C , D ) OCT - A imaging of LIF - treated mouse retina . Adult mice were intravitreally injected with 1 μl of LIF ( 50 ng , Sigma ) or vehicle solution ( PBS ) . Retinal OCT - A images were obtained 7 days after the injection and representative images are shown . Blood vessel density was determined as percentage of vessel - covered area / total area surface using ImageJ software and shown in D ) , n = 7 - 8 . Data information : Bars and error bars represent mean ± SEM . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical test .

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C, D) OCT-A imaging of LIF-treated mouse retina. Adult mice were intravitreally injected with 1 μl of LIF (50 ng, Sigma) or vehicle solution (PBS). Retinal OCT-A images were obtained 7 days after the injection and representative images are shown. Blood vessel density was determined as percentage of vessel-covered area/total area surface using ImageJ software and shown in D), n=7-8. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


E ) . STAT3 was phosphorylated in retinal ECs after LIF treatment . 50 ng of our in - house LIF with low - endotoxin levels was injected . Phospho - Stat3 was examined on cryosection of retina after 2 hour by IHC . Magnification in the dashed box is shown in the lower panel . n = 5 . Scale bar = 100 µm .

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E). STAT3 was phosphorylated in retinal ECs after LIF treatment. 50 ng of our in-house LIF with low-endotoxin levels was injected. Phospho-Stat3 was examined on cryosection of retina after 2 hour by IHC. Magnification in the dashed box is shown in the lower panel. n = 5. Scale bar = 100 µm.


F ) . Five - day old neonatal mice were intravitreally injected with LIF ( 50 ng ) or vehicle solution ( PBS ) . After 3 days , retinas were subjected to IF staining with Dyight - 488 - labeled lectin . Representative images for similar ocular loci and quantification of lectin - labeled area using ImageJ software were shown , n = 4 . Scale bar = 100 µm . Data information : Bars and error bars represent mean ± SEM . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical test .

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F). Five-day old neonatal mice were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days, retinas were subjected to IF staining with Dyight-488-labeled lectin. Representative images for similar ocular loci and quantification of lectin-labeled area using ImageJ software were shown, n=4. Scale bar = 100 µm. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


A ) . LIF enhanced laser - induced CNV . Intravitreal administration of LIF ( 10 ng and 100 ng , Sigma ) and OSM ( 10 ng ) after laser - induced CNV . PBS was used as vehicle control . OCT - A imaging and immunostaining of choroid flatmounts was carried out 10 days after laser induction . Quantification of CNV area is shown . Dashed line circles indicate the CNV in OCT - A images . n = 5 . Dot plot shows all data points from three independent experiments . Scale bar = 100 µm .

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A). LIF enhanced laser-induced CNV. Intravitreal administration of LIF (10 ng and 100 ng, Sigma) and OSM (10 ng) after laser-induced CNV. PBS was used as vehicle control. OCT-A imaging and immunostaining of choroid flatmounts was carried out 10 days after laser induction. Quantification of CNV area is shown. Dashed line circles indicate the CNV in OCT-A images. n = 5. Dot plot shows all data points from three independent experiments. Scale bar = 100 µm.


D ) . Avascular area in choroid in sodium iodate model was determined and quantified using Image J . E ) . Thickness of retina in H & E staining was quantified and bar graph was shown using ImageJ . Data information : Bars and error bars represent mean ± SEM . All experiments were carried out in three independent studies . Two - way ANOVA was used as statistical test .

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D). Avascular area in choroid in sodium iodate model was determined and quantified using Image J. E). Thickness of retina in H&E staining was quantified and bar graph was shown using ImageJ. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


( G ) Human choroidal EC ( HCEC ) and mouse retinal EC ( MREC ) were treated with LIF 10 ng , LIF 100 ng for 30 min . ( PBS was used as control ) . STAT3 phosphorylation was detected by western blot .

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(G) Human choroidal EC (HCEC) and mouse retinal EC (MREC) were treated with LIF 10 ng, LIF 100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation was detected by western blot.


( H ) H & E staining of LIFR and CD31 in mouse eye sections . The magnified images of the dashed line boxes , a and b , are shown on the right panel . Box a shows LIFR staining in retina and box b showed LIFR staining in RPE / choroid layer .

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(H) H&E staining of LIFR and CD31 in mouse eye sections. The magnified images of the dashed line boxes, a and b, are shown on the right panel. Box a shows LIFR staining in retina and box b showed LIFR staining in RPE/choroid layer.


( I ) Whole mount staining of LIFR and CD31 in mouse retina . The fluorescent staining for CD31 ( green ) and LIFR ( red ) is shown . White arrows indicate the co - staining of CD31 and LIFR on retinal ECs .

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(I) Whole mount staining of LIFR and CD31 in mouse retina. The fluorescent staining for CD31 (green) and LIFR (red) is shown. White arrows indicate the co-staining of CD31 and LIFR on retinal ECs.


Violin plots of the log scale normalized expression of LIF receptor subunits , IL6ST ( A ) , LIFR ( B ) , LIF ( C ) , IL6R ( D ) in all cell types in the human choroid Note the wide distribution of IL6ST ( gp130 ) , the common subunit to all receptors of the IL - 6 family ( A ) . In contrast , LIFR is strongly expressed in ECs and to a lesser extent in fibroblasts and pericytes ( B ) . LIF is expressed in fibroblasts , macrophages / monocytes and pericytes and minimally in ECs ( C ) . The specific IL - 6 receptor ( IL6R ) is highly expressed in macrophage / monocytes ( D ) , while LIFR has little expression in these cells ( B ) .

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Violin plots of the log scale normalized expression of LIF receptor subunits, IL6ST (A), LIFR (B), LIF (C), IL6R (D) in all cell types in the human choroid Note the wide distribution of IL6ST (gp130), the common subunit to all receptors of the IL-6 family (A). In contrast, LIFR is strongly expressed in ECs and to a lesser extent in fibroblasts and pericytes (B). LIF is expressed in fibroblasts, macrophages/monocytes and pericytes and minimally in ECs (C). The specific IL-6 receptor (IL6R) is highly expressed in macrophage/monocytes (D), while LIFR has little expression in these cells (B).


Violin plots of the log scale normalized expression of LIF receptor subunits KDR ( E ) in all cell types in the human choroid KDR shows , as expected , high expression selectively in EC ( E ) .

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Violin plots of the log scale normalized expression of LIF receptor subunits KDR (E) in all cell types in the human choroid KDR shows, as expected, high expression selectively in EC (E).


A , A targeted MAPPIT screen identifies several DUBs as putative RAS interactors . A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait . pSEL ( + 2L ) - HRAS G12V was expressed in HEK293T cells together with the indicated prey . BRAF served as a positive control . Each measurement was done in triplicate . The results are expressed as a mean of normalized luciferase activity ( leptin - treated cells vs leptin - untreated cells ) . The overall mean value + 2 s . d . served as a threshold .

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A, A targeted MAPPIT screen identifies several DUBs as putative RAS interactors. A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait. pSEL(+2L)-HRAS G12V was expressed in HEK293T cells together with the indicated prey. BRAF served as a positive control. Each measurement was done in triplicate. The results are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells). The overall mean value + 2 s.d. served as a threshold.


B , MAPPIT assay confirms the interaction between OTUB1 and RAS proteins . pSEL ( + 2L ) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey . Empty vector and two random baits , MAL and eDHFR , were used as negative controls . REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits . The results are expressed as a mean of normalized luciferase activity ± s . e . m ( leptin - treated cells vs leptin - untreated cells ) , n = 3 .

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B, MAPPIT assay confirms the interaction between OTUB1 and RAS proteins. pSEL(+2L) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey. Empty vector and two random baits, MAL and eDHFR, were used as negative controls. REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits. The results are expressed as a mean of normalized luciferase activity ± s.e.m (leptin-treated cells vs leptin-untreated cells), n=3.


C , NRAS Q61K mutant co - immunoprecipitates with OTUB1 . At 48 hours post - transfection with Flag - tagged NRAS Q61K and HA - tagged OTUB1 expression constructs , HA - tagged OTUB1 was immunoprecipitated with anti - HA - agarose followed by immunoblotting using anti - Flag or anti - HA antibodies .

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C, NRAS Q61K mutant co-immunoprecipitates with OTUB1. At 48 hours post-transfection with Flag-tagged NRAS Q61K and HA-tagged OTUB1 expression constructs, HA-tagged OTUB1 was immunoprecipitated with anti-HA-agarose followed by immunoblotting using anti-Flag or anti-HA antibodies.


D , OTUB1 interacts with wt NRAS and active NRAS mutant . Flag - tagged NRAS proteins were immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing HA - tagged OTUB1 or empty vector ( V ) .

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D, OTUB1 interacts with wt NRAS and active NRAS mutant. Flag-tagged NRAS proteins were immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V).


E , GTP binding does not affect the complex formation between NRAS and OTUB1 . Recombinant Flag - tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA - tagged OTUB1 in the excess of GTP - γ - S or GDP , followed by immunoblotting with the indicated antibodies .

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E, GTP binding does not affect the complex formation between NRAS and OTUB1. Recombinant Flag-tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA-tagged OTUB1 in the excess of GTP-γ-S or GDP, followed by immunoblotting with the indicated antibodies.


F , OTUB1 interacts with wt KRAS . Flag - tagged KRAS was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing HA - tagged OTUB1 or empty vector ( V ) . C , D , E , F , Immunoprecipitates , IP . Whole cell lysate , WCL .

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F, OTUB1 interacts with wt KRAS. Flag-tagged KRAS was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). C, D, E, F, Immunoprecipitates, IP. Whole cell lysate, WCL.


G , OTUB1 co - localizes with KRAS at the plasmamembrane . At 24 hours after co - transfection with GFP - tagged KRAS and HA - tagged OTUB1 , HeLa cells were immunostained with anti - HA antibody . The outlined areas are shown at higher magnification at the top of each image . Scale bar , 10 micron .

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G, OTUB1 co-localizes with KRAS at the plasmamembrane. At 24 hours after co-transfection with GFP-tagged KRAS and HA-tagged OTUB1, HeLa cells were immunostained with anti-HA antibody. The outlined areas are shown at higher magnification at the top of each image. Scale bar, 10 micron.


A , Suppression of OTUB1 expression increases NRAS mono - and di - ubiquitination . 6xHis - tagged ubiquitin and Flag - NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1 . Ubiquitinated NRAS was purified by Co2 + metal affinity chromatography and detected by anti - Flag antibody .

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A, Suppression of OTUB1 expression increases NRAS mono- and di-ubiquitination. 6xHis-tagged ubiquitin and Flag-NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1. Ubiquitinated NRAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.


B , C , D , Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination . 6xHis - tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA - OTUB1 , the catalytically dead mutant HA - OTUB1 C91S , or empty vector ( V ) . Ubiquitinated RAS was purified by Co2 + metal affinity chromatography and detected by anti - Flag antibody .

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B, C, D, Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination. 6xHis-tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA-OTUB1, the catalytically dead mutant HA-OTUB1 C91S, or empty vector (V). Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.


E , OTUB1 induces membrane RAS re - localization . Confocal imaging of HeLa cells expressing the indicated constructs . For each sample , z - stacks obtained by scanning the sample from the apical to the basal layer . Step - size , 2 micron .

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E, OTUB1 induces membrane RAS re-localization. Confocal imaging of HeLa cells expressing the indicated constructs. For each sample, z-stacks obtained by scanning the sample from the apical to the basal layer. Step-size, 2 micron.


F , RAS cellular distribution expressed as percentage of cells with specific RAS localization . For quantification of RAS localization , cells were randomly imaged using IN Cell Analyzer . RAS localization ( > 200 cells ) was scored as intracellular and diffused ( IN ) , mostly at the plasmamembrane ( PM ) , or both intracellular and plasmamembrane ( PM / IN ) . p - value = 0 . 0005 as determined by Chi2 - test , n = 3 . Representative images of HeLa cells expressing GFP - tagged KRAS are shown in Appendix Figure S1 . Pull - down , PD . Whole cell lysate , WCL . Not modified , n . m .

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F, RAS cellular distribution expressed as percentage of cells with specific RAS localization. For quantification of RAS localization, cells were randomly imaged using IN Cell Analyzer. RAS localization (>200 cells) was scored as intracellular and diffused (IN), mostly at the plasmamembrane (PM), or both intracellular and plasmamembrane (PM/IN). p-value=0.0005 as determined by Chi2-test, n=3. Representative images of HeLa cells expressing GFP-tagged KRAS are shown in Appendix Figure S1. Pull-down, PD. Whole cell lysate, WCL. Not modified, n.m.


A , B , OTUB1 overexpression promotes serum - induced activation of endogenous wt RAS ( A ) or wt NRAS ( B ) . GTP - bound RAS was pulled down from HEK293T cells expressing HA - tagged OTUB1 or empty vector ( V ) using recombinant RAF1 RBD conjugated to agarose beads . Input was controlled by immunoblotting using anti - panRAS or anti - Flag

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A, B, OTUB1 overexpression promotes serum-induced activation of endogenous wt RAS (A) or wt NRAS (B). GTP-bound RAS was pulled down from HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) using recombinant RAF1 RBD conjugated to agarose beads. Input was controlled by immunoblotting using anti-panRAS or anti-Flag antibodies


C , D OTUB1 enhances serum - induced ERK1 / 2 phosphorylation in endogenous wt RAS ( C ) or wt KRAS ( D ) . Levels of phosphorylated ERK1 / 2 and total ERK1 / 2 in HEK293T cells expressing HA - tagged OTUB1 or empty vector ( V ) were analyzed by Meso Scale assay . The results are expressed as a mean of pERK1 / 2 levels relative to total ERK1 / 2 ± s . e . m . n = 2 .

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C, D OTUB1 enhances serum-induced ERK1/2 phosphorylation in endogenous wt RAS (C) or wt KRAS (D). Levels of phosphorylated ERK1/2 and total ERK1/2 in HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m. n=2.


. E , F , Overexpression of wt OTUB1 ( E ) or the catalytically dead mutant OTUB1 C91S mutant ( F ) promotes serum - induced MAPK activation in cell expressing wt NRAS . Whole cell lysates were analyzed by immunoblotting using the indicated antibodies .

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. E, F, Overexpression of wt OTUB1 (E) or the catalytically dead mutant OTUB1 C91S mutant (F) promotes serum-induced MAPK activation in cell expressing wt NRAS. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies.


G , H , Overexpression of OTUB1 has no effect on serum - induced MAPK activation in cell expressing mutant NRAS Q61K or KRAS G12V . Whole cell lysates were analyzed by immunoblotting using antibodies as indicated . A , B , C , D , E , F , G , H Serum - starved HEK293T cells expressing the indicated constructs were stimulated with 10 % serum for the indicated time periods .

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G, H, Overexpression of OTUB1 has no effect on serum-induced MAPK activation in cell expressing mutant NRAS Q61K or KRAS G12V. Whole cell lysates were analyzed by immunoblotting using antibodies as indicated. A,B,C,D,E,F,G,H Serum-starved HEK293T cells expressing the indicated constructs were stimulated with 10% serum for the indicated time periods.


I , Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial cells ( HEK TEST ) , expressing empty vector ( V ) , myristoylated AKT1 ( myr - AKT ) , MEK1 D218 , D222 mutant ( MEKDD ) .

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I, Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial cells (HEK TEST), expressing empty vector (V), myristoylated AKT1 (myr-AKT), MEK1 D218,D222 mutant (MEKDD).


J , K , OTUB1 cooperates with active AKT1 to promote anchorage - independent growth . Representative images of soft - agar colonies formed by HEK TEST cells expressing the indicated constructs . The number of soft agar colonies formed by cells expressing OTUB1 compared to cells expressing an empty vector . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 2 .

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J, K, OTUB1 cooperates with active AKT1 to promote anchorage-independent growth. Representative images of soft-agar colonies formed by HEK TEST cells expressing the indicated constructs. The number of soft agar colonies formed by cells expressing OTUB1 compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=2.


B , C , OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13 . 1 copy number alteration . Pearson correlation of OTUB1 copy number ( log2 ratio ) with OTUB1 mRNA levels ( RNAseq normalized read counts , log2 transformed ) was analyzed .

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B,C, OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13.1 copy number alteration. Pearson correlation of OTUB1 copy number (log2 ratio) with OTUB1 mRNA levels (RNAseq normalized read counts, log2 transformed) was analyzed.


D , E , F , OTUB1 expression in TCGA lung adenocarcinoma ( LUAD ) and lung squamous cell carcinoma ( SCC ) patients . Patients were stratified according to their OTUB1 mRNA levels and / or their KRAS status as described in Materials and Methods . Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis . p - values were determined by two - sided t - Test . Total number of patients , n .

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D,E,F, OTUB1 expression in TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (SCC) patients. Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis. p-values were determined by two-sided t-Test. Total number of patients, n.


G , Gain of 11q13 . 1 locus is an early event in lung adenocarcinoma development . TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages ( T1 - T4 ) . Total numbers of patients , n . Statistical comparison of the sample distributions were compared using Chi - square test .

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G, Gain of 11q13.1 locus is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


H , OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development . TCGA lung adenocarcinoma patients were stratified by tumor stages ( T1 - T4 ) and OTUB1 expression levels as described in Materials and Methods . Total numbers of patients , n . Statistical comparison of the sample distributions were compared using Chi - square test .

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H, OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients were stratified by tumor stages (T1-T4) and OTUB1 expression levels as described in Materials and Methods. Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


I , KRAS mutation is a late event in lung adenocarcinoma progression . TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages ( T1 - T4 ) . Total numbers of patients , n . Statistical comparison of the sample distributions were compared using Chi - square test .

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I, KRAS mutation is a late event in lung adenocarcinoma progression. TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


, Suppression of OTUB1 expression in NSCLC cell lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1 .

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, Suppression of OTUB1 expression in NSCLC cell lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1.


B , OTUB1 suppression decreases serum - induced ERK1 / 2 phosphorylation . Serum - starved A549 cells expressing shRNAs against OTUB1 or GFP were stimulated with 10 % serum for the indicated time periods . Levels of phosphorylated ERK1 / 2 and total ERK1 / 2 were analyzed by Meso Scale assay . The results are expressed as a mean of pERK1 / 2 levels relative to total ERK1 / 2 ± s . e . m ; p - values were determined by two - sided t - Test , n = 2 .

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B, OTUB1 suppression decreases serum-induced ERK1/2 phosphorylation. Serum-starved A549 cells expressing shRNAs against OTUB1 or GFP were stimulated with 10% serum for the indicated time periods. Levels of phosphorylated ERK1/2 and total ERK1/2 were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m; p-values were determined by two-sided t-Test, n=2.


C , D , OTUB1 suppression impairs the anchorage - independent growth of Western blot cancer cell lines . Representative images of soft - agar colonies formed by Western blot cells expressing shRNAs against OTUB1 or GFP . The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 .

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C, D, OTUB1 suppression impairs the anchorage-independent growth of Western blot cancer cell lines. Representative images of soft-agar colonies formed by Western blot cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.


C , D , OTUB1 suppression impairs the anchorage - independent growth of lung cancer cell lines . Representative images of soft - agar colonies formed by lung cells expressing shRNAs against OTUB1 or GFP . The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 .

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C, D, OTUB1 suppression impairs the anchorage-independent growth of lung cancer cell lines. Representative images of soft-agar colonies formed by lung cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.


E , Xenograft tumor growth of A549 cells expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice . p - value was determined by 2 - way ANOVA .

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E, Xenograft tumor growth of A549 cells expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice. p-value was determined by 2-way ANOVA.


A , OTUB1 overexpression promotes serum - induced MAPK activation in wt KRAS H1993 lung adenocarcinoma cells .

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A, OTUB1 overexpression promotes serum-induced MAPK activation in wt KRAS H1993 lung adenocarcinoma cells.


B , OTUB1 overexpression does not affect serum - induced MAPK activation in mutant KRAS A549 lung adenocarcinoma cells . A , B , Serum - starved lung cells expressing Flag - OTUB1 or an empty vector ( V ) were stimulated with 10 % serum for the indicated time periods . Whole cell lysates were analyzed by immunoblotting using antibodies against pERK1 / 2 , ERK1 / 2 , Flag and vinculin .

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B, OTUB1 overexpression does not affect serum-induced MAPK activation in mutant KRAS A549 lung adenocarcinoma cells. A,B, Serum-starved lung cells expressing Flag-OTUB1 or an empty vector (V) were stimulated with 10% serum for the indicated time periods. Whole cell lysates were analyzed by immunoblotting using antibodies against pERK1/2, ERK1/2, Flag and vinculin.


C , Representative immunoblots of Flag - tagged OTUB1 overexpression in lung adenocarcinoma cell

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C, Representative immunoblots of Flag-tagged OTUB1 overexpression in lung adenocarcinoma cell lines


D , E , OTUB1 overexpression affects anchorage - independent growth of lung adenocarcinoma cell lines . Representative images of soft - agar colonies formed by the indicated cells expressing Flag - OTUB1 or empty vector ( V ) . The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 .

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D, E, OTUB1 overexpression affects anchorage-independent growth of lung adenocarcinoma cell lines. Representative images of soft-agar colonies formed by the indicated cells expressing Flag-OTUB1 or empty vector (V). The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.


F , G , Xenograft tumor growth of A549 , H1993 or H1437 cells expressing Flag - OTUB1 or an empty vector ( V ) subcutaneously injected into nude mice . p - values were determined by 2 - way ANOVA .

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F,G, Xenograft tumor growth of A549, H1993 or H1437 cells expressing Flag-OTUB1 or an empty vector (V) subcutaneously injected into nude mice. p-values were determined by 2-way ANOVA.


A , Representative examples of OTUB1 immunostaining in lung adenocarcinomas . Samples were scored as negative / low , medium or high staining as described in Materials and Methods . Scale bar , 100 micron .

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A, Representative examples of OTUB1 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Materials and Methods. Scale bar, 100 micron.


B , OTUB1 expression in lung non - small cell carcinomas with different KRAS mutation status . OTUB1 expression was assessed in TMA samples by IHC .

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B, OTUB1 expression in lung non-small cell carcinomas with different KRAS mutation status. OTUB1 expression was assessed in TMA samples by IHC.


C , OTUB1 up - regulation is an early event in lung adenocarcinoma development . Lung adenocarcinoma patients expressing different levels of OTUB1 were stratified by tumor stages ( T1 - T3 ) . Total number of patients , n .

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C, OTUB1 up-regulation is an early event in lung adenocarcinoma development. Lung adenocarcinoma patients expressing different levels of OTUB1 were stratified by tumor stages (T1-T3). Total number of patients, n.


D , Representative examples of phosphorylated - ERK1 / 2 immunostaining in lung adenocarcinomas . Samples were scored as negative / low , medium or high staining as described in Material and Methods . Scale bar , 100 micron .

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D, Representative examples of phosphorylated-ERK1/2 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Material and Methods. Scale bar, 100 micron.


E , OTUB1 levels correlates with increased levels of phosphorylated - ERK1 / 2 in wt KRAS lung adenocarcinomas . Association between OTUB1 and pERK1 / 2 expression levels was assessed in the same tumor samples by IHC .

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E, OTUB1 levels correlates with increased levels of phosphorylated-ERK1/2 in wt KRAS lung adenocarcinomas. Association between OTUB1 and pERK1/2 expression levels was assessed in the same tumor samples by IHC.


F , Ki67 expression in lung adenocarcinomas expressing different levels of OTUB1 . Ki67 expression was assessed by IHC . The percentage of Ki67 - positive cells was counted , and the samples were scored as negative / low , < 10 % ; medium , between 10 and 40 % ; high , > 40 % . See Figure EV4 for representative images of Ki67 staining .

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F, Ki67 expression in lung adenocarcinomas expressing different levels of OTUB1. Ki67 expression was assessed by IHC. The percentage of Ki67-positive cells was counted, and the samples were scored as negative/low, <10%; medium, between 10 and 40%; high, > 40%. See Figure EV4 for representative images of Ki67 staining.


G , Wt KRAS lung adenocarcinoma TCGA patients with medium OTUB1 expression have poorer overall survival .

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G, Wt KRAS lung adenocarcinoma TCGA patients with medium OTUB1 expression have poorer overall survival.


H , OTUB1 expression levels are not associated with survival rate of KRAS - mutant lung adenocarcinoma TCGA patients . G , H , Patients were stratified according to their OTUB1 mRNA levels and / or their KRAS status as described in Materials and Methods . Overall survival of lung adenocarcinoma patients expressing different levels of OTUB1 mRNA as measured by Kaplan - Meier curves . p - values were determined by Log Rank Test .

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H, OTUB1 expression levels are not associated with survival rate of KRAS-mutant lung adenocarcinoma TCGA patients. G,H, Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Overall survival of lung adenocarcinoma patients expressing different levels of OTUB1 mRNA as measured by Kaplan-Meier curves. p-values were determined by Log Rank Test.


( C ) Representative flow cytometry results showing CD71 , a cell surface marker , can be efficiently knocked - down in the majority of organoid cells after 5 days of Dox and TMP treatment . N = 3 different organoid lines and 3 different gRNAs for CD71 were used . Mean ± SEM is labelled .

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(C) Representative flow cytometry results showing CD71, a cell surface marker, can be efficiently knocked-down in the majority of organoid cells after 5 days of Dox and TMP treatment. N = 3 different organoid lines and 3 different gRNAs for CD71 were used. Mean ± SEM is labelled.


( A ) Human fetal lung tip organoids faithfully express in vivo tip progenitor markers , SOX2 and SOX9 . Upper panel : SOX2 / SOX9 dual expression in human fetal lungs . Lower panel : SOX2 / SOX9 in human fetal lung tip organoids . SOX9 green , SOX2 red , E - Cadherin white . Data information : Scale bars denote 50 μm (

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(A) Human fetal lung tip organoids faithfully express in vivo tip progenitor markers, SOX2 and SOX9. Upper panel: SOX2/SOX9 dual expression in human fetal lungs. Lower panel: SOX2/SOX9 in human fetal lung tip organoids. SOX9 green, SOX2 red, E-Cadherin white. Data information: Scale bars denote 50 μm (A


( B ) qRT - PCR showing that SOX9 gRNAs effectively knocked down SOX9 transcript levels using inducible CRISPRi after 5 days of Dox and TMP treatment . 3 different organoid lines and 2 different SOX9 gRNAs were used . Error bars : mean ± SEM . Two - sided Student ' s t - test with equal variance * * * p < 0 . 001 .

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(B) qRT-PCR showing that SOX9 gRNAs effectively knocked down SOX9 transcript levels using inducible CRISPRi after 5 days of Dox and TMP treatment. 3 different organoid lines and 2 different SOX9 gRNAs were used. Error bars: mean ± SEM. Two-sided Student's t-test with equal variance ***p < 0.001.


( D ) CD44 protein expression in SOX9 + tip progenitor cells . SOX9 green , SOX2 red , CD44 white . Data information : Scale bars = 50

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(D) CD44 protein expression in SOX9+ tip progenitor cells. SOX9 green, SOX2 red, CD44 white. Data information: Scale bars = 50 μm


( E ) LGR5 mRNA expression enrichment in SOX9 expressing tip progenitor cells . SOX9 red , LGR5 cyan . ( F ) Tip progenitor cells are of high WNT signalling activity . NOTUM yellow , SOX9 red . Data information : Scale bars = 50

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(E) LGR5 mRNA expression enrichment in SOX9 expressing tip progenitor cells. SOX9 red, LGR5 cyan. (F) Tip progenitor cells are of high WNT signalling activity. NOTUM yellow, SOX9 red. Data information: Scale bars = 50 μm


( H ) qRT - PCR showing rescue of SOX9 transcription after constitutively activated β - catenin overexpression in organoids cultured without WNT activators . N = 3 different organoid lines . Error bars : mean ± SEM .

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(H) qRT-PCR showing rescue of SOX9 transcription after constitutively activated β-catenin overexpression in organoids cultured without WNT activators. N = 3 different organoid lines. Error bars: mean ± SEM.


( I ) WB showing SOX9 protein was rescued after constitutively activated β - catenin overexpression in organoids cultured without WNT activators .

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(I) WB showing SOX9 protein was rescued after constitutively activated β-catenin overexpression in organoids cultured without WNT activators.


( A , B ) HCR images showing ETV5 ( A ) and ETV4 ( B ) expression in SOX9 expressing tip progenitors . ETV4 / 5 red , SOX9 cyan . Data information : Scale bars denote 50 μm ( A , B )

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(A,B) HCR images showing ETV5 (A) and ETV4 (B) expression in SOX9 expressing tip progenitors. ETV4/5 red, SOX9 cyan. Data information: Scale bars denote 50 μm (A, B)


( C ) qRT - PCR showing ETV4 and ETV5 dual knockdown in tip organoids . N = 3 different organoid lines and 2 different ETV4 and ETV5 gRNA combinations were used . Error bars : mean ± SEM . Two tailed paired t - test : * * p < 0 . 01 ; * * * p < 0 . 001 .

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(C) qRT-PCR showing ETV4 and ETV5 dual knockdown in tip organoids. N =3 different organoid lines and 2 different ETV4 and ETV5 gRNA combinations were used. Error bars: mean ± SEM. Two tailed paired t-test: **p < 0.01; ***p < 0.001.


( F ) qRT - PCR of ETV4 , ETV5 and SOX9 expression level in organoids in self - renewing medium and self - renewing medium without RTK ligands . N = 3 different organoid lines used . Error bars : mean ± SEM . Two - tailed paired T - test : * * p < 0 . 01 ; n . s . non - significant .

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(F) qRT-PCR of ETV4, ETV5 and SOX9 expression level in organoids in self-renewing medium and self-renewing medium without RTK ligands. N = 3 different organoid lines used. Error bars: mean ± SEM. Two-tailed paired T-test: **p < 0.01; n.s. non-significant.


Immunoblots and qRT - PCRs showing that expression levels of miR - 203 , miR - 200 , and E - cadherin are increased after ZEB1 knockdown in Panc1 , MDA - MB - 231 . Vice versa , the drug - resistant clones of BxPC3 , H358 , and DU - 145 show increased expression of ZEB1 and decreased expression of the miRNAs and E - cadherin . n = 3 , mean ± SEM , except for H358 ( data from microarray ) . Unpaired Student ' s t - test .

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Immunoblots and qRT-PCRs showing that expression levels of miR-203, miR-200, and E-cadherin are increased after ZEB1 knockdown in Panc1, MDA-MB-231. Vice versa, the drug-resistant clones of BxPC3, H358, and DU-145 show increased expression of ZEB1 and decreased expression of the miRNAs and E-cadherin. n = 3, mean ± SEM, except for H358 (data from microarray). Unpaired Student's t-test.


Lentiviral overexpression of miR - 200c and miR - 203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay . For the changes in EC50 values , see Table1 . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

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Lentiviral overexpression of miR-200c and miR-203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay. For the changes in EC50 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


Overexpression of miR - 203 decreases expression of the anti - apoptotic factor survivin and sensitizes to gemcitabine - triggered apoptosis as evaluated by cleaved caspase - 3 in Western blot and immunofluorescence . Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine , respectively , for 48 h . Scale bar 20 μm .

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Overexpression of miR-203 decreases expression of the anti-apoptotic factor survivin and sensitizes to gemcitabine-triggered apoptosis as evaluated by cleaved caspase-3 in Western blot and immunofluorescence. Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine, respectively, for 48 h. Scale bar 20 μm.


MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR - 203 or all miR - 200 members . For the changes in EC80 values , see Table1 . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

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MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR-203 or all miR-200 members. For the changes in EC80 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


Overexpression of miR - 203 shows reduced numbers of the CD24 / CD44 double - positive cancer stem cell population as determined by FACS analysis . The arrow indicates the reduction in the CD24 high subpopulation and reduction in CD133 by miR - 203 overexpression in hPaca1 cells .

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Overexpression of miR-203 shows reduced numbers of the CD24/CD44 double-positive cancer stem cell population as determined by FACS analysis. The arrow indicates the reduction in the CD24 high subpopulation and reduction in CD133 by miR-203 overexpression in hPaca1 cells.


Cancer stem cell sphere assay showing reduced sphere - forming capacity of Panc1 and hPaca1 in miR - 203 overexpression cells . Colonies with a diameter greater than 75 μM for Panc1 and greater 30 μM for hPaca1 cells were counted as spheres . n = 3 , mean ± SEM , Mann - Whitney U - test .

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Cancer stem cell sphere assay showing reduced sphere-forming capacity of Panc1 and hPaca1 in miR-203 overexpression cells. Colonies with a diameter greater than 75 μM for Panc1 and greater 30 μM for hPaca1 cells were counted as spheres. n = 3, mean ± SEM, Mann-Whitney U-test.


B , C Histone marks were analyzed using ChIP coupled to qRT - PCR for Panc1 control versus shZEB , MDA - MB - 231 control versus shZEB ( B ) , and BxPC3 control versus gemcitabine resistant ( gr ) ( C ) . In MDA - MB - 231 and Panc1 , the active histone marks H3K4me3 , H3ac , H4ac , and H3K9ac were enriched . Vice versa , in the drug - resistant clones of BxPC3 , the active marks were reduced in the CpG islands . The repressive histone mark H3K27me3 was not detectable in the miR - 200 loci , but in the loci of miR - 203 and E - cadherin in Panc1 and MDA - MB - 231 . DNA methylation status was determined by bisulfite sequencing . Depletion of ZEB1 in MDA - MB - 231 resulted in almost complete demethylation , whereas the selection of drug - resistant , ZEB1 - expressing clones in BxPC3 induced complete methylation . n = 2 ( Panc1 ) or 3 ( MDA - MB - 231 and BxPC3 ) , mean ± SEM ; unpaired Student ' s t - test .

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B, C Histone marks were analyzed using ChIP coupled to qRT-PCR for Panc1 control versus shZEB, MDA-MB-231 control versus shZEB (B), and BxPC3 control versus gemcitabine resistant (gr) (C). In MDA-MB-231 and Panc1, the active histone marks H3K4me3, H3ac, H4ac, and H3K9ac were enriched. Vice versa, in the drug-resistant clones of BxPC3, the active marks were reduced in the CpG islands. The repressive histone mark H3K27me3 was not detectable in the miR-200 loci, but in the loci of miR-203 and E-cadherin in Panc1 and MDA-MB-231. DNA methylation status was determined by bisulfite sequencing. Depletion of ZEB1 in MDA-MB-231 resulted in almost complete demethylation, whereas the selection of drug-resistant, ZEB1-expressing clones in BxPC3 induced complete methylation. n = 2 (Panc1) or 3 (MDA-MB-231 and BxPC3), mean ± SEM; unpaired Student's t-test.


Heat map showing the relative expression levels after drug treatment for 48 h in Panc1 . Values measured by qRT - PCR were depicted with the software GENE - E . Only mocetinostat upregulated the miRNAs and downregulated ZEB1 .

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Heat map showing the relative expression levels after drug treatment for 48 h in Panc1. Values measured by qRT-PCR were depicted with the software GENE-E. Only mocetinostat upregulated the miRNAs and downregulated ZEB1.


Relative expression of indicated genes in Panc1 measured by qRT - PCR after treatment with different HDAC inhibitors . Note the downregulation of ZEB1 and upregulation of miR - 203 , miR - 200 , and E - cadherin by mocetinostat . n = 3 , mean ± SEM ; unpaired Student ' s t - test . For significance , see Supplementary Table S1 .

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Relative expression of indicated genes in Panc1 measured by qRT-PCR after treatment with different HDAC inhibitors. Note the downregulation of ZEB1 and upregulation of miR-203, miR-200, and E-cadherin by mocetinostat. n = 3, mean ± SEM; unpaired Student's t-test. For significance, see Supplementary Table S1.


Immunoblot and immunofluorescence showing that mocetinostat treatment ( 1 μM , 48 h ) reduced ZEB1 expression and induced E - cadherin in Panc1 . Expression of histone deacetylases was not altered by mocetinostat , but histone acetylation was induced . Scale bar 10 μm .

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Immunoblot and immunofluorescence showing that mocetinostat treatment (1 μM, 48 h) reduced ZEB1 expression and induced E-cadherin in Panc1. Expression of histone deacetylases was not altered by mocetinostat, but histone acetylation was induced. Scale bar 10 μm.


Chromatin immunoprecipitation analysis validated mocetinostat - induced ( 1 μM , 48 h ) enrichment of the active histone marks H3ac , H4ac , H3K9ac , and H3K4me3 at ZEB1 target gene loci in Panc1 . n = 3 , mean ± SEM ; unpaired Student ' s t - test .

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Chromatin immunoprecipitation analysis validated mocetinostat-induced (1 μM, 48 h) enrichment of the active histone marks H3ac, H4ac, H3K9ac, and H3K4me3 at ZEB1 target gene loci in Panc1. n = 3, mean ± SEM; unpaired Student's t-test.


Mocetinostat treatment reduced expression of the anti - apoptotic miR - 203 target survivin and sphere - forming capacity in Panc1 when pre - treated with mocetinostat for 48 h . n = 3 , mean ± SEM ; Mann - Whitney U - test .

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Mocetinostat treatment reduced expression of the anti-apoptotic miR-203 target survivin and sphere-forming capacity in Panc1 when pre-treated with mocetinostat for 48 h. n = 3, mean ± SEM; Mann-Whitney U-test.


Immunoblot and immunofluorescence for cleaved caspase - 3 showing strong increase in apoptosis in gemcitabine - resistant Panc1 after combined treatment with mocetinostat ( 1 μM ) and gemcitabine ( 50 nM ) . Scale bar 20 μm .

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Immunoblot and immunofluorescence for cleaved caspase-3 showing strong increase in apoptosis in gemcitabine-resistant Panc1 after combined treatment with mocetinostat (1 μM) and gemcitabine (50 nM). Scale bar 20 μm.


MTT assay for Panc1 treated with the indicated concentrations of mocetinostat ( left ) or SAHA ( middle ) and gemcitabine ( 72 h ) . Combined treatment of mocetinostat and gemcitabine significantly reduced cell viability . In contrast , a combination with SAHA had no effect . For calculation of the CI and synergy between the drugs , see Table2 . Comparison of mocetinostat and SAHA alone ( right ) . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * * * P < 0 . 001 and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

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MTT assay for Panc1 treated with the indicated concentrations of mocetinostat (left) or SAHA (middle) and gemcitabine (72 h). Combined treatment of mocetinostat and gemcitabine significantly reduced cell viability. In contrast, a combination with SAHA had no effect. For calculation of the CI and synergy between the drugs, see Table2. Comparison of mocetinostat and SAHA alone (right). n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are ***P < 0.001 and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


Effects of mocetinostat on cleaved caspase expression and susceptibility to gemcitabine in the patient - derived pancreatic cancer cells . Note that hPaca1 behaves similar to Panc1 , but mocetinostat had no significant effect in hPaca2 . Scale bar 20 μm . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

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Effects of mocetinostat on cleaved caspase expression and susceptibility to gemcitabine in the patient-derived pancreatic cancer cells. Note that hPaca1 behaves similar to Panc1, but mocetinostat had no significant effect in hPaca2. Scale bar 20 μm. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU - 145 and the docetaxel - resistant subclone DU - 145 DR ( left ) . Mocetinostat treatment of DU - 145 DR downregulates ZEB1 , upregulates E - cadherin , miR - 200 , and miR - 203 expression . For relative miRNA expression , the expression levels in original DU - 145 were set to 1 ( middle panels , the immunoblot panel derives from the same experiment shown in Fig1A ) . Mocetinostat sensitizes DU - 145 DR to docetaxel ( right ) . For calculation of the CI and synergy between the drugs , see Table2 . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 ; for exact P - values , see Supplementary Table S4 ) .

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MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU-145 and the docetaxel-resistant subclone DU-145 DR (left). Mocetinostat treatment of DU-145 DR downregulates ZEB1, upregulates E-cadherin, miR-200, and miR-203 expression. For relative miRNA expression, the expression levels in original DU-145 were set to 1 (middle panels, the immunoblot panel derives from the same experiment shown in Fig1A). Mocetinostat sensitizes DU-145 DR to docetaxel (right). For calculation of the CI and synergy between the drugs, see Table2. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05; for exact P-values, see Supplementary Table S4).


Relative tumor volume ( RTV ) of Panc1 - derived tumors in NMRI nu / nu mice . Eleven days after implantation , mice were randomized according to tumor volume . Treatment with mocetinostat ( 60 or 90 mg / kg / day ) and gemcitabine ( 25 mg / kg / day ) was implemented ( day 0 ) as depicted in the scheme . Shown are the group medians of the RTVs over time ( left ) and the individual RTVs on day 32 ( right ) . n = 5 for each treatment group ; nonparametric Mann - Whitney U - test .

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Relative tumor volume (RTV) of Panc1-derived tumors in NMRI nu/nu mice. Eleven days after implantation, mice were randomized according to tumor volume. Treatment with mocetinostat (60 or 90 mg/kg/day) and gemcitabine (25 mg/kg/day) was implemented (day 0) as depicted in the scheme. Shown are the group medians of the RTVs over time (left) and the individual RTVs on day 32 (right). n = 5 for each treatment group; nonparametric Mann-Whitney U-test.


Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E - cadherin in tumor tissues of mice treated with mocetinostat . Scale bar 40 μm , inserts for higher magnifications 10 μm . Squares indicate magnified regions .

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Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E-cadherin in tumor tissues of mice treated with mocetinostat. Scale bar 40 μm, inserts for higher magnifications 10 μm. Squares indicate magnified regions.


Representative pictures of in situ hybridization for miR - 203 and control probe showing gain of miR - 203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat - treated xenograft tumors . Scale bar 40 μm , inserts for higher magnifications 5 μm . Squares indicate magnified regions .

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Representative pictures of in situ hybridization for miR-203 and control probe showing gain of miR-203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat-treated xenograft tumors. Scale bar 40 μm, inserts for higher magnifications 5 μm. Squares indicate magnified regions.


Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice . Panc1 cells were pre - treated with mocetinostat ( 1 μM ) and / or gemcitabine ( 50 nM ) for 48 h , followed by a 7 - day recovery period before being injected subcutaneously ( left ) . Equal numbers of viable cells were injected in 75 μl volume . At day 9 after injection , tumor growth was detectable in all groups ( lower right ) . To better visualize and compare tumor growth , the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted ( upper right ) . The individual absolute tumor volumes on day 37 ( lower left ) and the group medians of the absolute tumor volumes over time ( lower right ) are shown . For cells pre - treated with the combination of mocetinostat and gemcitabine , tumor growth was arrested . n = 4 for each treatment group ; nonparametric Mann - Whitney U - test .

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Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice. Panc1 cells were pre-treated with mocetinostat (1 μM) and/or gemcitabine (50 nM) for 48 h, followed by a 7-day recovery period before being injected subcutaneously (left). Equal numbers of viable cells were injected in 75 μl volume. At day 9 after injection, tumor growth was detectable in all groups (lower right). To better visualize and compare tumor growth, the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted (upper right). The individual absolute tumor volumes on day 37 (lower left) and the group medians of the absolute tumor volumes over time (lower right) are shown. For cells pre-treated with the combination of mocetinostat and gemcitabine, tumor growth was arrested. n = 4 for each treatment group; nonparametric Mann-Whitney U-test.


A , B Relative expression levels of miR - 203 and miR - 200c in pancreatic adenocarcinomas . ( A ) In normal versus tumor tissue of the same case ( n = 6 cases ) , ( B ) in the non - recurrence ( n = 10 cases ) versus the recurrence group ( n = 11 cases ) . The mean value of the lower group in each figure was set to 1 . Nonparametric Mann - Whitney U - test .

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A, B Relative expression levels of miR-203 and miR-200c in pancreatic adenocarcinomas. (A) In normal versus tumor tissue of the same case (n = 6 cases), (B) in the non-recurrence (n = 10 cases) versus the recurrence group (n = 11 cases). The mean value of the lower group in each figure was set to 1. Nonparametric Mann-Whitney U-test.


B The Na + and K + contents and Na + / K + ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d .

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B The Na+ and K+ contents and Na+/K+ ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d.


C Linear regression of root Na + / K + ratio under salt stress ( 150 mM NaCl for 1 d ) and fruit weight in 195 accessions from three groups . R2 , coefficient of determination . n is the accession number of the three groups .

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C Linear regression of root Na+/K+ ratio under salt stress (150 mM NaCl for 1 d) and fruit weight in 195 accessions from three groups. R2, coefficient of determination. n is the accession number of the three groups.


D The Na + / K + ratios in roots of TS - 21 , TS - 422 , TS - 577 , and TS - 670 after treatment with 150 mM NaCl for the indicated time ( n = 3 biological repeats ) .

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D The Na+/K+ ratios in roots of TS-21, TS-422, TS-577, and TS-670 after treatment with 150 mM NaCl for the indicated time (n = 3 biological repeats).


E , F Salt - resistance assay of two PIM accessions ( TS - 21 and TS - 422 ) and two BIG ( TS - 577 and TS - 670 ) . 20 - day - old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks ( E ) . The survival rates were obtained from at least 12 plants in three repeated experiments ( F ) .

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E, F Salt-resistance assay of two PIM accessions (TS-21 and TS-422) and two BIG (TS-577 and TS-670). 20-day-old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks (E). The survival rates were obtained from at least 12 plants in three repeated experiments (F).


A Manhattan plot displaying the GWAS results of Na + / K + ratio in root . The red - dashed line indicates the Bonferroni - adjusted significance threshold ( P = 1 . 0 × 10 - 7 ) . Red arrow indicates the significant SNP signal of Na + / K + ratio associated with SlHAK20 .

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A Manhattan plot displaying the GWAS results of Na+/K+ ratio in root. The red-dashed line indicates the Bonferroni-adjusted significance threshold (P = 1.0 × 10-7). Red arrow indicates the significant SNP signal of Na+/K+ ratio associated with SlHAK20.


B The nucleotide diversity ratios between PIM and CER , and between CER and BIG on chromosome 4 . The black - dashed horizontal lines indicate top 10 % threshold for entire chromosome 4 ( 1 . 82 πPIM / πCER for domestication and 4 . 27 πCER / πBIG for improvement ) . The red arrows indicate the position of SlHAK20 in the sweeps . π , nucleotide diversity .

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B The nucleotide diversity ratios between PIM and CER, and between CER and BIG on chromosome 4. The black-dashed horizontal lines indicate top 10% threshold for entire chromosome 4 (1.82 πPIM/πCER for domestication and 4.27 πCER/πBIG for improvement). The red arrows indicate the position of SlHAK20 in the sweeps. π, nucleotide diversity.


C SlHAK20 - based association mapping and pairwise LD analysis . Dots represent SNPs . Indel48 is highlighted in red . The indel - 2381 , indel - 1818 in the promoter region and five nonsynonymous variants are marked in blue . These eight variants are related to the pairwise LD diagram with dashed lines .

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C SlHAK20-based association mapping and pairwise LD analysis. Dots represent SNPs. Indel48 is highlighted in red. The indel-2381, indel-1818 in the promoter region and five nonsynonymous variants are marked in blue. These eight variants are related to the pairwise LD diagram with dashed lines.


D Haplotypes of SlHAK20 among tomato natural variations . The distribution of Na + / K + ratio in each haplotype group is exhibited by a box plot . The n indicates the number of accessions belonging to each haplotype . In the box plots , the middle line indicates the median , the box indicates the range of the percentiles of the total data using Turkey method , the whiskers indicate the interquartile range and the outer dots are outliers . Significant difference was determined by Student ' s t - test .

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D Haplotypes of SlHAK20 among tomato natural variations. The distribution of Na+/K+ ratio in each haplotype group is exhibited by a box plot. The n indicates the number of accessions belonging to each haplotype. In the box plots, the middle line indicates the median, the box indicates the range of the percentiles of the total data using Turkey method, the whiskers indicate the interquartile range and the outer dots are outliers. Significant difference was determined by Student's t-test.


E Allele distribution of the SlHAK20 locus at position in PIM , CER , and BIG groups . The n indicates the accession number .

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E Allele distribution of the SlHAK20 locus at position in PIM, CER, and BIG groups. The n indicates the accession number.


The expression pattern of SlHAK20 in tomato . The GUS activity was examined in seedling ( left ) and root ( right ) of 21 - days - old SlHAK20TS - 21pro : GUS transgenic tomato ( B ) . Bars , 2 cm .

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The expression pattern of SlHAK20 in tomato. The GUS activity was examined in seedling (left) and root (right) of 21-days-old SlHAK20TS-21pro:GUS transgenic tomato (B). Bars, 2 cm.


The expression pattern of SlHAK20 in tomato . Cross - sections of hypocotyl ( C ) of SlHAK20TS - 21pro : GUS transgenic plants . The scale bar is 400 μm in ( C )

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The expression pattern of SlHAK20 in tomato. Cross-sections of hypocotyl (C) of SlHAK20TS-21pro:GUS transgenic plants. The scale bar is 400 μm in (C)


The expression pattern of SlHAK20 in tomato . Cross - sections of root he scale bar is 200 μm in ( D ) .

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The expression pattern of SlHAK20 in tomato. Cross-sections of root he scale bar is 200 μm in (D).


E Subcellular localization of SlHAK20 - YFP in tomato . The SlHAK20Hap1 - YFP ( left ) and SlHAK20Hap2 - YFP ( right ) were localized in the plasma membrane . YFP , yellow florescence protein . FM4 - 64 , a lipophilic styryl compound as a red fluorescent marker of plasma membrane . Bar , 20 μm .

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E Subcellular localization of SlHAK20-YFP in tomato. The SlHAK20Hap1-YFP (left) and SlHAK20Hap2-YFP (right) were localized in the plasma membrane. YFP, yellow florescence protein. FM4-64, a lipophilic styryl compound as a red fluorescent marker of plasma membrane. Bar, 20 μm.


F SlHAK20 complements the K + uptake - defective yeast mutant assayed using a dose - response method . The K + uptake - defective yeast mutant R5421 transformed with SlHAK20Hap1 , SlHAK20Hap2 , and vector ( p416 - GPD ) were cultured in AP medium containing indicated concentrations of K + for 24 h . The data points represent three replicates .

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F SlHAK20 complements the K+ uptake-defective yeast mutant assayed using a dose-response method. The K+ uptake-defective yeast mutant R5421 transformed with SlHAK20Hap1, SlHAK20Hap2, and vector (p416-GPD) were cultured in AP medium containing indicated concentrations of K+ for 24 h. The data points represent three replicates.


G Na + uptake analysis of yeast cells expressing SlHAK20 . Shown are the Na + concentrations in the medium over the time of culture of the ANT3 yeast cells transformed with SlHAK20Hap1 , SlHAK20Hap2 , or empty vector . The initial NaCl concentration in the medium was 60 μM . The data are shown as the means ± SD ( n = 4 ) .

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G Na+ uptake analysis of yeast cells expressing SlHAK20. Shown are the Na+ concentrations in the medium over the time of culture of the ANT3 yeast cells transformed with SlHAK20Hap1, SlHAK20Hap2, or empty vector. The initial NaCl concentration in the medium was 60 μM. The data are shown as the means ± SD (n = 4).


H Na + uptake kinetic analysis of SlHAK20 in yeast cells . The curves show concentration - dependent Na + inﬂux in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in the presence of 0 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 100 , 150 or 200 μM NaCl in the medium . The data are expressed as mean ± SD ( n = 3 ) .

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H Na+ uptake kinetic analysis of SlHAK20 in yeast cells. The curves show concentration- dependent Na+ inﬂux in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in the presence of 0, 5, 10, 20, 30, 40, 50, 60, 100, 150 or 200 μM NaCl in the medium. The data are expressed as mean ± SD (n = 3).


Na + / K + ratio contents in roots of TS - 21 wild type and slhak20 mutants . Data are shown as means ± SD ( n = 4 ) .

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Na+/K+ ratio contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).


Na + contents in roots of TS - 21 wild type and slhak20 mutants . Data are shown as means ± SD ( n = 4 ) .

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Na+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).


K + contents in roots of TS - 21 wild type and slhak20 mutants . Data are shown as means ± SD ( n = 4 ) .

K+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).


Na + contents in the xylem sap of TS - 21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days . Data represent means ± SD ( n = 4 ) .

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Na+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).


K + contents in the xylem sap of TS - 21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days . Data represent means ± SD ( n = 4 ) .

K+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).


G Shoot height of TS - 21 , slhak20 - 1 , and slhak20 - 2 . Plant height of 24 - day - old TS - 21 , slhak20 - 1 , and slhak20 - 2 were measured after growing under normal conditions for 14 days , followed by salt treatment for 14 days , and recovery for 10 days . Values are means ± SD ( n = 10 plants of each genotype ) .

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G Shoot height of TS-21, slhak20-1, and slhak20-2. Plant height of 24-day-old TS-21, slhak20-1, and slhak20-2 were measured after growing under normal conditions for 14 days, followed by salt treatment for 14 days, and recovery for 10 days. Values are means ± SD (n = 10 plants of each genotype).


H Salt resistance of TS - 21 wild type and slhak20 mutants . TS - 21 , slhak20 - 1 , and slhak20 - 2 grown under normal growth conditions for 24 days , followed by a treatment with 200 mM NaCl for 14 days , and then recovered for 10 days . Control plants grown under normal growth conditions for 38 days . Scale bars , 5 cm .

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H Salt resistance of TS-21 wild type and slhak20 mutants. TS-21, slhak20-1, and slhak20-2 grown under normal growth conditions for 24 days, followed by a treatment with 200 mM NaCl for 14 days, and then recovered for 10 days. Control plants grown under normal growth conditions for 38 days. Scale bars, 5 cm.


Salt - tolerance assay of TS - 670 and slhak20 mutant plants . TS - 670 , slhak20 - 3 , and slhak20 - 4 grown under normal growth conditions for 23 days , and then treated with 150 mM NaCl for 5 days . Control plants grown under normal growth conditions for 28 days .

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Salt-tolerance assay of TS-670 and slhak20 mutant plants. TS-670, slhak20-3, and slhak20-4 grown under normal growth conditions for 23 days, and then treated with 150 mM NaCl for 5 days. Control plants grown under normal growth conditions for 28 days.


Plant height and fresh weight of TS - 670 , slhak20 - 3 , and slhak20 - 4 under normal and salt stress conditions . Shoot height f 23 - day - old TS - 670 , slhak20 - 3 , and slhak20 - 4 were quantified after growing under normal conditions for 5 days , or salt treatment with 150 mM NaCl for 5 days . Values are means ± SD ( n = 10 plants of each genotype ) .

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Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. Shoot height f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype).


Plant height and fresh weight of TS - 670 , slhak20 - 3 , and slhak20 - 4 under normal and salt

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Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress


Plant height and fresh weight of TS - 670 , slhak20 - 3 , and slhak20 - 4 under normal and salt stress conditions . resh weight f 23 - day - old TS - 670 , slhak20 - 3 , and slhak20 - 4 were quantified after growing under normal conditions for 5 days , or salt treatment with 150 mM NaCl for 5 days . Values are means ± SD ( n = 10 plants of each genotype ) .

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Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. resh weight f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype).


Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative plants ere analyzed among these genotypes . ontrol plants were grown under normal growth conditions for 28 days

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Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative plants ere analyzed among these genotypes. ontrol plants were grown under normal growth conditions for 28 days


Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative plants ere analyzed among these genotypes . ontrol plants were grown under normal growth conditions for 28

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Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative fresh weight of shoots were analyzed among these genotypes . Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured . Values are means ± SD ( n = 12 plants of each genotype for fresh weight

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Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative fresh weight of shoots were analyzed among these genotypes. Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured. Values are means ± SD (n = 12 plants of each genotype for fresh weight


Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative fresh weight of shoots were analyzed among these genotypes . Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured . Values are means ± SD ( n = 12 plants of each genotype for fresh

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Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative survival rate were analyzed among these genotypes .

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Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative survival rate were analyzed among these genotypes.


A The transcript levels of SlHAK20 in TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines grown under normal growth conditions .

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A The transcript levels of SlHAK20 in TS-670 and three independent SlHAK20Hap1-YFP transgenic lines grown under normal growth conditions.


B The protein levels of SlHAK20Hap1 - YFP in TS - 670 and transgenic plants . ACTIN was used as loading control .

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B The protein levels of SlHAK20Hap1-YFP in TS-670 and transgenic plants. ACTIN was used as loading control.


Phenotypes of transgenic lines in soil under salt stress . The survival rates were obtained from at least 8 plants in four repeated

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Phenotypes of transgenic lines in soil under salt stress. The survival rates were obtained from at least 8 plants in four repeated experiments


38 - day - old plants grown in soil as control . 20 - day - old TS - 670 and Hap1OE - 1 to - 3 were treated with 175 mM NaCl for 3 weeks and recovered for 2

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38-day-old plants grown in soil as control. 20-day-old TS-670 and Hap1OE-1 to -3 were treated with 175 mM NaCl for 3 weeks and recovered for 2 weeks


The Na + in shoots and roots of TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . The 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 and Hap1OE - 3 plants grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days .

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The Na+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


The K + in shoots and roots of TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . The 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 and Hap1OE - 3 plants grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days .

The K+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


The Na + / K + ratio in shoots and roots of TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . The 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 and Hap1OE - 3 plants grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days .

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The Na+/K+ ratio in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


( C ) Immunohistochemistry for C . burnetii in the lung on d7 after i . t . infection . Representative images and quantitation of lung tissue positive for C . burnetii ( n = 12 - 13 mice , pooled from three experiments , each normalized to the mean of WT or Acod1 + / - infected mice ) . Each dot represents one mouse .

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(C) Immunohistochemistry for C. burnetii in the lung on d7 after i.t. infection. Representative images and quantitation of lung tissue positive for C. burnetii (n=12-13 mice, pooled from three experiments, each normalized to the mean of WT or Acod1+/- infected mice). Each dot represents one mouse.


( E ) Immunohistochemistry for C . burnetii on day 7 after intraperitoneal infection . Each dot represents one mouse .

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(E) Immunohistochemistry for C. burnetii on day 7 after intraperitoneal infection. Each dot represents one mouse.


representative images of liver sections and quantification of immunohistochemistry for C . burnetii in liver sections ( G ) . n = 6 - 11 mice ( for NMII infections ) , pooled from two experiments . Each dot represents one mouse . ( Mann - Whitney test comparing treatment and control group of NMII infected Acod1 - / - mice in specified organs , *

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representative images of liver sections and quantification of immunohistochemistry for C. burnetii in liver sections (G). n=6-11 mice (for NMII infections), pooled from two experiments. Each dot represents one mouse. (Mann-Whitney test comparing treatment and control group of NMII infected Acod1-/- mice in specified organs, *


( B ) BAX / BAK - deficient HeLa cells reconstituted with BAX and where indicated HA - Parkin were treated with antimycin A and oligomycin ( AO ) for 2 h and then 1 µM of each of the BH3 mimetics ABT - 737 and S63485 for 6 h . Cell death was assessed at the indicated times by LDH assay . Data is mean + / - SD of four independent experiments .

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(B) BAX/BAK-deficient HeLa cells reconstituted with BAX and where indicated HA-Parkin were treated with antimycin A and oligomycin (AO) for 2 h and then 1 µM of each of the BH3 mimetics ABT-737 and S63485 for 6 h. Cell death was assessed at the indicated times by LDH assay. Data is mean +/- SD of four independent experiments.


( C ) BAX / BAK - deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin ( AO , 3 h ) , MG132 ( 20 µM , 3 . 5 h ) and BH3 mimetics ( 1 µM of each of ABT - 737 and S63485 , 1 h ) . Cytosol and membrane fractions were immunoblotted as indicated . Graph shows densitometric analysis of non - ubiquitinated membrane proteins ( top , relative to untreated control ) or cytochrome c release ( bottom ; cytosol / cytosol + membrane , relative to untreated controls ) from three independent experiments . Error bars represent mean + / - SD .

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(C) BAX/BAK-deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin (AO, 3 h), MG132 (20 µM, 3.5 h) and BH3 mimetics (1 µM of each of ABT-737 and S63485, 1 h). Cytosol and membrane fractions were immunoblotted as indicated. Graph shows densitometric analysis of non-ubiquitinated membrane proteins (top, relative to untreated control) or cytochrome c release (bottom; cytosol/cytosol+membrane, relative to untreated controls) from three independent experiments. Error bars represent mean +/- SD.


( D ) BAX / BAK - deficient HeLa cells ectopically expressing BAX were treated and

(D) BAX/BAK-deficient HeLa cells ectopically expressing BAX were treated and analysed


( E ) UBA pull - down of ubiquitinated proteins following antimycin A and oligomycin ( AO ) treatment of BAX / BAK - deficient HeLa cells ectopically expressing HA - Parkin and BAX for 3 h . Representative immunoblot from two independent experiments .

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(E) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment of BAX/BAK-deficient HeLa cells ectopically expressing HA-Parkin and BAX for 3 h. Representative immunoblot from two independent experiments.


( A ) Subcellular fractionation of cytosol and membrane fractions of HeLa cells expressing HA - Parkin following 2 h of antimycin A and oligomycin ( AO ) treatment .

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(A) Subcellular fractionation of cytosol and membrane fractions of HeLa cells expressing HA-Parkin following 2 h of antimycin A and oligomycin (AO) treatment.