Fig 2: Knockdown of TWIST1 or EP300 reduces resistance to apatinib in parental cells overexpressing COL1A2. GC parental cells transfected with oe-COL1A2 were further transfected with sh-TWIST1 or sh-EP300. (a) The mRNA expression of TWIST1, EP300, and COL1A2 in parental cells by RT-qPCR. (b) The protein expression of TWIST1, EP300, and COL1A2 in parental cells by western blot. (c) The IC50 values of parental cells by CCK-8 assay. (d) The number of colonies formed by parental MKN-45 and AGS cells treated with 1 µM apatinib. (e) Flow cytometry analysis of the proportion of apoptotic cells in parental MKN-45 and AGS cells. Data indicate means ± SD of three biological replicates. Two-way ANOVA and Tukey's multiple comparison test; **p < 0.01 (vs. oe - COL1A2 + Scr).

Fig 3: EP300 promotes COL1A2 expression through an H3K27ac-dependent manner. GC cells were treated with HAT inhibitor or DMSO. (a) ChIP-qPCR analysis of the H3K27ac modification level of COL1A2 promoter. (b) The mRNA expression of COL1A2 in parental cells by RT-qPCR. (c) The protein expression of COL1A2 in parental cells by western blot. (d) UCSC browser analysis of the binding of EP300 to the promoter of COL1A2. GC cells were transfected with oe-NC or oe-EP300. (e) ChIP-qPCR analysis of the H3K27ac modification level of COL1A2 promoter. (f) The mRNA expression of COL1A2 in parental cells by RT-qPCR. (g) The protein expression of COL1A2 in parental cells by western blot. (h) Colocalization of TWIST1 with EP300 in AGS and MKN-45 cells by double-labeled immunofluorescence staining. Data indicate means ± SD of three biological replicates. Two-way ANOVA and Tukey's multiple comparison test; ∗∗p < 0.01 (vs. DMSO or oe-NC).

Fig 4: Involvement of autophagy or EMT pathway in the miR-338-5p-related CRC migration in vitro. (a) LC3 puncta were labelled with anti-LC3 antibody and fluorescent isothiocyanate-conjugated goat anti-rabbit IgG (green fluorescence). SW480 cell nuclei were stained with Hoechst 33258 (blue fluorescence). Images were photographed using florescent microscopy (the scale bar represents 10 µm). Data are presented as median (IQR) (n = 5) (P value was analysed using Mann-Whitney tests). (b) SW480 cells were treated with 10 µM of amiodarone for 48 h and then transfected with 100 nM of miR-338-5p or N.C. PIK3C3 and LC3 expression was measured using Western blot. (c) For transwell assay, SW480 cells were first treated with 10 µM of amiodaron and then transfected with/without 100 nM of miR-338-5p. Data are presented as median (IQR) (n = 5) (P value was analysed using Mann-Whitney tests).The scale bar represents 50 µm. (d) After infection with lentivirus carrying miR-338-5p or sh-GFP, HCT116 stable cells were treated with amiodarone (10 µM) for 48 h. Expression of PIK3C3 and LC3 was assayed by Western blot. (e) Transwell invasion assay was performed on HCT116 cells after infection with miR-338-5p or shGFP lentivirus and counted for invaded cells for 48 h. The scale bar represents 50 µm. Data are expressed as means ± SEM (n = 8) (P value was analysed using t tests). (f) SW480 cells were infected with sh-ATG-5 lentivirus and then transfected with 100 nM of miR-338-5p or N.C. Expression of ATG5-ATG12 and LC3 was measured using Western blot. Representative results of transwell assays. The scale bar represents 50 µm. The number of migrated cells are shown as means ± SEM (n = 8) (P values were analysed using t tests). (g) After infection with lentivirus carrying miR-338-5p, western blot was used to analyse the expression of PIK3C3, LC3, p62, Snail, Twist, N-cadherin, E-cadherin, vimentin, fibronectin and ß-actin, respectively, in HCT116 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)