The H6PD gene is amplified in several types of malignancies, and earlier work pointed toward a potential involvement of the enzyme in cancer cell growth. In the present study, we demonstrated a pivotal role of H6PD in proliferation and migratory potential of 3 human breast cancer cell lines. Knockdown of H6PD decreased proliferation and migration in SUM159, MCF7, and MDA-MB-453 cells. To understand the mechanism through which H6PD exerts its effects, we investigated the cellular changes after H6PD silencing in SUM159 cells. Knockdown of H6PD resulted in an increase in ER lumen oxidation, and down-regulation of many components of the unfolded protein response, including the transcription factors activating transcription factor-4, activating transcription factor-6, split X-box binding protein-1, and CCAAT/enhancer binding protein homologous protein. This effect was accompanied by an increase in sarco/endoplasmic reticulum Ca2+-ATPase-2 pump expression and an decrease in inositol trisphosphate receptor-III, which led to augmented levels of calcium in the ER. Further characterization of the molecular pathways involving H6PD could greatly broaden our understanding of how the ER microenvironment sustains malignant cell growth.—Tsachaki, M., Mladenovic, N., Štambergová, H., Birk, J., Odermatt, A. Hexose-6-phosphate dehydrogenase controls cancer cell proliferation and migration through pleiotropic effects on the unfolded protein response, calcium homeostasis, and redox balance.Hexose-6-phosphate dehydrogenase (H6PD) produces reduced NADPH in the endoplasmic reticulum (ER) lumen. NADPH constitutes a cofactor for many reducing enzymes, and its inability to traverse biologic membranes makes  In addition, the levels of the UPR mediator split X-box-binding protein (sXBP)-1 were increased, and microarray analysis showed altered expression of several UPR proteins. Furthermore, muscles from H6PD−/− mice showed decreased expression of the sarco/ER Ca2+-ATPase (SERCA), which is responsible for pumping calcium from the cytosol into the ER at the expense of ATP.Hexose-6-phosphate dehydrogenase (H6PD)  is the oysiology , 8. MuscH6PD gene is amplified in 3–4% of pancreatic, sarcomatous, and ovarian tumors and in 1–2% of breast, lung adenocarcinoma, and melanoma tumors, supporting a role of the enzyme in cancer cell growth.Recently, down-regulation of H6PD with short interfering (si)RNAs in the murine cancer cell lines CT26 (colon) and 4T1 (breast) correlated with a reduction in the overall glucose uptake, as well as a decreased cell proliferation . In addi−), estrogen receptor (ER−), nor the Her2 receptor (Her2−)], the PR+, ER+, Her2− cell line MCF7, and the PR−, ER−, Her2+ cell line MDA-MB-453. We further demonstrated that H6PD knockdown dramatically reduces migration in all cell lines tested. Subsequently, we attempted to elucidate the mechanism through which H6PD influences cancer cell growth. Our results suggest a major role of H6PD in regulating UPR signaling proteins, as well as ER calcium balance. H6PD depletion also caused an increase in ER oxidation and a significant decrease in cellular oxygen consumption rate. The above findings highlight, for the first time, the consequences of NADPH depletion within the ER for cancer cell physiology, paving the way for further investigations into the ER-related molecular pathways that promote malignant cell proliferation and migration.In the present study, we showed that H6PD promotes cancer cell proliferation by using 3 different breast cancer cell models, each representing one of the main molecular subtypes of breast cancer: the triple-negative cell line SUM159 , followed by incubation for 1 h at 37°C. After 3 washes with HEPES buffer, fluorescence was monitored in real time using an inverted microscope . Once the signal was stabilized, 10 mM carbachol (Cch) was added, and fluorescence was monitored until return to baseline. For fluorescence intensity measurements, an entire field visualized under the ×20 objective was chosen, and signal was quantified with Suite X software .2 before transfer to the XF96 instrument. Three measurements of OCR and ECAR were performed before injection of 1 μM oligomycin and 0.5 μM FCCP, followed by 5 more measurements. During the experiment, the cells were kept in a humidified chamber at 37°C, under normal oxygen conditions. Data were processed using the Wave 2.0 software and the XF Cell Energy phenotype report generator (Agilent Technologies).The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in real-time using the Seahorse XF96 analyzer (Agilent Technologies). Cell line characterization was performed according to the manufacturer’s instructions, to achieve optimal cell densities and determine the concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) to be applied. Cells were transfected with siRNAs and seeded in 6 replicates for each sample on a 96-well microplate. On the day of the measurement, cells were washed twice with XF base medium (Agilent Technologies) and supplemented with the appropriate amounts of pyruvate, glutamine, and glucose, according to the culture medium concentrations of each cell line, and the pH was adjusted to 7.4. The cells were incubated in this medium for 1 h in an incubator without COet al. . Significance was accepted at P < 0.05.Data are presented as means ± To examine the impact of H6PD on cancer cell proliferation, we first silenced its expression by using specific siRNAs in the SUM159, MCF7, and MDA-MB-453 cell lines. Subsequently, we performed Western blot analysis at 24, 48, and 72 h after siRNA delivery, to evaluate the efficiency of protein level reduction over time . In SUM1After confirming the knockdown efficiency in our cell models, we down-regulated H6PD and monitored cell proliferation with the xCelligence RTCA. The transfection procedure did not affect proliferation, as observed when comparing nontransfected and mock siRNA-transfected cells A. To inv2/mitotic CCNB1 were greatly reduced after H6PD down-regulation  and p27 (gene CDKN1B), which control progression of the cell cycle at the G1 checkpoint, were increased. In accordance, cell cycle analysis at 48 h after H6PD down-regulation, showed a higher percentage of cells at the G1 phase , CCNA2 (regulating S phase and mitotic entry), and Ggulation A. The leG1 phase B. IncreaBesides increased proliferation potential, a central aspect of tumor cell physiology is their ability to migrate and invade distant tissues. To evaluate whether H6PD is involved in cancer cell migration, we performed a cell migration assay (Transwell) with a serum gradient as a chemoattractant. H6PD knockdown led to an 80% decrease in migration of SUM159 cells, which had the highest migratory potential of the 3 cell lines analyzed (CTNNB1), which was also found to be down-regulated -1α, which, upon activation and phosphorylation, leads to the splicing of the sXBP1, producing the active transcription factor sXBP1, that upon translocation to the nucleus, leads to the expression of target genes, often involved in ERAD , 29. OurThe UPR response can be elicited by a variety of stimuli, including defects in protein folding or protein-folding overload in the ER. To investigate whether the observed differences after H6PD down-regulation in the UPR protein levels are a consequence of changes in protein folding, we analyzed the expression of members from the 3 main protein-folding families , 31. Fir2 were used . We observed a significant increase in FRET efficiency both at 42 and 72 h after H6PD knockdown, which translates into increased ER luminal calcium concentrations, starting at least 42 h after siRNA treatment . Unfortunately, we were not able to successfully implement the ER-specific hyper probe in our cell system . Given tPrevious work showed that H6PD down-regulation in 2 murine cancer cell lines decreased overall ATP levels, without affecting mitochondrial oxygen consumption. In addition, supernatant lactate concentration, indicative of glycolysis, was found to be decreased . In the 1 phase. G1 block after H6PD down-regulation has been reported before, albeit with a more pronounced difference   were decMice lacking H6PD present with skeletal muscle defects and muscles cells from these animals exhibited increased Grp78 protein expression, in addition to alterations in different UPR proteins, as shown by microarrays, including an induction of CHOP . Using tCHOP and XBP1 expression   , 50 and and P62) , 52. Furand P62) , 54.The reliance of cancer cells on the UPR pathway has been well established, and increased levels of critical UPR components have been shown in a multitude of human cancer tissues, including those originating in breast, colon, lung, pancreas, skin, prostate, and brain , 55, 56.57In an attempt to elucidate the mechanism underlying the attenuation of UPR after H6PD silencing, we examined a potential involvement of impaired protein folding. We found significantly increased levels of calreticulin, a calcium-binding protein that surveils proper folding of glycoproteins in the ER lumen before exit to the Golgi . In addiAccording to a previous study, knockdown ofH6PDin 2 murine cancer cell lines caused a reduction in ATP content, which was attributed to a reduction in glucose consumption, whereas no difference in oxidative phosphorylationwas observed (9). In the present study, in analyzing SUM159 cells, we did not find differences in totalATP levels afterH6PDdown-regulation.As different cancer cells can display profound differences in their metabolic capacity and in using different energy sources, we determinedthe impact ofH6PDknockdown on energy metabolism in the 2 breast cancer cell lines SUM159 and MDA-MB-453, both in their basal state and under stressed conditions by treatment with the mitochondrial electron transport chain inhibitor antimycin and the oxidative phosphorylation uncoupler FCCP. Comparison of the baseline and stressed phenotype provides insight into the metabolic potential of each cell line. Knockdown of H6PD led to increased ECAR in the SUM159 cells and decreased OCR in the MDA-MB-453 cells at steady state. Analysis of the rate of OCR/ECAR utilization D showed The results presented highlight the importance of NADPH production in the ER through H6PD for cancer cells. We demonstrated a significant role for H6PD in promoting cell proliferation and migration. Moreover, we described the cellular changes after H6PD silencing that could underpin these effects, including an increased ER luminal oxidation state within the ER, an attenuation of the UPR pathway and an increase in ER calcium concentrations. The ER is a dynamic organelle where vital functions take place, encompassing, among others, protein synthesis and quality control, lipid synthesis, and calcium signaling. Our study opens new avenues into understanding how compromised ER homeostasis influences cancer cell physiology and proposes H6PD as a potential novel therapeutic target against cancer.http://www.fasebj.org to obtain this information.This article includes supplemental data. 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Mammalian patatin-like phospholipase domain–containing proteins (PNPLAs) are lipid-metabolizing enzymes with essential roles in energy metabolism, skin barrier development, and brain function. A detailed annotation of enzymatic activities and structure–function relationships remains an important prerequisite to understand PNPLA functions in (patho-)physiology, for example, in disorders such as neutral lipid storage disease, non-alcoholic fatty liver disease, and neurodegenerative syndromes. In this study, we characterized the structural features controlling the subcellular localization and enzymatic activity of PNPLA7, a poorly annotated phospholipase linked to insulin signaling and energy metabolism. We show that PNPLA7 is an endoplasmic reticulum (ER) transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine in mammalian cells. We found that transmembrane and regulatory domains in the PNPLA7 N-terminal region cooperate to regulate ER targeting but are dispensable for substrate hydrolysis. Enzymatic activity is instead mediated by the C-terminal domain, which maintains full catalytic competence even in the absence of N-terminal regions. Upon elevated fatty acid flux, the catalytic domain targets cellular lipid droplets and promotes interactions of PNPLA7 with these organelles in response to increased cAMP levels. We conclude that PNPLA7 acts as an ER-anchored lysophosphatidylcholine hydrolase that is composed of specific functional domains mediating catalytic activity, subcellular positioning, and interactions with cellular organelles. Our study provides critical structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes. PNPLA genes including neutral lipid storage disease, non-alcoholic fatty liver disease, ichthyosis, hereditary spastic paraplegia, and other neurodegenerative syndromes 1–6–10–yndromes , 13, 14.yndromes , 16.PNPLA6 gene have been linked to a complex spectrum of neuroendocrine disorders including ataxia, spastic paraplegia, chorioretinopathy, and hypopituitarism  of the closely related PNPLA7 . Both proteins share a highly conserved domain architecture that is assembled of the enzymatic patatin-like phospholipase domain and extensive “non-enzymatic” segments of poorly defined function including three putative cyclic nucleotide monophosphate (cNMP)-binding sites , 25. Initrations . In mammtrations . AlthougIn this study, we further characterized the enzymatic function of PNPLA7 in cellular lipid metabolism and established detailed structure–function relationships among domain architecture, subcellular positioning, and enzymatic activity of the protein. We confirm that PNPLA7 acts as a potent intracellular lysophospholipase and identify LPC as a major substrate of PNPLA7 in living cells. Moreover, we demonstrate that PNPLA7 is composed of specific functional parts mediating ER targeting, interactions with LDs, and substrate hydrolysis. Our study provides novel structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes.A). The lipid hydrolase activity of PNPLA7-EGFP was 2.6-fold higher toward C18:1 LPC as compared with C18:0 LPC, suggesting a preference of the enzyme for unsaturated compared with saturated species . Lipid analysis revealed that stable expression of PNPLA7-EGFP significantly reduced cellular LPC levels (−34%) as compared with EGFP-expressing control cells but did not affect total cellular levels of LPE, LPS, PC, PE, or PS . The decrease in LPC levels observed in PNPLA7-EGFP-expressing cells was mainly due to a reduction in unsaturated LPC species containing C16:1 (−35%), C18:1 (−43%), and C18:2 (−52%) fatty acids (FAs) . Although a trend toward decreased cellular levels was also observed for other LPC species, the difference was not significant when compared with controls . To further investigate the function of PNPLA7 in cellular LPC metabolism, we depleted endogenous PNPLA7 in murine AML12 cells by RNAi and assessed cellular LPC hydrolase activities and LPC levels. Stable expression of two independent shRNA constructs targeting PNPLA7 (shRNA1 and shRNA2) decreased cellular Pnpla7 mRNA concentrations by 59 and 63%, respectively, as compared with cells expressing scrambled shRNA (A). This was accompanied by decreased PNPLA7 protein levels in cells expressing either shRNA1 or shRNA2, whereas expression of the closely related PNPLA6 was unchanged (B). The reduction in PNPLA7 expression was associated with a 16–20% decrease in cellular LPC hydrolase activity  but did not affect total cellular LPC levels . Taken together, these data provide evidence that PNPLA7 acts as an intracellular LPC-preferring lysophospholipase that contributes to cellular LPC hydrolase activity in mammalian cells.To assess possible function(s) of PNPLA7 in cellular lipid metabolism, we first created cell lines stably expressing PNPLA7-EGFP or EGFP, respectively, and subjected them to enzyme activity assays and lipid analysis. Consistent with previous studies, cell homogenates expressing PNPLA7-EGFP exhibited increased hydrolytic activity toward several lysophospholipid species as compared with EGFP-expressing control homogenates including C18:1 LPC (4.6-fold), C18:1 lysophosphatidylethanolamine , and C18:1 lysophosphatidylserine  . In addiFig. 1ed shRNA A. This wnchanged B. The reA. The N-terminal half of the protein includes a single predicted transmembrane (TM) domain close to the N terminus and a putative “regulatory” (R) domain, which harbors three predicted cNMP-binding domains (CBDs). The C-terminal half (C domain) includes the patatin-like phospholipase domain predicted to mediate enzymatic activity . Subcellular fractionation of Neuro-2a postnuclear supernatants (PNSs) and subsequent immunoblotting showed that endogenous PNPLA7 was enriched in the membrane fraction but undetectable in the cytosol . Likewise, a PNPLA7-EGFP fusion protein transiently expressed in COS-7 cells was exclusively recovered in the membrane fraction , indicating a similar subcellular distribution of endogenous PNPLA7 and ectopically expressed PNPLA7-EGFP. Membrane-associated PNPLA7-EGFP was partially solubilized with Triton X-100 but not with NaCl or Na2CO3, similar to the integral membrane protein Calnexin but unlike the peripheral membrane protein DDHD2 . Next, we used proteinase K protection assays to assess the topological orientation of PNPLA7 within cellular membranes. Incubation of isolated microsomes with increasing concentrations of proteinase K provoked a dose-dependent depletion of the signal corresponding to full-length PNPLA7-EGFP in our immunoblots . This was accompanied by the generation of a ∼25-kDa fragment likely representing EGFP, which is known to be resistant to proteinase K digestion, indicating proteolytic degradation and therefore cytosolic orientation of the C terminus of PNPLA7 . Using antibodies directed toward the C domain or the R domain of PNPLA7, we failed to detect additional protein bands eluding proteolytic digestion of PNPLA7-EGFP, suggesting that these domains are also largely exposed to the cytosol and thus accessible to proteolytic degradation . Integrity of the isolated microsomes was confirmed by the apparent protection of the 66-kDa luminal domain of Calnexin from proteolytic degradation . To address whether the N terminus of PNPLA7 is oriented toward the cytosol or the lumen, we repeated the proteinase protection assay with microsomes isolated from COS-7 cells expressing PNPLA7 harboring an N-terminal HA tag (HA-PNPLA7). As shown in Fig. 3E, proteinase K treatment depleted the signal corresponding to full-length HA-PNPLA7 and led to the accumulation of an immunoreactive peptide of less than 10 kDa consistent with a luminal orientation of the N terminus. Addition of Triton X-100 to solubilize microsomal membranes rendered this peptide susceptible to proteolytic digestion, suggesting that microsomal integrity is indeed required to protect the N-terminal part of PNPLA7 from proteolysis. In an independent approach, we permeabilized COS-7 cells expressing HA-PNPLA7 either with Triton X-100 or digitonin and costained the cells with antibodies directed toward the HA tag and the C domain . Permeabilization with Triton X-100 rendered both epitopes accessible to antibody staining, resulting in a reticular staining pattern reminiscent of the ER and an apparent overlap of both signals. In contrast, digitonin treatment, which is used to selectively permeabilize the plasma membrane, rendered only the C region but not the N-terminal HA tag accessible to antibody staining . Taken together these results suggest that PNPLA7 is a bilayer-spanning TM protein that orients its N terminus toward the lumen but exposes its R and C domains largely toward the cytosol.To link the lipid-metabolizing function of PNPLA7 to a specific cellular site, we next assessed its subcellular localization and the topological orientation of its domains. The arrangement of possible functional domains within the PNPLA7 polypeptide is shown in Fig. 3in DDHD2 , 29  and assessed their subcellular localization in living COS-7 cells by confocal fluorescence microscopy. Consistent with the results described above, ectopically expressed full-length PNPLA7-EGFP exhibited a reticulate pattern that colocalized with an ER marker protein in COS-7 cells (B). A PNPLA7 mutant lacking the C domain (PNPLA7-N-EGFP) exhibited a similar subcellular distribution as full-length PNPLA7-EGFP and colocalized with the ER, indicating that the C domain is dispensable for ER association of PNPLA7. Furthermore, sole expression of the N-terminal TM domain (PNPLA7-TM-EGFP) was sufficient to target EGFP to the ER, whereas deletion of the TM domain (PNPLA7-ΔTM-EGFP) compromised ER localization of PNPLA7, suggesting an essential requirement of the TM domain for ER targeting of PNPLA7. Notably, expression of a construct harboring an internal deletion of the R but not the TM domain resulted in aggregation of the recombinant protein that failed to properly distribute to the ER network. This suggests that both the TM domain and the R domain contribute to the positioning of full-length PNPLA7 at the ER. Consistent with these findings, a mutant lacking both the TM and R domains (PNPLA7-C-EGFP) distributed diffusely in the cytoplasm and showed only minimal overlap with the ER marker (B). As a complementary approach, we used subcellular fractionation and immunoblotting to assess membrane association of PNPLA7 mutant proteins. As shown in Fig. 4C, PNPLA7-EGFP, PNPLA7-N-EGFP, PNPLA7-TM-EGFP, and PNPLA7-ΔR-EGFP were recovered exclusively in the membrane fraction, whereas PNPLA7-ΔTM-EGFP and PNPLA7-C-EGFP distributed between the soluble and particulate fractions, suggesting impaired membrane association of these mutants. Thus, our data suggest that the N-terminal TM domain mediates efficient membrane tethering of PNPLA7 and that both the TM and R domains but not the C domain control the subcellular positioning of PNPLA7 at the ER.We next addressed the functional contribution of individual protein domains to the subcellular positioning of PNPLA7. To this end, we constructed EGFP-tagged PNPLA7 variants lacking individual domains A and ass-7 cells B. A PNPLR marker B. As a cA). Likewise, neither PNPLA7-N-EGFP nor PNPLA7-ΔTM-EGFP exhibited apparent colocalization with LDs under these conditions. In contrast, PNPLA7-ΔR-EGFP showed punctate and semi-ringlike enrichments in close proximity to LDs, indicating a possible interaction of this truncation mutant with LDs. This phenomenon was even more pronounced for PNPLA7-C-EGFP, which presented as numerous ring-shaped structures surrounding cellular LDs . Moreover, PNPLA7-C-EGFP colocalized with ectopically expressed PLIN2-mCherry, a bona fide LD protein, suggesting that it indeed associates with the LD surface .In addition to the ER, PNPLA7 has been shown to localize to a subset of cellular LDs upon incubation of cells with FAs . To addrFig. 5 surface , 31 . Low levels of PNPLA7-EGFP, PNPLA7-ΔTM-EGFP, and PNPLA7-ΔR-EGFP were also detected in the LD fraction. However, the abundance of these proteins was much lower as compared with PNPLA7-C-EGFP.To further study the interactions of PNPLA7 protein domains with LDs, we fractionated transfected COS-7 cells by ultracentrifugation and assessed the abundance of each PNPLA7 variant in the LD fraction by immunoblotting. Consistent with the microscopy data, ectopically expressed PNPLA7-C-EGFP was markedly enriched in the LD fraction, resembling the distribution of endogenous PLIN2 C. Low leA. Mutants containing truncations at the C terminus retained the ability to localize to LDs, suggesting that the patatin-like phospholipase domain is largely dispensable for LD targeting. In contrast, even minor truncations at the N terminus of the C domain compromised LD targeting. As shown in A and B, a stretch between amino acids 681 and 967 was sufficient for LD targeting, whereas further N- or C-terminal truncations abolished LD association of the C domain. Thus, a stretch of 287 amino acids is essential for the interactions between the C domain of PNPLA7 and LDs.To identify the minimal domain required for LD targeting of PNPLA7-C-EGFP, we further truncated the protein and assessed the ability of these mutants to colocalize with LDs. Schematic drawings of the truncations and their effects on LD targeting are summarized in Fig. 6A), we next asked whether the presence of cNMP analogs would affect the subcellular distribution of PNPLA7. Incubation of cells with 8-CPT-cAMP provoked a subcellular redistribution of ectopically expressed PNPLA7-EGFP, which manifested as ring- and semi-ringlike enrichments of the protein close to the LD surface on top of its reticulate ER-like distribution . A similar response was elicited by 8-CPT-cGMP (data not shown). In contrast to PNPLA7-EGFP, PNPLA7-N-EGFP was not enriched at LDs under identical conditions. Furthermore, deletion of CBD3 but not CBD1 or CBD2 abolished LD interactions of PNPLA7-EGFP (A and B). In summary, these data show that the C domain localizes to cellular LDs and suggest an involvement of both the C domain and CBD3 in LD interactions of PNPLA7 in response to elevated cNMP levels. Noteworthy, the specific C18:1 LPC hydrolase activity of PNPLA7 was largely unchanged upon incubation of cells with FAs and/or cNMPs (C) or starvation (data not shown), suggesting that alterations in subcellular distribution do not alter PNPLA7 activity.Because the N-terminal region contains three putative CBDs A, we nexribution B, inset.LA7-EGFP , A and Bor cNMPs C or starA). As shown in Fig. 8B, ectopic expression of PNPLA7-EGFP or PNPLA7-C-EGFP increased cellular LPC hydrolase activity to a similar extent (2.1–2.3-fold) as compared with EGFP-expressing controls, suggesting that the C domain maintains full catalytic competence also in the absence of TM and R domains. The specific activities of PNPLA7-ΔTM-EGFP and PNPLA7-ΔR-EGFP were 3.0- and 3.3-fold higher than the activity of EGFP-expressing controls, thereby moderately exceeding the activity of PNPLA7-EGFP. In contrast, expression of PNPLA7-N-EGFP failed to increase cellular lysophospholipase activity, which is consistent with a requirement of the C domain for substrate hydrolysis . To assess whether the catalytic C domain is functional also in intact cells, we generated COS-7 lines stably expressing PNPLA7-C-EGFP or EGFP, respectively, and performed lipid analyses. Expression of PNPLA7-C-EGFP reduced cellular levels of LPC by 42% compared with EGFP-expressing control cells but did not affect cellular levels of PC, PE, PS, LPE, and LPS . Thus, the C domain is both required and sufficient to mediate catalytic activity of PNPLA7 in mammalian cells.We finally aimed to understand how specific protein domains regulate the enzymatic function of PNPLA7. To do so, we expressed PNPLA7-EGFP or truncated PNPLA7-EGFP mutant proteins in COS-7 cells and compared cellular lysophospholipase activities. Immunoblotting revealed similar expression levels of PNPLA7-EGFP and truncated versions of the protein A. As shoin vivo  and/or a specific subset of LDs (e.g. ER-associated or “nascent” LDs).We initially described PNPLA7 as a lipid hydrolase with a dual subcellular localization at the ER and LDs . Our newAdditional structure–function analyses revealed that the C domain constitutes the major catalytic part of PNPLA7 and thus serves a dual role in subcellular localization and substrate hydrolysis. Expression of full-length PNPLA7 or the isolated C domain increased cellular lysophospholipase activity to a similar extent, suggesting that N-terminal domains neither restrict nor promote catalytic activity of PNPLA7. Consistent with this finding, it has been shown that the C-terminal esterase domain of the homologous human protein PNPLA6 acquires catalytic competence also in the absence of N-terminal regions . Sole exsn-glycero-3-phosphocholine), C16:0 LPC , C18:1 LPC (1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine), C18:1 LPS (1-oleoyl-2-hydroxy-sn-glycero-3-phospho-l-serine), C18:1 LPE (1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), C18:1/C18:1 PC , C18:1/C18:1 PE , and C18:1/C18:1 PS  were obtained from Avanti Polar Lipids . l-α-Lysophosphatidylcholine (ART1809) was obtained from American Radiolabeled Chemicals . HCS LipidTOXTM Deep Red was obtained from Thermo Fisher Scientific . 8-CPT-cAMP was purchased from Abcam , and 8-CPT-cGMP was purchased from Sigma-Aldrich.C18:0 LPC (1-stearyl-2-hydroxy-TM sheep anti-mouse IgG antibody (NA931) linked to horseradish peroxidase (HRP) was purchased from GE Healthcare. An HRP-linked goat anti-rabbit IgG antibody (PI-1000) was purchased from Vector Laboratories . An HRP-linked goat anti-guinea pig IgG antibody (6090-04) was purchased from Southern Biotechnology . A Rhodamine RedTM-X-linked goat anti-mouse IgG antibody was obtained from Jackson ImmunoResearch Laboratories , and DyLight® 488-conjugated goat anti-rabbit IgG antibody was obtained from Thermo Fisher Scientific.A rabbit anti-GFP antibody (ab6556) was purchased from Abcam. A rabbit anti-PNPLA7 antibody (HPA009130), which is directed to the C domain of PNPLA7, and a mouse anti-HA antibody (H3663) were obtained from Sigma-Aldrich. A guinea pig anti-PLIN2 antibody (GP40) was obtained from PROGEN . A rabbit anti-DDHD2 antibody (25203-1-AP) was obtained from Proteintech . Rabbit anti-Calnexin (2679), rabbit anti-GAPDH (2118), and rabbit anti-IRE1α (3294) antibodies were obtained from Cell Signaling Technology . Antisera against the R domains of murine PNPLA7 and PNPLA6 were generated by repetitive immunizations of rabbits with the peptides CSVPLPSNHGEVDELRQSQGSGSNT and PAGDPVKPTSLEAPPAPLLSRC, respectively, which were coupled to keyhole limpet hemocyanin as carrier . An ECLProtein domains were analyzed using the protein family database (Pfam), an online database containing collections of protein domains and families . TM domaThe vectors pDsRed2-ER, pmCherry-N1, and pEGFP-N3 were obtained from Clontech. pEGFP-N1 containing the coding sequence (cds) of human PLIN2 was a kind gift from Stefan Höning . To generate a plasmid encoding PLIN2-mCherry, the cds of PLIN2 was amplified using the primers 5′-GCG AAT TCG CCA TGG CAT CCG TTG CAG TTG A-3′ and 5′-CAA CCG GTC GAT GAG TTT TAT GCT CAG ATC G-3′, and the PCR product was digested with EcoRI and AgeI and ligated into the multiple cloning site of pmCherry-N1. The cds of PNPLA7 was amplified by PCR using cDNA of murine cardiac muscle as template and the primers 5′-TTC TCG AGG CCA TGG AGG AGC AGT CCC AGT CC-3′ (F1) and 5′-GCG AAT TCG ACG AAG GAT GTT CCA GTC TTG G-3′ (R1). PNPLA7 truncation mutants were generated by PCR using the primers 5′-TTC TCG AGG CCA TGG CAG AAC CCA CTC CTC AGT AC-3′ (F2) and R1 for ΔTM-PNPLA7 (Δ1–40), 5′-TTC TCG AGG CCA TGG TTG TAA CGC GAC TGA TTC ATC TC-3′ (F3) and R1 for PNPLA7-C (Δ1–680), 5′-GCG AAT TCG CCA TGG AGG AGC AGT CCC AGT C-3′ (F4) and 5′-CAG GAT CCC CGT CGT AAT CTT CTC ACT C-3′ (R2) for TM-PNPLA7 (Δ41–1326), F1 and 5′-GCG AAT TCG ACT GTG GGT ACC TGC GCT TGA TAG-3′ (R3) for PNPLA7-N (Δ681–1326), F3 and 5′-GCG AAT TCG ATC TTG CCA CAT CCG CTG GGA G-3′ (R4) for PNPLA7-C1 , F3 and 5′-GCG AAT TCG AGA AGA TGT TGC TGA TGC TGC-3′ (R5) for PNPLA7-C2 , F3 and 5′-GCG AAT TCG AAG CAA ACA GAG CAC CCA TGA A-3′ (R6) for PNPLA7-C3 , F3 and 5′-GCG AAT TCG AGG CAC ACC CTC TAG CTC CAC C-3′ (R7) for PNPLA7-C4 , 5′-TTC TCG AGG CCA TGT TTG CTC TGG AGC TCC AAC A-3′ (F4) and R1 for PNPLA7-C5 (Δ1–741), 5′-TTC TCG AGG CCA TGT TGG TGC TTG GAG GGG GTG GA-3′ (F5) and R1 for PNPLA7-C6 (Δ1–923), 5′-TTC TCG AGG CCA TGT CCA TGG GGG CAA AGG TTG TG-3′ (F6) and R1 for PNPLA7-C7 (Δ1–1090), and F4 and R6 for PNPLA7-C8 . The PCR products were purified by agarose gel electrophoresis and inserted into the multiple cloning site of pEGFP-N3 using the XhoI and EcoRI restriction sites to create C-terminal in-frame fusions with EGFP. Internal deletion mutants of PNPLA7 were generated using the Q5® site-directed mutagenesis kit with PNPLA7-EGFP as template and the primers 5′-CCG TCG TAA TCT TCT CAC-3′ and 5′-GTT GTA ACG CGA CTG ATT C-3′ for ΔR-PNPLA7 (Δ41–680), 5′-CAG ATC ATC ATG GTC CGG-3′ and 5′-CCG AAC ATT TTT CAA CAT GTA C-3′ for ΔCBD1 (Δ144–265), 5′-GTC CTG GGC GTG GCA CAC-3′ and 5′-GTC CTT TGT GGC AGC TCT GAA G-3′ for ΔCBD2 (Δ455–565), and 5′-GTT GTA ACG CGA CTG ATT C-3′ and 5′-CAT CCT CTT CAC AAC TGT G-3′ for ΔCBD3 (Δ578–680). A DNA fragment encoding for PNPLA7 harboring an N-terminal HA tag was created by PCR using the primers 5′-TAA TCT CGA GGC CAC CAT GTA CCC ATA CGA TGT TCC AGA TTA CGC TAT GGA GGA GCA GTC CCA GTC-3′ and 5′-GAC GAA TTC GTC AGG AAG GAT GTT CCA GTC-3′. The PCR product was purified as described above and inserted into the multiple cloning site of pEGFP-N3 using the XhoI and EcoRI restriction sites. To generate lentiviral expression vectors, pEGFP-N3 constructs encoding the open reading frames for EGFP, PNPLA7-EGFP, and PNPLA7-C-EGFP were digested with XhoI and NotI, and the resulting fragments were inserted into the multiple cloning site of pLVX IRES Puro (Clontech). Mission® lentiviral pLKO.1 vectors encoding for scrambled shRNA or shRNAs targeting murine PNPLA7 were obtained from Sigma-Aldrich. Constructs used for RNAi experiments targeted the sequences CCAAGAGGATTCTGCGCTTTA (shRNA1) and CATGTCCTTGTCAGGCTATAT (shRNA2) of the PNPLA7 cds.ml-glutamine, 1 mm sodium pyruvate, non-essential amino acids, 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. COS-7 cells (ATCC CRL-1651) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. AML12 cells (ATCC CRL-2254) were cultured in a 1:1 mixture of DMEM and Ham's F-12 medium supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone. All cells were maintained at 37 °C, 95% humidity, and 5% CO2. Cells were transfected with Metafectene®  according to the manufacturer's instructions and used for experiments 24 h thereafter. Cells were treated with 400 μm oleic acid bound to bovine serum albumin , 1 mm 8-CPT-cAMP, or 1 mm 8-CPT-cGMP for 16 h or with Hanks' buffered salt solution for 4 h before being processed for imaging or lipid hydrolase assays.Neuro-2a cells (ATCC CCL-131) were maintained in minimum essential medium supplemented with 2 mLentiviral particles harboring pLVX IRES Puro vectors for the expression of EGFP-tagged proteins or pLKO.1 vectors for the expression of shRNAs were generated in HEK293T cells (ATCC CRL-3216) according to the manufacturer's instructions (Clontech). Before transduction, COS-7 or AML12 cells were seeded into 6-well plates at a density of 300,000 cells/well. Cells were incubated for 24 h with lentivirus-containing supernatants in the presence of 8 μg/ml Polybrene. To select for stable expression, cells were maintained for 7 days in medium containing 2 μg/ml puromycin.6 cells) were extracted twice according to Folch et al.  for UPLC-triple quadrupole analysis. Chromatographic separation was modified after Knittelfelder et al.  starting with a 25-min gradient with 100% solvent A , 10 mm ammonium acetate, 0.1% formic acid). An EVOQ EliteTM triple quadrupole mass spectrometer (Bruker) equipped with an electrospray ionization source was used for detection. Lipid species were analyzed by selected reaction monitoring . Data acquisition was done by MS Workstation (Bruker). Data were normalized for recovery and extraction and for ionization efficacy by calculating analyte/internal standard ratios.Total lipids of cell pellets , by passing 30 times through a 26-gauge needle on ice. PNSs were obtained by centrifugation at 4 °C and 1,000 × g for 10 min. PNS was further fractionated into cytosol and membrane fractions by ultracentrifugation at 4 °C and 100,000 × g for 60 min. For the isolation of lipid droplets, cells were homogenized on ice with a Dounce homogenizer in 20 mm Tris/HCl, 1 mm EDTA, pH 7.4, and lipid droplets were isolated by ultracentrifugation using a discontinuous sucrose gradient according to Brasaemle and Wolins (TM Protein Assay kit according to the manufacturer's protocol (Thermo Scientific).COS-7 cells were washed with phosphate-buffered saline (PBS), scraped from tissue culture plates, and collected by brief centrifugation. For the preparation of membrane and cytosol, cells were homogenized in 10 md Wolins  with minm NaCl, 0.1 m Na2CO3, or 1% Triton X-100 for 30 min on ice. Afterward, soluble and insoluble components were separated by centrifugation at 4 °C and 100,000 × g for 30 min and subjected to immunoblotting. For membrane protection assays, isolated membrane fractions were incubated in the presence of 0–10 μg/ml proteinase K in the absence or presence of 1% Triton X-100 for 15 min at room temperature. The reactions were stopped by the addition of PMSF at a final concentration of 2 mm, further incubated for 5 min, and subjected to immunoblotting.Membrane fractions of COS-7 cells were resuspended in buffer A and incubated in the presence of 1 m Tris/HCl, 150 mm NaCl, 0.1% Tween 20, pH 7.5 (TBST), containing 10% milk to block unspecific binding sites. Primary and secondary antibodies were diluted in TBST containing 5% milk and incubated with the membrane for 1 h at room temperature followed by extensive washing with TBST.Samples were mixed with Laemmli buffer, denatured for 5 min, subjected to SDS-PAGE, and electroblotted onto PVDF membranes. Membranes were incubated for 1 h with 20 mTM, Life Technologies) according to the manufacturer's instructions. RT-qPCR was performed as described previously  and the MaximaTM SYBR Green/ROX PCR reaction mixture . The following primers were used: Pnpla7 fw, 5′-CGT GTT TTC CAA CGA CCA CC-3′; Pnpla7 rv, 5′-TCT GCT AGT GCC CTG AGG AT-3′; 36B4 fw, 5′-GCT TCA TTG TGG GAG CAG ACA-3′; 36B4 rv, 5′-CAT GGT GTT CTT GCC CAT CAG-3′. Relative mRNA levels were quantified according to the ΔΔCt method using 36B4 as reference gene.Cells were harvested and snap frozen in liquid nitrogen. RNA was extracted using the TRIzol® reagent  followed by centrifugation at 4 °C and 1,000 × g. Lipid substrates were prepared by sonication of glycerophospholipids in 200 mm Bistris propane buffer, pH 7, containing 10 mm CHAPS, 1 mm EDTA, and 600 mm NaCl. Unless otherwise indicated, substrates contained lysophospholipids at final concentrations of 3 mm or diacylglycerophospholipids at final concentrations of 1 mm. Assays were started by the addition of 50 μl of substrate to 50-μl samples in solution A and incubated under steady shaking at 37 °C in a water bath. Substrate hydrolysis is expressed as release of FAs, which was determined routinely using a commercially available kit (non-esterified FA (NEFA) reagent, Wako, Neuss, Germany) according to the manufacturer's instructions. For the determination of endogenous LPC hydrolase activities in AML12 cells, 3H-labeled LPC at a specific activity of 0.33 μCi/μmol was included in the substrate. After incubation, assays were extracted with 0.5 ml of chloroform/methanol/acetic acid , and extracts were separated by thin layer chromatography using Silica G plates and hexane/diethyl ether/acetic acid  as solvent. FAs were identified by comigrating authentic standards, and the associated radioactivity was determined by liquid scintillation counting. All assays were performed in triplicates.Protein samples for lipid hydrolase assays were prepared by sonication of COS-7 or AML12 cells in 0.25 COS-7 cells were seeded in chambers mounted onto coverslips  and transfected as described above. Cells expressing EGFP-tagged proteins were fixed with 4% paraformaldehyde for 20 min at room temperature, and LDs were counterstained using HCS LipidTOX Deep Red. Cells expressing HA-PNPLA7 were fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized for 15 min with PBS containing either 0.1% Triton X-100 or 0.0005% digitonin. Cells were washed three times with PBS and incubated at room temperature with PBS containing 1% BSA for 1 h to block unspecific binding. Primary antibodies were diluted in blocking solution and incubated with cells overnight at 4 °C. Cells were washed with PBS and incubated with secondary antibodies diluted in blocking solution at room temperature for 1 h followed by extensive washing with PBS. Cells were imaged using a Leica SP5 confocal microscope equipped with a Leica HCX 63× 1.4 numerical aperture oil immersion objective. EGFP and DyLight 488-conjugated antibodies were excited at 488 nm, and emission was detected between 500 and 530 nm. DsRed2, mCherry, and Rhodamine Red were excited at 561 nm, and emission was detected between 580 and 610 nm. HCS LipidTOX Deep Red was excited at 633 nm, and emission was detected between 650 and 700 nm.t test. Group differences were considered statistically significant for p < 0.05 (*).All measurements were performed in triplicates. Data are presented as means ± S.D. Statistical significance was determined by the Student's unpaired C. H., P.-A. C., and R. Z. conceived and coordinated the study and wrote the paper. H. X. and F. H. performed molecular biology work. C. H., B. K., and P.-A. C. performed all cellular, biochemical, and imaging experiments. T. O. E. performed lipid analyses. All authors reviewed the results and approved the final version of the manuscript.


Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (sleeping sickness), yet little is known about which PKs are essential for survival in mammals. A recent kinome-wide RNAi screen with 176 individual bloodstream form Trypanosoma brucei lines identified PKs required for proliferation in culture. In order to assess which PKs are also potential virulence factors essential in vivo, lines were pooled, inoculated into mice, and screened for loss of fitness after 48 h RNAi. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to growth in culture. We identified 49 PKs with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. Nine PKs had a more pronounced growth defect in vivo, than in vitro. Amongst these PKs were several with putative functions related to stress responses mediated through the PI3K/TOR or MAPK signaling cascades, which act to protect the parasite from complement-mediated and osmotic lysis. Identification of these virulence-associated PKs provides new insights into T. brucei-host interaction and reveals novel potential protein kinase drug targets.Protein kinases (PKs) are a class of druggable targets in  Trypanosoma brucei is an obligate parasite in the class kinetoplastida, a group of flagellated protozoa characterized by a dense complex of circular mitochondrial DNA called the kinetoplast. T. brucei spp infect humans and animals in sub-Saharan Africa, causing diseases that remain major threats to health and economies in the region. T. brucei exhibits a complex life cycle that requires adaptation to many different environments, including different compartments within the blood-feeding insect vector responsible for transmission between different mammalian hosts: the tsetse fly. Once in the mammal, T. brucei resides extracellularly in the bloodstream, tissue fluids, central nervous system and adipose tissue1. Bloodstream T. brucei undergo antigenic variation and express a single Variant Surface Glycoprotein (VSG) per cell, which can be switched upon expansion of the population to create diversity2. The host builds an adaptive immune response against at least the most abundant variants, leading to their clearance and enabling outgrowth of cells that have switched to an antigenically distinct VSG. Iteration of this process leads to the characteristic waves of parasitemia3.T. brucei protein kinome represents 2% of the parasite’s protein-coding capacity and comprises 157 conserved eukaryotic PKs (ePKs), 12 non-catalytic pseudokinases and 20 atypical PKs (aPKs)6. Substantial differences exist between the T. brucei and the human protein kinomes, as the parasites lack receptor-linked tyrosine kinases and tyrosine-like kinases. Despite this, tyrosine phosphorylation has been reported, possibly due to dual-specificity kinases5. T. brucei also has a relatively reduced representation of AGC and CAMK families, while CMGCs, STEs and NEKs are comparatively expanded. In addition, several highly divergent PKs are likely to play parasite-specific functions that may present targets for selective inhibition by small molecules5.Protein kinases (PKs) are key signalling proteins in eukaryotes, playing critical roles as central regulators in many biological functions, as well as being validated drug targets. The 7. Several high-throughput screening campaigns with compound libraries have been published linking in vitro trypanocidal activity with PK inhibition10, though the specific PK target in each case is unknown. Over 40 T. brucei PKs have been shown to be essential for normal cell proliferation in vitro11. Many biological roles for PKs have been revealed in T. brucei, but they have been observed mainly in the artificial conditions of in vitro culture. In this paper we use a kinome-focused RNAi library in a 72 h mouse infection model to address a key question of both biological and pharmaceutical relevance: which PKs are required for survival of the parasite in the environment of the mammalian bloodstream?PKs are a promising source of druggable targets, with more than 100 inhibitors already in clinical trials and successful drugs in the market, such as the prototypical compound Imatinib® for chronic myeloid leukemiaT. brucei RNAi cell lines to identify PKs essential for proliferation of bloodstream form parasites in culture, cell cycle regulators and negative regulators of BSF to PCF differentiation6. In order to increase the capacity for screening the kinome RNAi library, we made a pool of the 177 available cell lines, which targeted 183 of the PKs (6 were double knockdowns)6. This pool allowed parallel phenotyping of the population in a single culture (in vitro) and in a single mouse (in vivo) through RNAi target sequencing  with a bootstrap analysis including 1000 repeats, which enabled calculation of the median and estimation of 95% confidence intervals for each gene (Data sets in vivo. When the 0.6 (induced/uninduced) threshold was applied to the in vitro data set it was determined to be discriminatory, showing a progressive increase of the number of targets identified with time  and are likely to be kinetoplastid-specific. All of the 49 PKs have orthologues in either Leishmania major or Trypanosoma cruzi or both, though with variable levels of sequence identity between the kinetoplastid parasites  and Tb927.10.10350 (STE11/Bck1p), experienced growth retardation in vivo from 48 h, indicating a loss of fitness. For three other genes, Tb927.2.2430 (Other/CK2A2), Tb927.7.960 (CMGC/SRPK1) and Tb927.10.14300 (STE11/MRK1), the loss of fitness after in vivo RNAi was even more pronounced, since a decrease in parasitemia or clearance was seen before 96 h  to fresh rat serum for 3 h (10% and 50% concentration) and analyzed relative survival of RNAi induced and uninduced cells when compared to controls that were not exposed to rat serum , which caused impaired cell growth in vitro, caused no serum sensitivity. In contrast, RNAi against Tb927.2.1820 (CAMKL), Tb927.3.3920 (AUK2) and Tb927.7.960 (CMGC/SRPK1) resulted in significant, concentration-dependent sensitivity to serum, manifest in survival rates 50% lower than the equivalent uninduced samples. To understand whether such sensitivity was due to complement-mediated lysis, these three cell lines -RNAi induced and uninduced- were further exposed to 50% serum complement-inactivated with 20 mM EDTA17. For Tb927.2.1820 (CAMKL) and Tb927.7.960 (CMGC/SRPK1), the EDTA treatment ablated the serum sensitivity, strongly suggesting that the RNAi phenotype was due to increased complement-mediated killing. AUK2, however, was more difficult to interpret as Tet− cells showed increased survival when EDTA was added.One of the most obvious and distinctive elements encountered by parasites during infection in mice when compared with rum Fig. . In addiin vivo than in vitro  have a significantly more pronounced loss of fitness phenotype tro Fig. ; these htro Fig. . To testols Fig. . RNAi agT. brucei to survive in the mammalian bloodstream. We modified whole-genome parallel phenotyping based on RITseq technology12 and applied it to a defined collection of individually designed and validated RNAi cell lines. This method was used to analyze in vitro and in vivo experiments to permit the identification of 49 essential PKs, including 9 PKs that appear more important for survival in the environment of the mammalian circulatory system than in culture. Bioinformatic assessment of each gene suggested these PKs are involved in parasite stress-response pathways. Validation of individual cell lines identified PKs that play roles in protecting the parasite from mammalian serum factors and osmotic stresses. Our findings expand the repertoire of essential and potentially druggable PKs in this pathogen and begin to explore the mechanisms by which the parasite survives the multiple stresses of life inside its host.In this study we aimed to define the cohort of protein kinases that are essential for in vivo experiments for several reasons. The coefficient of determination between the induced/uninduced read mapping ratios obtained between the two experiments was very good, especially when focused on those with a loss of fitness RNAi phenotype , elements involved in stress management are also ‘indirect’ or ‘contributory’ elements required for establishment of the infection19. A connection between stress resistance and virulence has also been found for many other infectious eukaryotic organisms24, including Leishmania26. Sequence similarity between eight of the T. brucei in vivo-specific PKs and kinases in other eukaryotes suggests diverse functions, including gene expression, alternative splicing, protein synthesis and translational regulation in response to extracellular signals, and stress adaptation linked with autophagosome formation  PKs were identified amongst the in vivo cohort. Tb927.10.14300 (STE11/MRK1) has features in common with members of the yeast High Osmolarity Glycerol (HOG) pathway, which responds to osmotic challenge through actin recovery29, while the MORN motifs-containing protein Tb927.10.10350 (STE11/Bck1p) resembles human MEKK3, a positive regulator of the stress-activated protein kinase (SAPK) pathway30 putatively involved in cell integrity maintenance under stress conditions. The increased susceptibility to osmotic shock seen after RNAi induction of these two STE11 PKs  were not identified in this screen, which may suggest redundancy amongst the T. brucei STE7 cohort, or perhaps a non-canonical mode of action for the T. brucei STE11 PKs. Two other MAPKs (ERK-like), often involved in transcriptional and non-transcriptional regulation in response to external stimuli, were found to be essential both in vivo and in vitro: KFR1, previously reported to be regulated by interferon γ in order to promote proliferation in T. brucei32; and MAPK6 (ERK8), which regulates cytokinesis33.Trypanosomes are highly adaptable organisms, capable of withstanding immune assault by the host and responding to the many changing conditions they experience during their life cycles, including temperature, pH, osmotic imbalance, shear-forces, and availability of carbon and nitrogen sources. This study reveals that depletion of at least nine PKs limits the capacity of Data set . One of PKs Fig.  indicatein vivo cluster together within the PI3K/TOR pathway: Tb927.11.9190 (VPS15), Tb927.11.850 (Bud32) and Tb927.7.960 (SRPK1). VPS15 is required for stress-induced and developmentally-triggered autophagosome formation in other eukaryotes and assembles within the PI3K complexes34. PI3K signalling in yeast involves Bud32 , which acts as a transducer for TOR activation that, in turn, has also been related with autophagy and endocytosis under stress conditions. SRPK1 regulates splicing factors in response to stress and can be activated via PI3K signaling in other eukaryotes35. The identification of these TOR-related PKs is of interest, because TOR pathways are central for parasite-host interaction in T. brucei36. While most eukaryotes encode two TOR paralogues, trypanosomatids are the only known organisms to contain four, each integrated in a different protein complex, of which three are required for normal fitness both in vivo and in vitro . The three showed an increased sensitivity to serum upon RNAi induction, related to complement-mediated lysis. In what way, if at all, the two phenotypes might overlap is unclear but the potentially common function of each T. brucei PK in providing serum resistance . The final gene that is only essential for in vivo growth, CK2A2, has validated protein kinase activity and localizes primarily to the nucleolus42. CK2 is mainly controlled by distribution and abundance, with stress-induced mobilization extensively reported43. CK2 has also been associated with autophagy through intersection with PI3K/AKT/TOR and MAPK pathways44.It is intriguing that three PKs with a pronounced loss of fitness nce Fig.  might guT. brucei protein kinases are druggable45 and this work provides genetic validation for 49 protein kinases that are required for in vivo fitness. A phylogenetic comparative analysis between the T. brucei and Homo sapiens kinomes showed that 13 out of the 49 PKs had close human orthologues targeted by at least one of the compounds in the ‘HAT box’  + 10% v/v foetal bovine serum (GIBCO), Pen/Strep solution ] at 37 °C/5% CO2 in vented flasks. Selective antibiotics were used as follows: 0.2 μg ml−1 puromycin, 0.5 μg ml−1 phleomycin (InvivoGen), 2.5 μg ml−1 hygromycin B . RNAi was induced in vitro with tetracycline (Sigma Aldrich) in 70% ethanol at 1 μg ml−1.Monomorphic 6 cells ml−1 were exposed to fresh rat serum for 3 h (10% and 50% concentration). Relative survival was measured of induced (for 48 h) and uninduced clones compared to no-serum controls. The whole experiment was performed in triplicate.To test for serum resistance 200 μl of log-phase parasites in HMI11 at 1018 2 × 106 cells were resuspended for 5 min at 4 °C in 0.5 ml of a solution 55 mM KCl/1 mM glucose to induce cell swelling. 0.5 ml of a 263 mM KCl/1.75 mM Mg2Cl shrinking solution was added on top and cells incubated for 5 min at 4 °C. Remaining cells were washed once and resuspended in 1 ml of HMI11 media before counting. Survival of the induced cells compared to uninduced controls was plotted and significant results (p < 0.05) after T-tests indicated with a star.To test for response to osmotic shock6. RNAi lines were pooled into 9 ‘Mixed Stabilate Trypanosome Libraries’ (MSTL 1–9), each containing 19–25 cell lines. Then MSTLs were further pooled to make the final culture.All RNAi cell lines in this work were generated as previously describedin vitro RITseq was defrosted, grown for 24 h and diluted to contain 1 × 105 cells ml−1 in 100 ml. In triplicate the culture was split into two 50 ml flasks (tetracycline induced and uninduced control) and grown for 120 h, reducing cells to 1 × 105 cells ml−1 every 24 h. 1 × 107 cells were sampled daily for analysis.The library used for the ad libitum. Infections were performed by intraperitoneal injection and were evaluated daily by assessing parasitemia levels with a hemocytometer by tail vein sampling diluting blood in anticoagulant (CBSS/Heparin) 0.83% w/v. If parasitemia exceeded 108 cells ml−1 mice were euthanized with carbon dioxide and cervical dislocation. RNAi was induced in mice with doxycycline hyclate (Sigma Aldrich) added in sugared drinking water (0.2 g/L plus 50 g/L sucrose). For the in vivo RITseq, blood from euthanized mice 72 h after infection was collected and immediately applied to DEAE-cellulose columns as described50. DNA was isolated from the parasites using a QIAamp DNA kit .Female CD1 outbred mice (6–8 weeks old) were obtained from Charles River . Animals were given water and food A-3′) was used to PCR enrich RNAi inserts amplified from 10 ng of genomic DNA obtained per sample in a 50 µl reaction using Q5® High-Fidelity DNA polymerase . The PCR program was: 3 minutes at 98 °C; followed by 28 cycles comprising: 10 seconds at 98 °C, 10 seconds at 61 °C and 30 seconds at 72 °C; with a final extension step at 72 °C for 10 minutes. PCR products were cleaned up with Minielute PCR purification kit , 400 ng of DNA per sample were fragmented, size selected (average read length after sequencing ~106 nucleotides) and processed according to Ion Torrent library prep protocols. The multiplexed sample was subjected to emulsion PCR and Ion Torrent sequencing. The library pool used at Experiment 2 was processed as above with the following exceptions, a mix of MSTLs 1–9, PCR annealing temperature was elevated to 64 °C, as additional 6 nucleotide-long labels were added to the single universal primers 51, using default parameters. For in vivo Exp.1, single end reads generated from each sample were selected by the presence of a 9 base diagnostic tag [GCCAACTTT] present within the universal primers, permitting 1 mismatch . Approximately 20% of the reads contained the RNAi cassette. For in vivo Exp.2 and in vitro RITseq, single end reads were identified and clustered according to 6 base-long barcodes before being processed as in Exp.1 (without the fragmentation/size selection step) finding 65–70% of reads containing the RNAi target. Selected reads were mapped to the artificial chromosomes with Bowtie2 . The “.sam” format files generated by Bowtie2 were parsed and the coordinates of the appropriate chromosome to which the read mapped were recorded. The mapped reads were assigned to the appropriate PK sequence by reference to the index generated above. The read was assigned if it lay entirely within the PK sequence or overlapped the 3′ or 5′ end of the PK sequence. In each replicate, accumulated read abundances were normalized by multiplying raw counts 106 times, dividing by the sum of total valid reads accepted for the analysis in the whole sample and rounding to the next integer, i. e. reads per million (RPM). Bowtie2 mapping was used also to compare whole-genome/kinome-wide RITseq coverage as depicted in Fig. Artificial chromosomes were generated Supplementary informationDataset S1Dataset S2Dataset S3Dataset S4Dataset S5


The combination of different analysis techniques allowed us to describe intermediate/hybrid and complete EMT phenotypes respectively in HCC827- and HCC4006-derived drug-resistant human cancer cell lines. Interestingly, intermediate/hybrid EMT phenotypes, a collective cell migration and increased stem-like ability associate to resistance to the epidermal growth factor receptor inhibitor, erlotinib, in HCC827 derived cell lines. Moreover, the use of three complementary approaches for gene expression analysis supported the identification of a small EMT-related gene list, which may have otherwise been overlooked by standard stand-alone methods for gene expression analysis.Increasing evidence points to a key role played by epithelial-mesenchymal transition (EMT) in cancer progression and drug resistance. In this study, we used  Severtastasis , 4. Moretastasis , 6. The  studies , 8. In t studies , 8.Multiple signaling pathways and complex genetic and epigenetic mechanisms regulate the EMT program in normal and neoplastic epithelial tissues , 9–12. ILung cancer is the most frequent cause of cancer-related mortality worldwide leading to over a million deaths each year . Based oIn order to investigate the mechanisms of resistance to TKI, we have recently reported the establishment and characterization of NSCLC cell lines resistant to the EGFR inhibitor erlotinib .wet and in silico approaches to investigate whether E/M phenotypes are associated to erlotinib-resistance in our cellular model system. The combination of different analysis techniques allowed us to describe intermediate and complete EMT phenotypes in HCC827- and HCC4006-derived erlotinib-resistant cell lines respectively. Interestingly, EMT intermediate phenotypes, collective cell migration and increased stem-like ability associate to resistance to target therapy in the erlotinib-resistant HCC827-derived cell lines. Moreover, the use of three complementary approaches for gene expression analysis supported the identification of a small EMT-related gene list, which may have otherwise been overlooked by standard stand-alone methods for gene expression analysis.The effect of TKI target therapy on the selection of intermediate E/M phenotypes in cancer cells is still poorly investigated. Therefore, in this study, we used in vitro models of acquired resistance to the EGFR TKI erlotinib [50 > 10 μM) [Recently, in order to investigate mechanisms leading to resistance to EGFR-targeted therapy, two NSCLC cell lines (HCC827 and HCC4006) have been used to derive rlotinib . Both pa> 10 μM) . Charact> 10 μM)  and is sInterestingly, morphological analysis of the erlotinib-resistant NSCLC cells showed the presence of cells with a fibroblast-like cell shape reminiscent of EMT, especially in the RA1, RB1, RB2 and RC2.2 cell lines . Indeed,ZEB [Vimentin and Cadherin-1 despite generally used to characterize the EMT phenotype, could be not sufficient to identify and describe complex EMT features. Therefore, the expression levels of other EMT-related markers, such as Cadherin-2 , Claudin-1 (CLDN1), SNAI1 , SNAI2  and ZEB1, were also investigated by western blotting Figure  and qPCRZEB –35, we aA more complex picture was found for some EMT markers. In particular Cadherin-2 and Claudin-1 are observed respectively upregulated and downregulated in the HCC4006-derived RC2.2 cell line while in the HCC827-derived cell lines, Cadherin-2 appears to be upregulated only in RA1, RA2 and RB1 cell lines Figure  and ClauOverall the data suggest that the total population of the RC2.2 cell line, that does not express Cadherin-1 and expresses high levels of Vimentin, is in a mesenchymal state while the majority of HCC827-derived erlotinib-resistant cell lines might likely contain subpopulations with intermediate states of EMT.We used wound-healing assays in the erlotinib-resistant cell lines in order to investigate the effect of the observed EMT phenotypes on cell migration. The RC2.2 (HCC4006-derived) and the RA2 (HCC827-derived) erlotinib-resistant cell lines show an increased rate of wound closure, while for all other erlotinib-resistant cell lines quantitative differences in terms of wound closure capacity were not observed by comparison with their parental cell line Figure . ImportaMany cancer cells are known to exhibit collective invasion potential wherein cell–cell adhesions remain intact. Accordingly, in EMT phenotypes, marked by an heterogeneous cancer cell population with epithelial, E/M and mesenchymal features, the cells with intermediate E/M phenotypes could potentially contribute to a collective cell migration due to their ability to interact with epithelial cells by cell-cell contacts while also being migration-prone for their mesenchymal features. Indeed, in the wound healing assays of the HCC827-derived erlotinib-resistant cell lines, E/M Vimentin and Cadherin-1 double positive cells are detected at the wound leading edge in cell-cell contact with Cadherin-1 positive and Vimentin negative cells Figure . In partErlotinib-resistance and EMT phenotypes can associate with more aggressive behaviours, such as increased cell invasion and stem-like features. Therefore to further study the functional phenotype of the erlotinib-resistant NSCLC cell lines, matrigel-coated transwell invasion and sphere-forming assays were performed. The matrigel assays showed an increased cell invasion of the RA2 and RC2.2 NSCLC cell lines when compared to their parental erlotinib-sensitive cell lines Figure . InsteadRegarding the sphere-forming assays, we observed that the number of spheroids of the RA1, RB1, RB1.1 and RB2 cell lines with a diameter ≥ 50 μm was 2-3 fold higher than of the HCC827 parental cell line Figure  while thin vitro cell migration and cell invasion features of the RA2 and RC2.2 erlotinib-resistant cell lines do not associate with an increased stem-like ability. On the contrary the majority of HCC827-derived erlotinib resistant NSCLC cell lines with intermediated E/M phenotypes that do not show an increase in cell migration or invasion are more prone to form spheroids in vitro when compared to their parental erlotinib-sensitive cell line.Overall the results show that the increased To identify genes and pathways involved in erlotinib-resistance, microarray gene expression analysis was performed. Thousands of genes differentially expressed in the erlotinib-resistant cell lines were identified Table . InteresIn order to analyze the EMT phenotypes associated to erlotinib-resistance, we collected five lists of genes whose expression has been previously associated to EMT Table  Byers e. The firCO_59, SVM_85, and PROF_58 according to the related selection method  and the number of selected genes  (http://www.proteinatlas.org/ENSG00000104892-KLC3/cancer). KLC3 while donwregulated in the majority of our erlotinib-resistant cell lines is upregulated in the RA2 cell line  cut-off on gene expression (CO); (ii.) support vector machine (SVM); (iii.) expression profile analysis (PROF). Specifically, we used these supervised methods to identify subsets of genes having similar expression levels within the epithelial cell lines (HCC827 and HCC4006) while exhibiting different expression in the mesenchymal (RC2.2) and the cell lines with E/M features  (see the methods section for details). The gene subsets resulting by application of the above three methods, hereafter also referred to as ctively) , were exctively) . More inctively) . Similarctively)  as shownctively)  and the ctively) . Remarkactively) . Five ouctively) . CTSL2 octively) . KLC3 ore Figure . Interese Figure . STEAP1,e Figure . Finallyin NSCLC , is stroAnalysis of enriched functional annotations for the three subsets of EMT-related genes highlighted pathways associated to the EMT process, including cell adhesion molecules, tight junction, Cadherin and TGF β signaling pathway. Interestingly the TGF β pathway was found enriched in all three gene lists Tables -7.Prompted by the functional enrichment analysis, we decided to further investigate the TGF β signaling pathway. Indeed, it has been previously reported that NSCLC cells undergo EMT phenotypic changes upon short exposure to TGF β  or long Finally, growth inhibition assays indicated that LY-364947, alone or in combination with erlotinib, does not impair the viability of HCC827-derived erlotinib-resistant cell lines .Overall these results suggest that the observed EMT stable phenotypes associated to erlotinib-resistance likely appear independent or only partially mediated by TGF β-1 activation.Increasing evidence points to a key role played by EMT in cancer progression and drug resistance. Here we concentrated our study on the intermediate E/M phenotypes and their association to target therapy resistance in NSCLC cell lines. As observed in both physiology and pathology, a cell population can be highly heterogeneous with respect to its EMT phenotype. Indeed, we consistently observed distinct subpopulations of cells representing the three phenotypes previously described as epithelial, mesenchymal and intermediate E/M in all HCC827-derived erlotinib-resistant cell lines. Notably, the HCC827-derived cell lines studied in this work include cell subpopulations co-expressing Cadherin-1 and Vimentin, common markers for epithelial and mesenchymal phenotypes, respectively. The percentage of E/M cells was remarkably stable within each cell line, suggesting the existence of regulatory networks that preserve the cell phenotypes. It is also interesting to note that these intermediate E/M phenotypes may be selected by erlotinib treatment because the number of E/M cells was found increased in the erlotinib-resistant cell lines derived from the HCC827 cell line. On the contrary, the only established HCC4006-derived erlotinib-resistant cell line (ie: RC2.2) was found to be in a complete and stable mesenchymal state, with essentially all cells negative for Cadherin-1 and positive for Vimentin. Interestingly, only the RA2 and RC2.2 cell lines showed an increase in cell migration and cell invasion rate, suggesting that a qualitative rather than a quantitative variation of cell migration and eventually invasion may deserve further investigation in the other erlotinib-resistant cell lines. Remarkably HER3 a receptor suggested to be a strong epithelial marker and regulator of EGFR TKI sensitivity , was fouin vitro cell migration and cell invasion of the RA2 and RC2.2 erlotinib-resistant cell lines do not associate with increased stem-like features. On the contrary the majority of HCC827-derived erlotinib-resistant NSCLC cell lines, with intermediated E/M phenotypes, have similar or decreased cell migration and cell invasion rate when compared with their parental cell line but show an increased sphere-forming ability. Importantly, the mRNA expression profile of some largely used stemness markers could not predict such stem-like behavior  genes  genes . ImportaFurthermore, wound healing cell migration assays demonstrated a collective cell migration (reviewed in ) propertNext, we used our NSCLC cell lines microarray data to evaluate the expression of publicly available EMT-related gene signatures and applied different supervised analytical methods for selecting novel gene sets able to partition our cancer cell lines according to their observed EMT phenotypes. Notably, despite the heterogeneous composition, all the selected gene sets showed significant enrichment of the TGF β signaling pathway, confirming the relevance of this pathway with respect to the EMT phenotypes –55.Finally we investigated the effect of the modulation of the TGF β1 signaling pathway on cell survival and EMT phenotypes. We observed that the inhibition of TGFR-1, alone or in combination with EGFR inhibition, did not affect NSCLC cell lines survival, but it was able to partially decrease the number of Vimentin positive cells in some HCC827-derived erlotinib-resistant cell lines. Further investigation of the TGF β1 and of other enriched signaling pathways (i.e. the Wnt pathways) will be required to understand their contribution to the erlotinib resistance and the intermediate E/M phenotypes in our cellular model system.Overall these results confirmed cellular heterogeneity during EMT in cancer cell lines upon drug treatment. Importantly, our findings highlighted the association an intermediate E/M phenotypes to resistance to EGFR TKI in NSCLC cell lines. Treatment of NSCLC patients sensitive or resistant to EGFR inhibitors should attempt to impact not only oncogene-driven cancer cell growth but also the EMT phenotype in early NSCLC development stages.2 humidified incubator. For the treatment with human transforming growth factor beta-1 (TGF β-1) (Sigma-Aldrich) (2.5 ng/ml) and LY-364947 (Sigma-Aldrich), a TGF β receptor type-1 (TGFR-1) inhibitor (1 μM), cells were cultured in Opti-MEM reduced serum media (Thermo Fisher Scientific) for 48 hours. For the sphere assays the cells were cultured in DMEM/F12  supplemented with commercial hormone mix B27 (Gibco), EGF , FGF (20 ng/mL), purchased from ThermoFisher Scientific, and heparin (10U/mL) (Sigma-Aldrich). The primary antibodies used Vimentin (D21H3), E-cadherin (24E10), N-cadherin (D4R1H), Claudin-1 (D5H1D), Snail (C15D3), Slug (C19G7), ZEB1 , GAPDH (D16H11) were from Cell Signaling Technology; Vimentin (V9 and V9Cy3), α-Tubulin (DM1A), β-actin (AC15) and horseradish peroxidase (HRP)-β-actin (AC15) were purchased from Sigma-Aldrich; GAPDH (clone 1D4) was obtained from Novus Biologicals; Phalloidin Alexa Fluor-488 and E-cadherin (HECD-1) were from Thermo Fisher Scientific; N-Cadherin (32/N-Cadherin), E-cadherin (CD324)-Alexa Fluor-647, Vimentin Alexa Fluor-488 and isotype control antibodies: Alexa Fluor-647 Mouse IgG1k (Clone MOPC-21), Alexa Fluor-488 Mouse IgG1k (Clone MOPC-21) were from BD Biosciences; STEAP (B-4) and KLC3 (E-7) were from Santa Cruz Biotechnology. Secondary antibodies goat anti-rabbit IgG (H+L)-HRP and goat anti-mouse IgG (H+L)-HRP were purchased from Bio-Rad; goat anti-mouse Alexa Fluor-488 and 546 and Hoechst 33258 were obtained from Thermo Fisher Scientific. Erlotinib hydrochloride salt, an EGFR tyrosine kinase inhibitor, was from LC Laboratories, USA; Corning matrigel basement membrane matrix growth factor reduced was from BD Biosciences. Stock solutions (10 mM) for all inhibitors were prepared in DMSO and stored at -20°C or -80°C as suggested by the product datasheet. MTT, 3--2,5-diphenyltetrazolium bromide and bovine serum albumin were from Sigma-Aldrich. MTT stock solution  was stored at 4°C for 1 month.The human cell lines: HCC827 (ATCC CRL-2868) and HCC4006 (ATCC CRL-2871), kindly provided by Oreste Segatto, and the erlotinib-resistant cell lines , previously described  were culPower SYBR Green PCR master mix was from Applied Biosystems. Trizol reagent was from Life Technologies, the GoScript reverse transcription system kit was from Promega.4 cells/well) plated in 96-well plates at day 0 were treated at day 1 with increasing concentrations of erlotinib or LY-364947 (from 64 pM to 10 μM) or their combination in the ratio (1:3) in complete tissue culture medium and cultured for 72 hours at 37°C in 5% CO2. Next, cells were gently washed with 1x PBS, incubated for 4 hours with MTT and processed for color detection with DMSO. The resulting purple solution was spectrophotometrically measured at 570 nm and 630 nm [MTT assay was performed as previously described . Brieflyd 630 nm , 63.t0) and then cells were allowed to migrate from the edge of the scratch at 37°C/5% CO2. The monolayer was imaged at different time points to record the size of the wound closure at 5-24 (time t) hours with an Olympus BX41 microscope using a 4-10x lens and 8-10 images were recorded with an Olympus SP-350 camera. The area of wound coverage was calculated using the ImageJ software [The HCC827 and HCC4006 parental cell lines and the erlotinib-resistant derived cell lines were plated onto 35 mm plastic petri dishes and grown until confluence. A cell-free area was introduced in a monolayer of cells using a sterile yellow tip (time software . The deg35mm with Culture Insert (Ibidi) and an equal number of cells (0.5-1 × 105) in 70 μl of culture medium was seeded into the two reservoirs of the same insert and incubated at 37°C/5% CO2. After appropriate cell attachment (24 h) the culture insert was gently removed using sterile tweezers, and a 500 μm wide cell-free gap (wound) was generated. The dish was then filled with 2 mL of FBS containing medium and incubated for 8-16 hours. Then cells were processed for immunofluorescence and confocal microscopy analysis.For immunofluorescence and confocal microscopy analysis the cells were also subjected to wound healing assay with the μ-Dish5), HCC4006 (2.5x104) or RC2.2 (2.5x104) were seeded onto the upper surface of a filter with a volume of 100 μl RPMI 1640 medium containing 0.5% BSA and placed into the lower wells containing 600 μl of complete medium with 10% FBS or 0.1% BSA (negative control) to induce cell migration. The migration transwell chambers were incubated for 16h at 37°C in a humidified atmosphere containing 5% CO2. Following incubation, cells were fixed in 3.7% PFA for 10 min at room temperature, stained with 0.1% crystal violet (20% methanol solution) for 10 min, then washed 3 times with PBS. Next, the cells on the upper side of the membrane were removed by cotton swabs, and the crystal violet staining on the bottom surface of the membrane was eluted in 10% acetic acid and read at an optical density of 590 nm with a Varioskan Lux instrument.Assays were performed using a transwell  containing a polycarbonate membrane filter (8μm pore size) for 24-well plates according to the manufacturer’s instructions. HCC827 and RA2 (1x105/w) in RPMI 1640 containing 0.5% BSA were seeded in the upper chamber, and 500 μl RPMI 1640 containing 10% FBS was added to the lower chamber. After 24 h incubation at 37°C in a 5% CO2 atmosphere, cells that remained in the upper chamber were removed by cotton swabs and penetrating cells were stained with 1% crystal violet in 20% methanol. Then, the membranes were rinsed several times with water and images were recorded using a stereomicroscope Leica MZ12 served by an Olympus SP-350 camera. Next images were processed with the Image software in order to calculate the area of the membrane filter occupied by invasive cells.Matrigel invasion assays using transwell chambers  containing a polycarbonate membrane filter (8μm pore size) for 24-well plates coated with matrigel was used to determine NSCLC cell invasion properties. In brief, 1 mg/ml matrigel (50 μl) was added to each insert and solidified in a 37°C incubator for 5 h to form a thin gel layer. Next, 24 h serum-starved cells , FGF (20 ng/mL), and heparin (10 U/mL), and seeded into 6-well ultra- low attachment plates . Culture medium was replaced every 3 days; the number and size of the spheroids were recorded at 6-7 days after the start of the culture period using a stereomicroscope Leica MZ12 served by an Olympus SP-350 camera. The number of spheroids exceeding a diameter of 50 μm and their surface area were evaluated by the ImageJ software [The assay was performed essentially as previously described . Brieflysoftware .50 values were determined using a sigmoidal dose response inhibition variable slope method. The synergy of erlotinib and LY-364947 was evaluated by the Chou-Talalay method [Data were analysed by GraphPad Prism software v6.0c. The optical density values of cells treated with drugs, obtained by MTT assay reading, were expressed as percentage of cell survival and normalized with the value of cells treated with vehicle (DMSO). The regression trendline were fitted using a non-linear regression method and ICy method  using Coy method  as previy method .HCC827 parental and erlotinib-resistant cells, grown on poly-L-lysine coated coverslips or on 35 mm tissue culture plates or on the Ibidi culture dish and HCC4006 and RC2.2 cells, on 35 mm tissue culture plates, were fixed with 3.7% PFA for 10 minutes at room temperature. Next cells were permeabilized  for 6 minutes at room temperature, treated with blocking solution  and stained with primary antibodies in blocking solution, for 1 hour, washed with 1x PBS for 10 times and stained with fluorochrome-labeled secondary antibodies for 45 min at room temperature and Hoechst 33258 for 5 min. HCC827 samples were analysed with laser scanning confocal microscope, TCS SP5  using a 63x (NA=1.4) or 40x (NA=1.25) oil-immersion lens with optical pinhole at 1AU. For fluorescence images Argon ion laser at 488 nm, HeNe ion laser at 543 nm and blue diode laser at 405 nm were used as excitation sources. Confocal Z-stacks were collected at 0.4 μm intervals to a total optical depth of 8-12 μm. Confocal images (at least 10 images for each sample in three independent experiments) were processed with Volocity  and Adobe Photoshop CS4 software for image rendering and representation of x/y view. Images for direct comparison were all collected under same parameters and representative images were chosen. HCC4006 and RC2.2 cells, stained in tissue culture plates, were analyzed with an Olympus AX70 fluorescence microscope using a 20x or 40x lens and images were recorded with an Olympus XM10 camera and processed using Olympus CellSens Standard 1.8.1 software. The following setting were used for fluorescence microscopy images acquisition: exposure time at 500 ms, gain at 4 dB, and thresholds of the fluorescence intensity on dynamic range in order to include the overall histogram area. Next, the immunoreactivity for Vimentin, was independently evaluated by two investigators and Vimentin positive cells were independently counted. When discrepancies in counting occurred, a consensus interpretation was reached after reexamination. All cell counts were carried out using the ImageJ software . All ima6 cells were stained in 5ml round bottom tubes  with Alexa Fluor-conjugated antibodies for 45 min at room temperature. Cells were then washed twice with PBS+1% FBS. Finally samples were analyzed using a BD FACS Calibur flow cytometer (Becton Dickinson). Unspecific background of individual channels was determined with isotype matched controls. Plots were generated using FlowJo v10.2 software.Adherent cells were detached from tissue cell culture plates by treatment with a cell dissociation buffer enzyme-free PBS (Thermo Fisher) at 37°C. Cells were collected in 15 ml tubes, washed with ice-cold RPMI 1640 medium supplemented with 10% FBS and centrifuged at 400g for 5 min at 4°C. Next cell pellets were first washed with ice-cold PBS supplemented with 1% FBS to block unspecific antibody binding and fixed with PFA (3.7%) for 15 min on ice. After a wash with ice-cold PBS supplemented with 1% FBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature and washed with PBS+1%FBS. Then 0.5-1x10Cells were lysed in ice-cold RIPA buffer  containing a combination of protease and phosphatase inhibitor cocktails or in SDS Laemmli sample buffer. Cell lysates (RIPA buffer) were quantified for proteins content with the Bio-Rad DC Protein Assay kit. Proteins (25-50 μg) were then separated by 8-12% SDS-PAGE and transferred to nitrocellulose membranes 0.45 μm . The membranes were blocked with 5% non-fat milk (EuroClone) or 2% BSA (Sigma-Aldrich) in 1x TBS pH 7.6-8.0 containing 0.1 or 0.2% Tween 20 (TBST) for 2 hour at room temperature (RT) and subsequently probed with primary antibodies in 5% non-fat milk or 2-5% BSA in TBST overnight at 4°C. Then membranes were washed 10 minutes for 3 times with TBST and probed with horseradish peroxidase-conjugated secondary antibodies in 5% non-fat milk in TBST for 1 hour at RT. Chemidoc XRS Bio-Rad was used for images acquisition with a chemi-luminescent camera, band signals were quantified using ImageLab 4.0 Bio-Rad software.Ribosomal protein L31 (rp-L31) was used as a reference gene to normalize quantitation of target genes for differences in the amount of total RNA in each sample.For mRNA analysis, total RNA was extracted using the TRIzol (Life Techologies) reagent and retro-transcribed with the GoScript Reverse Transcription System (Promega) using oligo (dT) and random primers. qPCR analysis was performed with specific primers Table  and analmicroRNA levels were analyzed from total RNA using the following TaqMan MicroRNA Assays: miR-200a ID:000502, miR-200b ID:002251, miR-200c ID:002300 and RNU24 ID:001001. Relative expression was calculated using the comparative Ct method. The small nucleolar RNA U24 (RNU24) was used as a reference gene.Both for mRNA and microRNA analysis, total RNA of HCC827 and HCC4006 parental cell lines were used as calibrator samples, relative to which differences in the RNA amount of resistant cell lines have been calculated. The data were analyzed using SDS (Ver. 1.4) software (Applied Biosystems).For microarray gene expression analysis, total RNA was isolated using a total RNA purification kit . RNA quantity was determined by absorbance at 260 nm using a NanoDrop ND-1000 UV-VIS spectrophotometer . RNA quality was assessed with Agilent Bioanalyzer RNA 6000 Nano kit; 200 ng of RNA were labeled with LowInput QuickAmp Labeling Kit One-Color (Agilent Technologies), purified and hybridized overnight onto an Agilent SurePrint G3 Human Gene Expression Array (8x60K v2 G4851B - 039494) before detection, according to the manufacturer’s instructions. Agilent DNA Microarray scanner was used for slide acquisition and spot analysis was performed with Feature Extraction software (Agilent Technologies).limma package [Microarray raw data, provided by the European Brain Research Institute  were background corrected, log2 transformed and quantile normalised by using  package . A totalhttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE80344.The whole Agilent microarray dataset used in this study is publicly available from the NCBI's Gene Expression Omnibus (GEO) database  under acPCA of cell lines was performed using gene expression values as variables that were scaled to have unit variance before the analysis took place. Unsupervised hierarchical clustering was performed using the complete linkage method and Euclidean distances applied to the normalized expression data matrix .The expression values of biological sample duplicates were averaged and mean expression differences were calculated as positive and negative fold-changes of gene abundance between erlotinib-resistant and -sensitive cell lines. We used -1 and 1 log fold-change cutoffs in order to select genes with relevant down- and up-regulation, respectively. Recurrence in gene expression modulation of erlotinib-resistant cell lines were evaluated by selecting genes with down- or up-regulation in at least five of the six derived vs parental cell lines comparisons. The recurrent differentially expressed genes were ranked according their log fold-change mean in the 5/6 erlotinib-resistant cell lines.Five gene lists previously associated to EMT . This method selects genes differentially expressed between epithelial and mesenchymal cell lines (fold-change ≥ 2) and with no modulation between the subset of epithelial cell lines . This method selected a list of 59 genes .Support Vector Machines classification (SVM). A supervised classification of cell lines was performed by using the SVM implemented by the Weka software . Based oExpression profile analysis (PROF). A hierarchical clustering was firstly performed to group genes of the COMBO_270 list according to their expression profiles. The resulting cluster dendrogram was cut at N=38 clusters and expression trends of each cluster separately evaluated. Next, genes populating clusters showing modulation between epithelial and mesenchymal cell lines and/or between E/M phenotype cell lines and both epithelial and mesenchymal cell lines were selected (hereafter the PROF_58 list).Finally, intersection of the above subsets of selected genes yielded a shared list of 25 genes related to EMT (hereafter INT_25 list).Differentially expressed genes between erlotinib-resistant and -sensitive cell lines and EMT-related gene sets were submitted to the DAVID tool suite  to analyStatistical analysis was performed using Microsoft Excel and GraphPad Prism software 6.0c. Two-tailed t-test was used for all indicated comparisons if not otherwise stated and p-values lower than 0.05 were considered significant. In particular, the effects of treatment with LY-364947, TGF β1 and their combination were evaluated by analyzing the fold-changes of Vimentin positive cells in vehicle (control) vs LY-364947, vehicle vs TGF β1, and TGF β1 vs LY-364947+ TGF β1 comparisons. In the wound healing assay, differences between leading edge and inner side were evaluated by comparing the counts of total Vimentin positive, Vimentin/Cadherin-1 double positive, and Vimentin positive/Cadherin-1 negative cells. In qPCR experiments resistant cell lines were compared with their corresponding parental cell lines. Statistical analysis of the invasion and sphere-forming assays data was performed using the non-parametric Mann-Whitney U test.