Fig 2: miR-195-5p and JUP expression in CRC patients. Data from GEO dataset (GSE126092, GSE126093), which includes the miRNA and mRNA expression profile of 10 CRC patients with tumor tissue and the relative adjacent normal samples, demonstrated a considerable downregulation of miR-195 in tumor (A). Inversely, JUP was significantly overexpressed in CRC tissue compared with adjacent normal colon (B). ** p < 0.001, *** p < 0.0001. Pearson correlation analysis in all samples revealed a negative correlation between miR-195-5p and JUP expression. r = −0.689, R2 = 0.475, p < 0.0001. Each point represents a sample (C).

Fig 3: miR-195-5p (A) and JUP expression (B) in Caco2 and Lovo cell lines after transient transfection. miR-195-5p was markedly overexpressed at 30 nM and 50 nM concentrations compared with mock control. The gain of function of miR-195-5p regulates JUP mRNA expression. The increased intracellular miR-195-5p at 30 nM and 50 nM concentrations led to a significant decrease of JUP in all cell lines. Expression data were normalized to the housekeeping gene GAPDH and are representative of four independent experiments (n = 4; mean ± SEM) * p < 0.05, ** p < 0.001, *** p < 0.0001.

Fig 4: JUP interaction with HIF2α. A and B, HEK293T cells were transfected with Flag‐HIF2α and HA‐JUP. The cell lysates were immunoprecipitated with anti‐Flag (A) or anti‐HA antibody (B), and co‐immunoprecipitated proteins were detected by Western blotting analysis with anti‐HA or anti‐Flag antibody, respectively. C, Purified GST or GST‐JUP was incubated with Flag‐HIF2α‐expressing 786‐O lysates and precipitated with glutathione‐agarose beads. Pulled‐down Flag‐HIF2α proteins by GST‐JUP were detected by immunoblotting with the HIF2α antibody. Purified GST or GST‐JUP was visualized by Western blotting analysis using the GST antibody. D and E, 786‐O cells were harvested and subjected to endogenous Co‐IP analysis with JUP antibody. Rabbit IgG was used as a control. F, Schematic diagram of the various domains and relevant mutant fusion expression constructs of HIF2α (upper). HA‐JUP and Flag‐HIF2α full‐length and deletion mutants were ectopically expressed in HEK293T cells, followed by Flag‐immunoprecipitation and immunoblotting with HA antibody. Inputs are shown in the bottom panels. G, GST pull‐down assay detecting the regions of JUP that bind to HIF2α. Schematic diagram of the various domains and relevant mutant fusion expression constructs of JUP (upper). Flag‐HIF2α and GST‐JUP deletion mutants were ectopically expressed in HEK293T cells, followed by Western blotting analysis of Flag‐HIF2α pulled down by GST or GST‐JUP fusion proteins. Inputs are shown in the bottom panels. Abbreviations: HIF, hypoxia‐inducible factor; JUP, junction plakoglobin; bHLH, basic helix‐loop‐helix; PAS, Per‐Arnt‐Sim; PAC, PAS‐associated C‐terminal; ODD, oxygen‐dependent degradation; N‐TAD, N‐terminal transactivation domain; C‐TAD, C‐terminal transactivation domain; IH, inhibitory domain; ARM, armadillo; NTD, N‐terminal domain; CTD, C‐terminal domain; GST, glutathione‐S‐transferase; Co‐IP, co‐immunoprecipitation

Fig 5: A. Enriched pathways represented in differentially abundant proteins. Enriched pathways were identified using Pathway Studio from the set of differential proteins (p<0.05). Pathways with significance p < 0.01 are shown and include pathways involved in focal adhesion, hemidesmosomes as well as glycogen metabolism. B. Western blot analysis and mass spectrometry analysis of cell-cell adhesion proteins. 15 μg of total protein lysates from HCT116-GSK3β-WT and HCT116-GSK3β-KO cells were analysed by western blot analysis for protein expression of GSK3β, PKP2, PKP3, and JUP. Protein abundance of Actin was used as a loading control. n = 3. C. Western blot analysis and mass spectrometry analysis of cell-cell adhesion proteins. 15 μg of total protein lysates from HCT116-GSK3β-WT and HCT116-GSK3β-KO cells were analysed by western blot analysis for protein expression of DSG2 and GSK3β. Protein abundance of GAPDH was used as a loading control. n = 2. D. Western blot analysis and mass spectrometry analysis of cell-cell adhesion proteins. 15 μg of total protein lysates from HCT116-GSK3β-WT and HCT116-GSK3β-KO cells were analysed by western blot analysis for protein expression of ITGA6 and GSK3β. Protein abundance of GAPDH was used as a loading control. n = 2.