Fig 2: Effects of ALDH1A3 via functions of STAT1 and STAT3. (A) The gene signatures of KKU-100 cells cultured for 6 h with or without low doses (1 µM) or high doses (10 µM) of ruxolitinib were analyzed using Ingenuity Pathway Analysis (IPA). (B) The scheme of different gene signatures of KKU-100 cells was validated by IPA. (C) The nine putative transcription factors that correlated with ALDH1A3 and ruxolitinib were predicted using the PROMO 3.0 software. The lists of transcription factors are listed in the right panels. (D) Total RNA (5 µg) was isolated from the KKU-100 and KKU-M213 cells and subjected to RT–qPCR analyses to determine the mRNA levels of predicted transcription factors, including STAT1, ATF1, FOXP3, HIF1A, SRF, IRF-1, JUN, RELA, and SP1. Data are the mean ± standard deviation of three independent experiments. * p < 0.05 and ** p < 0.01 compared with the respective control group by the Student’s t test. (E) Schematic of the upstream promoter region of the ALDH1A3 gene with six predicted STAT1-binding sites (1–6). The region amplified in the re-ChIP assay is indicated by arrows (primers 1–3). The DNA samples represent yields of the secondary immunoprecipitation (second IP), control, and drug-treated groups (anti-pSTAT1 and p-STAT3). Data are the mean ± standard deviation of three independent experiments. * p < 0.05 and ** p < 0.01 compared with the respective control group by the Student’s t test.

Fig 3: Expression of STAT1, STAT3, and ALDH1A3 in patients with cholangiocarcinoma. (A) Relative mRNA expressions of ALDH1A3, STAT1, and STAT3 from TCGA databank in cholangiocarcinoma (n = 36) and control samples (n = 9). * p < 0.05 by Mann–Whitney U test. (B) Spearman’s rank correlation between the mRNA levels of ALDH1A3, STAT, and STAT3. The correlation coefficients r and P values were obtained using GEPIA and are shown in each panel. (C) Upper: Representative imaged of immunohistochemical nuclear staining of ALDH1A3, pSTAT1 (Y701-phosphorylated STAT1), and pSTAT3 (Y705-phosphorylated STAT3) in human cholangiocarcinoma specimens. Scale bar = 50 µm. Lower: Spearman’s rank correlation coefficient r and P values are shown in the panel (n = 24).

Fig 4: Schematic representation of the effects of ruxolitinib on malignancy of cholangiocarcinoma. A JAK2 inhibitor, ruxolitinib, can downregulate ALDH1A3 expression and inhibit cell proliferation and migration through STAT1/STAT3 in cholangiocarcinoma cell lines.

Fig 5: ALDH1A3 expression influences the migration ability in cholangiocarcinoma cell lines. (A) Total lysates (20 µg) from the normal cholangiocyte cell line (MMNK-1) and three cholangiocarcinoma cell lines (KKU-M055, KKU-100, and KKU-M213) were subjected to Western blot analysis using an anti-ALDH1A3 antibody as the probe, and ß-actin signals were used as loading controls. (B) The cell migration ability of all cell lines was analyzed using Transwell migration assays. (C) Correlation of protein expression levels of ALDH1A3 with migration abilities. (D) Total lysates (20 µg) prepared from the vector- and ALDH1A3-overexpressing KKU-M055 cells and vector- and ALDH1A3-knockdown KKU-M213 cells, respectively, were subjected to Western blot analyses using antibodies against ALDH1A3. ß-actin signals were used as loading controls. (E) The cell migration abilities of all KKU-M055 and KKU-M213 cells were analyzed using Transwell migration assays. The quantitative results shown in the bottom panels are the mean ± standard deviation of three independent experiments. * p < 0.05 compared with the respective vector cells by the Student’s t test.