondria from tumor cells to a peptide containing the BH3-domain of Bim is associated with chemotherapy response in patientsIn summary, our findings demonstrate a novel antitumor mechanism of idelalisib through Bim-mediated apoptosis. Idelalisib-induced Bim upregulation may serve as a biomarker for the clinical trials and may provide important implications for the future development and application.Supplemental material


AML-blasts induce HLA-DRlo cells from healthy donor-derived monocytes in vitro that suppress T-cells and express indoleamine-2,3-dioxygenase (IDO). We investigated whether a CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with pre-clinical activity against AML-blasts by redirection of T-cells can eradicate CD33+ MDSCs. In fact, T-cells eliminate IDO+CD33+ MDSCs in the presence of AMG 330. Depletion of total CD14+ cells (including MDSCs) in peripheral blood mononuclear cells from AML patients did not enhance AMG 330-triggered T-cell activation and expansion, but boosted AML-blast lysis. This finding was corroborated in experiments showing that adding MDSCs into co-cultures of T- and AML-cells reduced AML-blast killing, while IDO inhibition promotes AMG 330-mediated clearance of AML-blasts. Taken together, our results suggest that AMG 330 may achieve anti-leukemic efficacy not only through T-cell-mediated cytotoxicity against AML-blasts but also against CD33+ MDSCs, suggesting that it is worth exploring the predictive role of MDSCs for responsiveness towards an AMG 330-based therapy.Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 30%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. Here, we demonstrate increased frequencies of CD14The online version of this article (10.1186/s40425-018-0432-9) contains supplementary material, which is available to authorized users. Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults. The disease course is typically aggressive and despite therapeutic advances only 30% of the patients will be long-term survivors. Emerging evidence suggests that immune evasion in AML favors relapse and could antagonize novel immunotherapeutic concepts .Over the last years, myeloid derived suppressor cells (MDSCs) have been gaining momentum in cancer research as promoters of tumor immune escape. MDSCs represent a heterogeneous population that morphologically resembles monocytes or granulocytes sharing some features: myeloid origin, immature phenotype, and T-cell suppressive activity. Accumulating MDSCs have been described in AML patients , in myel+ AML-blasts and CD33+ MDSCs thereby further enhancing anti-leukemic immune activity.Bispecific T-cell engaging (BiTE®) antibody constructs simultaneously target tumor antigens of interest and the T-cell receptor complex. T-cells can be recruited in an antigen-independent manner . The fir+CD11b+CD33+ monocytic cells expressing low levels of HLA-DR (HLA-DRlo) and resembling one of the most established human MDSC-like phenotype [+ and/or CD34+ cells during initial AML diagnosis. The proportion of HLA-DRlo cells among monocytes was significantly increased in AML patients as compared to healthy controls (HD)  in line with previous observations [First, CD14henotype , 12 werervations , B-NHL [+ monocytes isolated from HD were co-cultured for three to five days with AML cell-lines  or primary AML-blasts (pAML) that were previously labeled with a vital dye for better discriminating both populations in ultra-low attachment surface plates allowing full recovery of monocytes. Presence of AML-blasts led (at day five) to a significant reduction of HLA-DR expression in CD14+ monocytes  and HLA-DRhi CD14+ subsets  iMDSCs. Noticeably, previous studies have shown that AMG 330-mediated lysis is co-determined by the cell surface CD33 levels , 9. The ets Fig. . Purifieion Fig.  and whicion Fig. .+ AML-blasts AML-PBMC samples from newly diagnosed patients were used for short-term (three to six days) cell cultures in presence of control BiTE® constructs or AMG 330. In line with previous reports [+ AML-blasts and the concomitant expansion of residual autologous T-cells  competing over the target antigen CD33 or by (B) suppressing successfully recruited T-cells (by IDO). Using a FACS-based indirect QIFIKIT® immunofluorescence assay we quantified the CD33 cell surface antigen density  CD33 levels on CD14+ cells  in AML-PBMCs prior to AMG 330 treatment did not have a detectable effect on T-cell activation or their production of cytokines as well as T-cell expansion , we cultured MOLM13 cells (cAML) with HD-derived T-cells and added AMG 330 in the presence of autologous HD-derived CD33+ monocytes or CD33+ iMDSCs. Co-cultures were performed for three days for preventing a reprogramming of the conventional monocytes into MDSCs that was occurring at day five  were treated with AMG 330 for three days. The median fluorescence intensity (MFI) of granzyme B (Grz B), CD107, perforin, CD69, CD137, CD25, CD154, IL2, IFNγ, and the cells’ expansion index was assessed by FACS in CD4+/CD8+ CD3+ T-cells as indicated. The association between those variables and the PBMCs’ initial frequency of HLA-DRlo cells among CD14+ cells was calculated using a Pearson correlation analysis. Abbreviations: p, p-value; r, Pearson correlation. Table S3. AML-derived PBMCs (n=12) were treated with AMG 330 for three days. The median fluorescence intensity (MFI) of granzyme B (Grz B), CD107, perforin, CD69, CD137, CD25, CD154, IL2, and IFNγ was assessed by FACS in CD4+/CD8+ CD3+ T-cells. The association between those variables and the PBMCs’ initial frequency of CD3+ T-cells was calculated using a Pearson correlation analysis. Abbreviations: p, p-value; r, Pearson correlation. (DOCX 50 kb)


MDMX (MDM4) is emerging as an important breast cancer (BC) biomarker, and oncoprotein, that can be targeted in combination with its well-known family member MDM2. While MDM2 has previously been implicated in driving BC metastasis, information about the role of MDMX in driving circulating tumor cells (CTCs) and BC metastasis is lacking. BCs often have alterations of MDM2, MDMX, and mutant p53 (mtp53). Therefore, the role of MDM2 and MDMX in the context of mtp53 in BCs requires further clarification. Our group has recently reported that triple negative breast cancer (TNBC) metastasis is dependent on both MDM2 and MDMX, and depleting MDM2 results in increased MDMX, but depleting MDMX does not cause an increase in MDM2. In the context of human TNBC expressing mtp53 in an orthotopic mouse model the down-regulation of MDMX virtually cleared CTCs from the blood. Contemplations, using the available literature, suggest that disrupting the stability and/or function of MDMX protein (and its downstream targets), in the context of mtp53 expressing BCs, might be beneficial for patient survival. It remains to be determined if blocking mtp53-MDMX pathways can inhibit early stage TNBC and eliminate CTCs that have the potential to form metastatic lesions. Tp53 gene) [MDMX (MDM4) directly promotes tumor formation and promotes genomic instability , 2. A nu53 gene) . A detai53 gene) . As MDMX53 gene) –10. When53 gene) . However53 gene) . MDM2 pr53 gene) . MDMX in53 gene) . This inin vivo (see , thereby suggesting signaling events regulated by a tumor stroma interaction.We found that TNBC metastasis depends on both MDM2 and MDMX, and depleting MDM2 in this context results in increased MDMX, while depleting MDMX does not cause an increase in MDM2 . This isvivo see , therebyThe current state of the field suggests that disrupting the stability and/or function of MDM protein family members, in the context of mtp53 expressing BCs, might be beneficial for targeting early stage TNBC and eliminating CTCs that can go on to form metastatic lesions , 13. Fur


Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations. 2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system , we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.The mitochondrial Ca MICU1 was originally reported to act as a gatekeeper by inhibiting MCU-mediated mitochondrial Ca2+ uptake at low [Ca2+]cyt  and TcMICU2 behave as the animal orthologs in modulating mitochondrial Ca2+ uptake. The results of this study indicate that both proteins are important for activating MCU, but do not form oligomers and do not behave as gatekeepers at low [Ca2+]cyt as the animal orthologs do. Our results support the presence of these proteins in the last eukaryotic common ancestor (LECA) but the development of lineage-specific functions in the Excavata and Opisthokonta supergroups.In this work, we investigated whether T. cruzi genomic database (www.tritrypdb.org): TcMICU1 (TcCLB.511391.210) and TcMICU2 (TcCLB.510525.130)  . The TcM525.130) . In turn10.1128/mBio.00348-19.1FIG S1Trypanosoma cruzi MICU1 (A) and MICU2 (B) protein sequences. Identical and similar residues are highlighted in dark and light blue, respectively. The predicted mitochondrial targeting signal is shown in orange. EF-hand domains are shown in magenta. In panel A, the polybasic sequence of MICU1 involved in the electrostatic interaction with the polyaspartate tail of EMRE (Homo sapiens MICU1 (HsMICU1) is shown in red. Vertical red arrows indicate two arginine residues of HsMICU1 involved in potential salt bridges with the D-ring of MCU. The alignment also shows basic residues in the C terminus of TcMICU1 that potentially could have this role , except those indicated with an asterisk, which were obtained from the TriTryp database (http://tritrypdb.org/). EF1 and EF2 represent EF-hand calcium-binding domains that were predicted with the highest scores according to PROSITE (http://prosite.expasy.org). #, the Bodo saltans MICU2 sequence in the database is incomplete: as a consequence, the first EF-hand is absent. Download FIG S2, TIF file, 2.1 MB.Sequences used in Copyright © 2019 Bertolini et al.2019Bertolini et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TcMICU1-OE and TcMICU2-OE) in T. cruzi epimastigotes. Whereas TcMICU2-2×HA was overexpressed using the pTREX-n vector following standard procedures, to obtain the TcMICU1-overexpressing cell line, we transfected epimastigotes with a construct in which the gene is linked to the blasticidin-resistant gene (Bsd) via the 2A peptide sequence, in order to coexpress the epitope-tagged TcMICU1 protein with the selection marker. Western blot analyses of total epimastigote extracts showed protein bands of the expected sizes, 48.9 and 55.4 kDa, for TcMICU1 and TcMICU2, respectively  their hemagglutinin HA)-tagged versions  and TcMICU2 (MICU2-OE) overexpressing epimastigotes were subjected to Western blot analysis under reducing (with β-mercaptoethanol [A and B]) and nonreducing (without β-mercaptoethanol [C and D]) conditions. Expression of HA-tagged proteins was confirmed using anti-HA monoclonal antibodies. Tubulin was used as a loading control. Download FIG S3, TIF file, 1.4 MB.Western blots of TcMICU1-OE and TcMICU2-OE under nonreducing conditions. To study if TcMICU1 and TcMICU2 form oligomers Copyright © 2019 Bertolini et al.2019Bertolini et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TcMICU1-OE and TcMICU2-OE cell lines to take up Ca2+, we monitored changes in fluorescence of Calcium Green-5N in digitonin-permeabilized epimastigotes in the presence of succinate as the substrate  decreases. Addition of the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) leads to an increase of fluorescence due to depolarization of the mitochondrial membrane potential (Δψm) and consequent Ca2+ release. There were no significant differences in mitochondrial Ca2+ uptake from either TcMICU1-OE or TcMICU2-OE epimastigotes compared with control cells , evaluated by changes in safranin O fluorescence in digitonin-permeabilized epimastigotes of TcMICU1-OE and TcMICU2-OE cells compared to control cells  Quantification of Ca2+ uptake rates at low extramitochondrial (0.8 and 1.5 μM) estimated free Ca2+ of control EV (empty vector) and TcMICU1-OE (MICU1-OE) epimastigotes. (B) Quantification of Ca2+ uptake rates at high estimated [Ca2+]ext  of control (EV) and MICU1-OE epimastigotes. In panels A and B, values are means ± SD (n = 3). ns, no significant differences (one-way ANOVA with multiple comparisons). (C) Changes in mitochondrial membrane potential (Δψm) of digitonin-permeabilized epimastigotes as detected by changes in safranin O fluorescence in control (EV) and MICU1-OE epimastigotes. Cells (5 × 107) were added to the reaction buffer (2 ml) containing 0.2% BSA, 5 mM succinate, 50 μM EGTA, and 5 μM safranin O. The reaction was started with 50 μM digitonin (DIG), and 250 μM ADP, 1.5 μM carboxyatractyloside (CAT), and 4 μM FCCP were added where indicated. (D) Changes in safranin fluorescence after addition of ADP from three experiments like that shown in panel C. Values are means ± SD (n = 3). ns, no significant differences (Student’s t test). (E) Quantification of Ca2+ uptake rates at low estimated [Ca2+]ext (0.8 and 1.5 μM) of control (EV) and MICU2-OE epimastigotes. (F) Quantification of Ca2+ uptake rates at high estimated [Ca2+]ext  of control (EV) and MICU2-OE epimastigotes. In panels E and F, values are means ± SD (n = 3). ns, no significant differences (one-way ANOVA with multiple comparisons). (G) Changes in mitochondrial membrane potential (Δψm) of digitonin-permeabilized epimastigotes as detected by changes in safranin O fluorescence in empty vector (EV) and MICU2-OE epimastigotes. The experimental conditions are the same as in panel C. (H) Changes in safranin O fluorescence after addition of ADP from 3 experiments as that shown in panel G. Values are means ± SD (n = 3). ns, no significant differences (Student’s t test). Fluorometric assays were performed using either the Hitachi F-4500 (TcMICU1-OE) or F-7000 (TcMICU2-OE) spectrofluorometer. Download FIG S4, TIF file, 0.7 MB.Analysis of mitochondrial CaCopyright © 2019 Bertolini et al.2019Bertolini et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TcMICU1 and TcMICU2, we generated null (knockout [-KO]) mutants for these two genes (TcMICU1-KO and TcMICU2-KO) using the CRISPR/Cas9 method , which has been successfully adapted recently to T. cruzi (33–T. cruzi epimastigotes were transfected with specific molecular constructs for the constitutive expression of Cas9 nuclease and single guide RNA (sgRNA) to target TcMICU1 or TcMICU2 genes    was dete10.1128/mBio.00348-19.7TABLE S1Table S1, DOCX file, 0.1 MB.Oligonucleotides used in this work. Download Copyright © 2019 Bertolini et al.2019Bertolini et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TcMICU1-KO and TcMICU2-KO epimastigotes in LIT (liver infusion tryptose) medium was significantly lower than that in the control cell line transfected with a scrambled sgRNA . Cultivation of epimastigote forms is carry out in the highly nutritive LIT medium supplemented with 10% fetal bovine serum (FBS), but to induce metacyclogenesis in vitro, we used a chemically defined medium, TAU 3AAG, that mimics triatomine urine (TAU) medium  medium . While Togenesis . Moreoveol cells  to O.TcMICU1-KO and TcMICU2-KO epimastigotes to take up Ca2+ was first evaluated using the Calcium Green-5N probe in the presence of 20 μM Ca2+ (2+ is taken up by mitochondria of control (scrambled) cells, but not of epimastigotes in which the MCU gene was ablated (TcMCU-KO) , indicating that Ca2+ uptake was mediated by MCU.The capacity of mitochondria from digitonin-permeabilized  μM Ca2+ . Under tcMCU-KO) . Subsequ+ uptake . Mitocho2+ uptake in the null mutant cells was secondary to mitochondrial membrane depolarization, we measured the mitochondrial membrane potential (Δψm) of digitonin-permeabilized epimastigotes, in the presence of succinate as a mitochondrial substrate, using safranin O. Knockout of either TcMICU1 or TcMICU2 did not affect the Δψm , we also performed these experiments in the absence of BSA. BSA protects or restores mitochondrial functions by reducing the amount of free fatty acids, which can alter the mitochondrial surface charge, affecting the permeability to ions  Changes in mitochondrial membrane potential (Δψm) of digitonin-permeabilized epimastigotes as detected by changes in safranin O fluorescence in control (scrambled), TcMICU1-KO (MICU1-KO), and TcMICU2-KO (MICU2-KO) epimastigotes. Cells (5 × 107) were added to the reaction buffer (2 ml) without BSA and with 5 mM succinate, 50 μM EGTA, and 5 μM safranin O. The reaction was started with 50 μM digitonin (DIG). (B) Changes in safranin O fluorescence after digitonin addition (between 100 and 200 s) in cells used in panel A. Values are means ± SD (n = 3). ns, no significant differences (one-way ANOVA with multiple comparisons). (C) Changes in mitochondrial membrane potential (Δψm) of digitonin-permeabilized epimastigotes as detected by changes in safranin O fluorescence in scrambled, MICU1-KO, and MICU2-KO epimastigotes. Cells (5 × 107) were added to the reaction buffer (2 ml) with 0.2% BSA, 5 mM succinate, 50 μM EGTA, and 5 μM safranin O. The reaction was started with 50 μM DIG. (D) Changes in safranin fluorescence after digitonin addition (between 100 and 200 s) in cells used in panel C. Values are means ± SD (n = 3). ns, no significant differences (one-way ANOVA with multiple comparisons). (E) Representative traces of Ca2+ uptake by digitonin-permeabilized scrambled, MICU1-KO, MICU2-KO, and MCU-KO epimastigotes, expressed in relative fluorescence units (RFU). The reaction was started after adding 50 μM DIG in the presence of 20 μM free Ca2+, 5 mM succinate, 0.2% BSA, and 0.5 μM Calcium Green-5N probe. (F) Quantification of data from experiments like that shown in panel E. Values are means ± SD (n = 3). ***, P < 0.001 by one-way ANOVA with multiple comparisons. ns, no significant differences. Download FIG S5, TIF file, 0.6 MB.Analysis of mitochondrial membrane potential in Copyright © 2019 Bertolini et al.2019Bertolini et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TcMICU1 and TcMICU2 affected Ca2+ uptake in the same way at low and high [Ca2+]ext (2+ uptake was measured at high [Ca2+]ext (5 to 50 μM), a similar significant reduction of Ca2+ uptake rate in the null mutants compared with control cells was observed , we were not able to detect mitochondrial Ca2+ uptake by either control or mutant cells  . Then we proceeded to analyze the level of pyruvate dehydrogenase (PDH) phosphorylation in these parasites by Western blotting as described previously (T. cruzi PDH (TcPDH) phosphorylation in TcMICU1-KO and TcMICU2-KO epimastigotes is significantly higher than in control cells (scrambled)  and in consequence an increase in the inactive form (phosphorylated) of TcPDH. We also measured citrate synthase activity as an indicator of mitochondrial integrity   under bachondria . Respiraeviously . Our resrambled)  and D. Wrylation . These rntegrity , 41 and ol cells . Finallyol cells , 33 . In addition, the threshold for Ca2+ uptake was affected by loss of either TcMICU1 or TcMICU2, as indicated by impaired uptake when given a [Ca2+]ext below the apparent control threshold. While permeabilized control cells were able to take up Ca2+ when the extramitochondrial Ca2+ concentration was between 800 nM and 1.5 μM, for permeabilized TcMICU1-KO and TcMICU2-KO epimastigotes, 5 μM [Ca2+]ext was still not sufficient to stimulate Ca2+ uptake. It has been reported that the mitochondria of permeabilized epimastigotes  that has been reported to bind the polyaspartate tail of EMRE by electrostatic interactions  gene  gene . TcPDP-KT. cruzi. TcMICU1 and TcMICU2 have significantly different properties from those of their animal orthologs: (i) they seem to have redundant roles, unless the loss of one of them could affect the other, (ii) they apparently do not form dimers linked by disulfide bonds, (iii) their overexpression does not affect mitochondrial Ca2+ uptake, (iv) their absence significantly decreases mitochondrial Ca2+ uptake and increases the [Ca2+]ext set point needed for Ca2+ uptake, suggesting that they could not function as gatekeepers but rather in stabilization of the MCU complex, and (v) their ablation results in alterations in the oxidative metabolism of the parasites, probably resulting in decreased replication, as either epimastigotes or amastigotes, and decreased host invasion by trypomastigotes. Our results suggest that although MICU1 and MICU2 were present in LECA, they develop different lineage specific properties in the Excavata and Opithokonta supergroups.In summary, our results indicate that the EF-hand-containing proteins TcMICU1 and TcMICU2 are essential components of the MCU complex of Taq DNA polymerase, Calcium Green-5N, Fluo-4, MitoTracker deep red FM, Alexa-conjugated secondary antibodies, an ATP determination kit, Pierce ECL (enhanced chemiluminescence) Western blotting substrate, a bicinchoninic acid (BCA) protein assay kit, the North2South biotin random prime labeling kit, the North2South chemiluminescent hybridization and detection kit, and HA epitope tag monoclonal antibody were from Thermo Fisher Scientific . Blasticidin S HCl, the BenchMark prestained protein ladder, the BenchMark protein ladder, Alexa Fluor-conjugated secondary antibodies, and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Life Technologies . The Wizard Plus SV miniprep purification system, Wizard SV gel and PCR cleanup system, GoTaq DNA polymerase, and T4 DNA ligase were from Promega . Anti-serine 293 antibodies to phosphorylated PDH-E1α were from Abcam . Antarctic phosphatase, restriction enzymes, and Q5 high-fidelity DNA polymerase were from New England Biolabs . Fluoromount-G was from SouthernBiotech . DNA oligonucleotides were purchased from Exxtend Biotecnologia, Ltd. . The protein assay reagent, Precision Plus protein dual color standards, anti-rabbit HRP-conjugated secondary antibodies, and nitrocellulose membranes were from Bio-Rad . Anti-c-Myc monoclonal antibody (clone 9E10) was from Santa Cruz Biotechnology . Antitubulin monoclonal antibody, carboxyatractyloside (CAT), oligomycin, safranin O, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), Benzonase nuclease, puromycin, G418, mammalian cell protease inhibitor cocktail (Sigma P8340), other protease inhibitors, and all other reagents of analytical grade were from Sigma .Rabbit polyclonal antibody against TcATG8.1 was a gift from Vanina Alvarez . High-fidelity Platinum T. cruzi epimastigotes (Y strain) were grown in liver infusion tryptose (LIT) medium    containid Murray  as descrd Murray . Vero cehttp://www.lactad.unicamp.br) or Helixxa facility (http://www.helixxa.com.br). Primer design and sequence analysis were carried out using DNAMAN software .Molecular constructs were verified by sequencing at the LaCTAD facility (MICU1 and MICU2 genes were identified in the T. cruzi genome database (http://www.tritrypdb.org) (https://www.ncbi.nlm.nih.gov) databases using the ClustalW method in MEGA7 (Putative pdb.org) . Both prin MEGA7  softwarein MEGA7  using thin MEGA7  and the in MEGA7 . The evoin MEGA7 . The ratin MEGA7  and PROSin MEGA7  were useTcMICU1 in T. cruzi epimastigotes, we generated a version of this gene linked with Bsd to the C terminus via a P2A peptide sequence. The full-length TcMICU1 sequence from the T. cruzi Y strain , tagged with 2×HA, was previously cloned into pTREX-n vector. Subsequently, TcMICU1-2×HA fragment was amplified using primers 1 and 2  was PCR amplified using a reverse primer that includes a 2×HA epitope tag coding sequence , resultstigotes . Gene clTcMICU1 gene (TryTripDB identifier [ID] TcCLB.511391.210) were PCR amplified (primers 7 and 8 [http://grna.ctegd.uga.edu/]). The protospacer sequence was included into the forward primer, while a common reverse primer was used for sgRNA amplification. These primers also contained a BamHI restriction site for cloning into Cas9/pTREX-n (TcMICU1-sgRNA/Cas9/pTREX-n construct. The sgRNA orientation was verified by PCR using the specific TcMICU1-sgRNA forward primer and the HX1 reverse primer (primers 7 and 9 ) obtained by PCR .For Southern blot analysis of TcMICU2-KO by Southern blotting, approximately 25 μg of gDNA was digested with HindIII enzyme and separated on a 0.8% agarose gel. Restriction fragments were transferred to nylon membrane and hybridized with a biotin-labeled probe, which spans the 436 nt (TcMICU2 [nt +195 to +631]) obtained by PCR , amplified by PCR, and labeled using the North2South biotin random prime labeling kit. Hybridization, posthybridization washes, and detection were performed with North2South chemiluminescent hybridization and detection kit, following the manufacturer’s recommendations. Signal detection was performed using the UVItec Alliance gel documentation system .Another strategy was the use of probes of 568 nt that recognize the T. cruzi Y strain epimastigotes (4 × 107 cells) were washed with phosphate-buffered saline (PBS) at pH 7.4 at room temperature (RT) and transfected in ice-cold CytoMix  containing 25 μg of each plasmid construct in 4-mm electroporation cuvettes with three pulses  delivered by a Gene Pulser Xcell electroporation system (Bio-Rad). Stable cell lines were established and maintained under drug selection with appropriate antibiotic(s) . Transfectant epimastigotes were cultured in LIT medium supplemented with 20% heat-inactivated FBS until stable cell lines were obtained.Transfections were performed as previously described . Brieflytrans-epoxysuccinyl)-l-leucine 4-guanidinobutylamide (E64), and Benzonase nuclease (25 U/ml of culture). For Western blots to detect the phosphorylated PDH-E1α subunit, phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF) were also added to the RIPA buffer. The cells were then incubated for 1 h on ice, and the protein concentration was determined by BCA protein assay. Thirty micrograms of protein from each cell lysate was mixed with 6× Laemmli sample buffer  before application to 10% SDS–polyacrylamide gels. When nonreducing conditions were required, protein extracts were mixed with sample buffer lacking β-mercaptoethanol. Separated proteins were transferred onto nitrocellulose membranes with a Bio-Rad Trans-blot apparatus. Membranes were blocked with 5% nonfat dried skim milk in PBS-T (PBS containing 0.1% [vol/vol] Tween 20) overnight at 4°C. Next, blots were incubated for 1 h at room temperature (RT) with a primary antibody: i.e., monoclonal anti-HA , rabbit polyclonal anti PDH-E1α (phospho-Ser-293) , and monoclonal antitubulin . After three washes with PBS-T, blots were incubated with the secondary antibody . Membranes were washed three times with PBS-T, and Western blot images were obtained and processed with a C-DiGit blot scanner (LI-COR Biosciences).Western blot analyses were performed as previously described , 69, 70.l-lysine-coated coverslips and then were permeabilized for 5 min with 0.1% Triton X-100. Permeabilized cells were blocked overnight at 4°C with PBS containing 3% BSA, 1% fish gelatin, 50 mM NH4Cl, and 5% goat serum. Next, cells were incubated with a monoclonal anti-HA  or rabbit anti-TcATG8.1 (1:100) diluted in 1% BSA in PBS (pH 8.0) for 1 h at RT. Cells were washed three times with 1% BSA in PBS (pH 8.0), and then cells were incubated for 1 h at RT in the dark with Alexa Fluor 488-conjugated goat anti-mouse or Alexa Fluor 546-conjugated goat anti-rabbit secondary antibodies . Then, cells were washed and mounted on slides using Fluoromount-G mounting medium containing 5 μg/ml of 2-(4-aminophenyl)-1-indole-6-carboxamidine (DAPI) to stain DNA. Controls were treated as described above, but in the absence of a primary antibody. Differential interference contrast and fluorescence optical images were captured with a Leica TCS SP5 II confocal microscope, with a 100× objective (1.44 aperture) under nonsaturating conditions, that uses photomultiplier tubes (PMTs) for detection of emission, and LAS AF software  for acquisition and processing of digital images.Cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 1 h at RT. To determine mitochondrial localization of proteins, epimastigotes were incubated with 100 nM MitoTracker deep red FM for 30 min at 28°C in culture medium before the fixing procedure. Cells could adhere to poly-g for 7 min and washed twice with buffer A with glucose . Epimastigotes were resuspended to a final density of 1 × 109 cells/ml in BAG and kept on ice. Before each experiment, a 50-μl aliquot of T. cruzi epimastigotes (5 × 107 cells) was added to the reaction buffer  containing 5 mM succinate, 50 μM EGTA, and 0.5 μM fluorescent cell-impermeable Ca2+ indicator Fluo-4  or 0.5 μM Calcium Green-5N . Mitochondrial Ca2+ uptake was initiated by the addition of different concentrations of free calcium, which were calculated using the software Maxchelator Calculator v1.2 (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaEGTA-NIST.htm). This calcium addition was followed by 50 μM digitonin and 4 μM FCCP. Fluorescence changes were monitored in an F-4500 or F-7000 fluorescence spectrophotometer (Hitachi) with excitation at 494 nm and emission at 520 nm using Fluo-4 or excitation at 506 nm and emission at 532 nm using Calcium Green-5N. The relative of calcium uptake was calculated as the absolute value of the slope of the linear regression fit in the linear range of the fluorescent signal . The relative rates of Ca2+ uptake were normalized for the control strains. The hyperbolic equation [Ca2+] = dK × [(F − Fmin)/(Fmax − F)] was used to convert the raw fluorescence readings measured during mitochondrial Ca2+ transport assays into Ca2+ concentration levels, where dK is the dissociation constant, F is any given fluorescence value, Fmin is the lowest fluorescence reading after addition of 0.5 mM EGTA, and Fmax is the maximal fluorescence obtained after two sequential additions of 1 mM CaCl2. These additions were performed at the end of each trace. dK for Ca2+ indicator probes in our conditions was determined according Chweih et al. .Cells were collected by centrifugation at 1,000 × h et al. . Mitochoin situ was done spectrofluorometrically using the indicator dye safranin O, as described previously (T. cruzi epimastigotes (5 × 107 cells) were incubated at 28°C in reaction buffer  containing 5 mM succinate, 0.2% BSA, 50 μM EGTA, and 5 μM safranin O, and the reaction was started with digitonin (50 μM). ADP (250 μM), carboxyatractyloside (1.5 μM), and FCCP (4 μM) were added to the medium at different time points. Fluorescence changes were monitored using the Hitachi F-4500 or F-7000 spectrofluorometer (excitation of 495 nm and emission of 586 nm).Estimation of mitochondrial membrane potential eviously , 72. Bri8) were incubated at 28°C in a 2-ml chamber containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES-KOH (pH 7.2), 2.5 mM K2PO4, 1 mM MgCl2, 50 μM EGTA, 5 mM succinate, and 25 μM digitonin. OCR was calculated as the negative time derivative of the oxygen concentration measured in the close respirometer chambers and expressed per milligram of protein. Data were recorded at 2-s intervals, and 10 data points were used to calculate the slope of the OCR plot through a polynomial fit with DatLab 4 software, as described previously  with DatLab 4 software for data acquisition and analysis. The equipment was calibrated as reported by its manufacturer. Cells  were washed twice with PBS and incubated in lysis buffer  for 10 min on ice. Then, proteins were quantified by BCA protein assay, and 260-μl reactions were set up in buffer containing 5 μg protein, 250 μM oxaloacetate, 50 μM acetyl-CoA, 100 μM 5,5′-dithio-bis (2-nitrobenzoic acid), and 10 mM Tris-HCl (pH 8.0). The increase in absorbance at 412 nm was monitored for 20 min at 28°C using a microplate reader . Vmax values were normalized taking the scrambled cell line as the reference value.Citrate synthase activity was measured using a previously described protocol  adapted T. cruzi epimastigotes grown in LIT medium and under starvation conditions were estimated by immunofluorescence analyses using anti-TcATG8.1 antibody as described previously . After being washed, 1 × 108 cells per treatment were centrifuged and resuspended in 100 μl of buffer A and then lysed on ice for 30 min by addition of 150 μl of 0.5 M HClO4. The lysates were neutralized (pH 6.5) by addition of 60 μl of 0.72 M KOH–0.6 M KHCO3. Samples were centrifuged at 1,000 × g for 5 min, and the supernatant was separated for adenine nucleotide determination. ATP, ADP, and AMP in extracted samples were quantified by a luciferin-luciferase bioluminescence assay in a luminometer as described previously (Control (transfected with scrambled sgRNA), eviously , with so2, 2 mM CaCl2, 0.035% sodium bicarbonate, and 8 mM phosphate at pH 6.9). After this stress, parasites were incubated for 96 h in TAU 3AAG medium . To increase the number of metacyclic forms to infect Vero cells, the contents of the flask were collected and resuspended in medium containing fresh fetal bovine serum and incubated at 37°C for 20 h. The complement in the FBS kills epimastigotes, while metacyclic trypomastigotes survive. Samples were harvested from the TAU 3AAG plus FBS-containing medium at days 5 and 10 of cultivation.For metacyclogenesis, we followed the protocol described by Bourguignon et al.  with min5 cells) were plated onto sterile coverslips in a 12-well plate and incubated overnight at 35°C in 7% CO2 in RPMI medium plus 10% fresh fetal bovine serum. Tissue culture-derived trypomastigote collections were incubated at 4°C overnight to allow amastigotes to settle from swimming trypomastigotes. Trypomastigotes from the supernatants of these collections were counted and used to infect the coverslips at a ratio of 50 parasites to 1 host cell. At 4 h postinfection, coverslips were washed extensively with Hanks’ solution, followed by phosphate-buffered saline (PBS) at pH 7.4 to remove any extracellular parasites. Coverslips were fixed immediately in 4% paraformaldehyde in PBS (pH 7.4) at 4°C for 30 min. Coverslips were washed once with PBS and mounted onto glass slides in Fluoromount-G containing 15 μg/ml of 2-(4-aminophenyl)-1H-indole-6-carboxamidine (DAPI), which stains host and parasite DNA. Coverslips were viewed on an Olympus BX60 microscope to quantify the number of host cells that contained intracellular parasites and the number of intracellular parasites per cell in randomly selected fields. Three hundred host cells were counted per sample in three independent experiments. To quantify amastigote replication, the following modifications were used: host cells were infected at a ratio of 10 parasites to 1 host cell, and coverslips were allowed to incubate for 48 h postinfection at 35°C in 7% CO2, prior to fixation and DAPI staining.Gamma-irradiated  Vero cells  for comparisons between more than two cell lines, and two-way ANOVA with multiple-comparison tests for analyses of grouped data.Statistical analyses were performed with GraphPad Prism software version 7.4 . Reported values are means ± standard deviation (SD) from 10.1128/mBio.00348-19.6FIG S6FIG S6, TIF file, 1.6 MB.Complete Western blots shown in this work. Download Copyright © 2019 Bertolini et al.2019Bertolini et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the