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: TWIST1 is positively correlated with COL1A2 and transcriptionally activates COL1A2. (a) The promoter of COL1A2 was queried in the Ensembl website at Chr7:94394582~94394928. (b) Analysis of the top 30 genes with the highest association with COL1A2 in the Chen Gastric, Cho Gastric, Cui Gastric, Deng Gastric, and Wang Gastric datasets in the Oncomine website. (c) Spearman correlation analysis of TWIST1 with COL1A2 in TCGA-STAD database. (d) The mRNA expression of TWIST1 in parental and drug-resistant cells by RT-qPCR. (e) The protein expression of TWIST1 in parental and drug-resistant cells by western blot. (f, g) The binding of TWIST1 to the COL1A2 promoter in parental cells examined using ChIP-qPCR. (h) Schematic diagram of the pGL3-Luc luciferase reporter vector containing the COL1A2 promoter. (i) The luciferase reporter vector was cotransfected into 293T cells together with different doses of TWIST1 overexpression plasmids to detect changes in luciferase activity in the cells. Data indicate means ± SD of three biological replicates. Two-way ANOVA and Tukey's multiple comparison test; ∗∗p < 0.01 (vs. parental cells, IgG, or 0).

Fig 4: COL1A2 is highly expressed in GC patients and is associated with poor prognosis and apatinib resistance. (a) Genes with differential expression in the Chen Gastric, Cho Gastric, Cui Gastric, Deng Gastric, and Wang Gastric datasets analyzed by cancer vs. normal analysis in the Oncomine database. (b) Analysis of COL1A2 expression in normal tissues and GC tissues in TCGA-STAD database. (c) Kaplan-Meier analysis of the relationship between COL1A2 and survival in patients with GC in TCGA-STAD database. (d) Analysis of COL1A2 staining intensity in GC tissues versus normal gastric tissues measured using the HPA website. (e) IC50 values of parental and drug-resistant cells examined using the CCK-8 assay. (f) Detection of COL1A2 mRNA expression in parental and drug-resistant cells by RT-qPCR. (g) Detection of COL1A2 protein expression in parental and drug-resistant cells by western blot. Data indicate means ± SD of three biological replicates. Two-way ANOVA and Tukey's multiple comparison test; ∗∗p < 0.01 (vs. parental cells).

Fig 5: Knockdown of COL1A2 inhibits the sensitivity of drug-resistant cells to apatinib. GC-resistant cells were transfected with Scr or sh-COL1A2 #1 or #2. (a) The mRNA expression of COL1A2 in resistant cells by RT-qPCR. (b) The protein expression of COL1A2 in resistant cells by western blot. (c) The IC50 values of resistant cells by CCK-8 assay. (d) The number of colonies formed by resistant MKN-45 and AGS cells treated with 1 μM apatinib. (e) Flow cytometry analysis of the proportion of apoptotic cells in resistant MKN-45 and AGS cells. (f) TUNEL analysis of the number of apoptotic bodies in resistant 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. Scr).