Fig 1: ETV5, VEGFA, and CCL2 show positive expression correlations with angiogenesis and are positively correlated with poor prognosis in CRC.a Representative images of IHC staining for ETV5, VEGFA, CCL2, and CD31 in the 75-patient cohort. b Expression of ETV5, VEGFA, CCL2, and CD31 was up-regulated in CRC tissues compared to levels in normal tissues (all p < 0.0001). c Expression of ETV5 was positively related to expression of VEGFA (p < 0.0001), CCL2 (p = 0.0342), and CD31 (p < 0.0001) in CRC tissues. d VEGFA (p < 0.0001) and CCL2 (p = 0.0002) were significantly associated with CD31 in CRC tissues. e, f Disease-free survival (DFS) and overall survival (OS) curves of the 75 patients in the cohort, as stratified by ETV5 and VEGFA expression patterns or by ETV5 and CCL2 expression patterns. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2: Deficiency of CDK6 or ETV5 combined with PLX inhibits cell proliferation of BRAFi-resistant cellsA. TFs with the potential to regulate CDK6 expression. The Y axis represents the regulatory potential score which were calculated by Cistrome DB Toolkit [39]. The X axis represents the different TFs. Each dot represents one ChIP-seq sample. B. Browser representation of the region near JUN from ATAC-seq of M238 and M238R1 under the different treatments. C. Interaction of TF JUN and genes whose essentiality increased after PLX treatment. Interacting partners of TF JUN were predicted using STRING database. JUN and ETV5 were individually labeled by the different colors to distinguish from the other proteins. Colored lines indicate different sources of evidence for each interaction. D. Rank plot of the TFs whose motif was enriched in the ETV5 ChIP-seq peaks. The Z scores were calculated according to their sequence logo similarity using MDSeqPos, available in Cistrome. For the negative “Z score”, the smaller ones mean significantly enriched. E. Validation of ETV5 knockout in M238R1 cells by Western blotting using indicated antibodies. For gene knockout experiments, 3 independent CRISPR guides targeting ETV5 were used, with one CRISPR guide targeting AAVS1 for control. GAPDH is the loading control for Western blotting.
Fig 3: ETV5 affects ESCC metastasis in vivo. (A) Four days after lentivirus infection, more than 90% of ECA109 cells were GFR positive in both Lv-shRNA and Lv-shETV5 groups, as shown by fluorescence microscopy. (B) Western blot indicated that ETV5 was significantly inhibited by Lv-shETV5 in ECA109 cells. (C-E) Effects of ETV5 knockdown on colonization in the mice lungs after tail vein injection of ECA109-derived cells, including shETV5 or negative control group. Lung images were performed to detect metastasis foci (C). Metastasis quantification was evaluated (D). Representative hematoxylin and eosin (H&E) staining of lung sections (E). *P<0.05; **P<0.01; ***P<0.001. - represents 100 µm.
Fig 4: p21 was bound to CDKs and changed the phosphorylation of p130.A Effects of ETV5 overexpression and knockdown on p130 phosphorylation in HCT116 and RKO cells, detected by western blot. B Endogenous p21 was knocked down after transfection with siRNA in both HCT116 and RKO cell lines. C and D Co-IP was performed to examine whether p21 interacted with CDK2, CDK4, and CDK6 in both HCT116 and RKO cell lines. E and F CCK-8 assay was performed to test cell viability with the CDK inhibitors Palbociclib and Dinaciclib. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 5: ETV5 suppressed the expression of p21 by a p53-independent pathway.A RT-qPCR and western blotting were performed to detect the changes of mRNA and protein in p21 and phosphorylation of p130 in HT29 cell lines expressing the mutant p53 gene. B Effects of ETV5 overexpression and knockdown on p53 expression and the phosphorylation of p53 in HCT116, RKO, and HT29 cell lines, detected by western blot. C Amplification of p21 promoter regions (a, b, and c) after ChIP using an ETV5 antibody on the 293 T cell line. D Luciferase reporter assay of three p21 promoter region mutations in the 293 T cell line. A specially designed plasmid was used to overexpress ETV5 in the 293T cell line. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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