Fig. 7: Combined overexpression of ZEB1 and ALKBH5 can enhance the therapeutic effects of GEM and RA.

A, B WB analyses using anti-FLAG, anti-HA, anti-CRABP2 and anti-FABP5 antibodies in ZEB1-FLAG, Del-CP-ZEB1, ALKBH5-HA, Del-P#2-ALKBH5 and ALKBH5 + ZEB1-overexpressing THJ-16T cells with or without 5 μM GEM and 10 μM RA (GEM + RA) treatment for 48 h. C, D CCK8 assays were performed on THJ-16T cells overexpressing ZEB1, Del-CP-ZEB1, ALKBH5, Del-P#2-ALKBH5 and ALKBH5 + ZEB1 following the same treatments described in (A, B). E, F Caspase 3/7 activity was measured in THJ-16T cells overexpressing ZEB1, Del-CP-ZEB1, ALKBH5, Del-P#2-ALKBH5 and ALKBH5 + ZEB1 following the same treatment described in (A, B). G, H Representative images from soft agar colony formation assays of THJ-16T cells overexpressing ZEB1, Del-CP-ZEB1, ALKBH5, Del-P#2-ALKBH5 and ALKBH5 + ZEB1 following the same treatment described in (A, B) are shown. Colony diameter was measured and graphed on the right; scale bar, 100 µm. I WB analyses using anti-ZEB1 and anti-ALKBH5 antibodies in ZEB1- and ALKBH5-overexpressing THJ-16T cells following the same treatment described in (A, B). J, K Mouse with a CDX of THJ-16T cells overexpressing ALKBH5 and ZEB1 were treated with or without RA (1.5 μg/kg) plus GEM administered 4 h later (100 mg/kg) for 18 days starting on Day 18. Representative images of the tumors at the end of the experiments were shown in (J), and the tumor growth curves were shown in (K). L Survival of mouse bearing xenografts formed by THJ-16T cells with or without ZEB1 and ALKBH5 knockout was monitored after inoculation for the indicated number of days. Mouse were administrated DMSO or RA (1.5 μg/kg) plus GEM 4 h later (100 mg/kg) for 18 days starting on Day 18 (n = 5/group). The data in (C–H) were analyzed by one-way ANOVA. The data in (K, L) were analyzed by two-way ANOVA. Images in (G, H) are representative of three independent experiments. *p < 0.05, **p < 0.01 indicate statistical significance.