EMBO/SourceData huggingface dataset:
Figure 4A. MCF10A cells were either mock or treated with 10 μM XL413 and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from three biological repeats and statistical significance was assessed by the student t test (** p˂0.01B. U2OS cells were either mock or treated with 10 μM XL413, 50 μM Mirin or both, and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413 and/or Mirin. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from four biological repeats and statistical significance was assessed by the student t test (*p<0.05, ** p˂0.01C. U2OS cells were either mock or pretreated for the indicated times with 10 μM XL413, 50 μM Mirin or both, and labelled with CldU (red). Then 50 nM CPT was added and cells labelled with of IdU (green) for further 30 minutes. A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.D. U2OS cells were transfected with control (siLuc) or MRE11 targeting siRNA (siMRE11). After 48 hours cells were either mock or pretreated with 10 μM XL413 and labelled with CldU (red) for 30 minutes. Where indicated 50 nM CPT was added and then cells were labelled with IdU (green). A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentileswith data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.
Figure 5B. Representative western blot of indicated proteins obtained from whole cell extracts. pS40/41 on MCM2 indicates effective CDC7 inhibition by XL413.C. Ratios of IdU/CldU tracts length is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. Mean values of IdU/CldU ratios are indicated above the plot. At least 100 replication forks were analyzed for each condition.D. The mean values of IdU/CldU tract length ratios from three independent experiments are plotted, with the standard deviation (blue lines) and mean (red line). Statistical analysis: student t-test; ns, not significant; **P<0.01, ***P < 0.001E. Electron micrographs of representative replication forks from U2OS cells A regressed arm is labelled: P=parental strand; D=daughter strand; and R=reversed fork; the four-way junction at the reversed fork is magnified in the inset. The red arrows indicates single stranded DNA at the fork. Scale bar is 200 nm ( = 460 bp), 100 nm ( = 230 bp) in the inset.F. Frequency of reversed replication forks isolated from mock-depleted (siLuc) and BRCA2-depleted (siBRCA2) U2OS cells upon 5 hour treatment with 4 mM HU in the presence and absence of 10 μM XL413. The number of replication intermediates analyzed is indicated in parentheses. Similar results were obtained in two independent experiments (Appendix TableG. Amount of ssDNA length at the junction (Red arrows in Figure 4D) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 hours in the presence or absence of XL413. N indicates the number of forks observed, and only molecules with detectable ssDNA stretches are included in the analysis. The lines show the mean length of ssDNA regions at the fork and the value is displayed above the plot
Figure 6A. Representative metaphase spread from BRCA2 depleted U2OS cell treated with HU. Scale bar = 5 μM. Arrows indicate the chromosomes enlarged in the insets, two of which show chromatid breaks (*B-C. The graphs show the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (** p˂0.01, *** p<0.001D-E. U2OS and AS-CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2) targeting siRNAs. 48 hours post transfection cells were either mock-treated or treated with 10 μM XL413 or 10 μM 3MB-PP1 respectively for 24 hours with Nocodazole added for the last 16 hours of the experiment. The graph shows the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (*p<0.05, *** p<0.001
Figure 1B) VEGF neutralizing antibody (B20) failed to suppress BCE cell growth induced by LN-229 CM, n=3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ns, not statistically significant.G) LIF and VEGF synergistically stimulated BCE cell growth. Cell proliferation was analyzed after 6 days using alamar blue as described in Materials and Methods, n=3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ns, not statistically significant.
Figure 2A) The JAK inhibitor baricitinib (Ba) blocked LIF-induced STAT3 phosphorylation. BCE cells were pre-incubated with DMSO, baricitinib (2 μM), cobimetinib (Co) (150 nM) or BEZ235 (BE) (5 nM) for 1 hour and were then treated with vehicle or LIF (10 ng/ml, Sigma) for 15 minutes. Ctrl, no pre-incubation with inhibitors.C) and D) STAT3 knockdown in BCE cells. BCE cells were transfected with siNegative and siRNAs targeting STAT3. qRT-PCR were performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and are presented in C. In D, cells transfected with siRNAs were treated with LIF (10 ng/ml) or vehicle for 15 minutes. Whole-cell lysates were subjected to Western blotting with the indicated antibodies. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.E) LIF-induced BCE cell growth was abolished by STAT3 knockdown. BCE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) or vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading at 590 nm for each vehicle group was set as 1, n=3. siNegative, negative control siRNA not targeting any known genes. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.
Figure 3B). JAK inhibitor baricitinib blocked activation of STAT3 by LIF. BAE cells pre-incubated with DMSO and inhibitors for 1 hour were treated with vehicle and LIF (10 ng/ml) for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies. Ctrl, no pre-incubation with inhibitors; Ba, baricitinib (2 μM); Co, cobimetinib (150 nM); BE, BEZ235 (5 nM).E). Knockdown of STAT3 in BAE cells. BAE cells were transfected with siRNAs targeting STAT3. In E, cells transfected with siRNAs were treated with LIF (10 ng/ml, Sigma) and vehicle for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies.F). LIF-induced BAE cell growth inhibition was abolished by knockdown of STAT3. BAE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) and vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading for each vehicle group was set as 1, n=3. Data information: Bars and error bars represent mean±SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test.
Figure 4C, 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.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.
Figure 5A). 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.
Figure 6(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.(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.
Figure 7Violin 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).
Figure 1A, 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.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).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.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.
Figure 2A, 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.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.
Figure 3A, 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 antibodiesC, 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. 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).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.
Figure 4B,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.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.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.
Figure 5, 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.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.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.
Figure 6A, 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.C, Representative immunoblots of Flag-tagged OTUB1 overexpression in lung adenocarcinoma cell linesD, 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.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.