Fig. 3. THZ1 exhibits inhibitory potency against CCA in vitro and in vivo.

a Five CCA cell lines (HuCCT1, HuH28, RBE, HCCC9810, and OZ) were plated in 96-well plates and treated with THZ1 or THZ1R (a THZ1 analog with no significant inhibitory activity of CDK7) at different concentrations for 72 h. Cell viability was measured by CCK-8 assay and dose–response curves show percent cell viability to that of DMSO-treated cells. IC₅₀ values were calculated using nonlinear regression analysis in Prism 7.0. Data represent mean ± SEM of three independent replicates. b HuCCT1, HuH28, and RBE cells were treated with THZ1 at two concentrations (100 nM and 200 nM) for 72 h and cell apoptosis was measured by Annexin V-FITC/PI staining followed by flow cytometry. Data represent mean ± SEM of three independent replicates. (*P < 0.05; **P < 0.01; ***P < 0.001) c HuCCT1, HuH28, and RBE cells were treated with three concentrations (100 nM, 200 nM, and 400 nM) of THZ1 for 48 h for measuring Caspase 3/7 activation. Data represent mean ± SEM of three independent replicates. (*P < 0.05; **P < 0.01; ***P < 0.001) d HuCCT1, HuH28, and RBE cells were treated with THZ1 for 24 h and Cleaved PARP was analyzed by western immunoblotting. β-actin served as an internal control. e Nude mice were injected subcutaneously with HuCCT1 cells and randomly separated into two groups when tumor size reached around 200 mm³. Two groups of mice were treated with THZ1 (10 mg/kg, bid, i.p.) or vehicle (bid, i.p.) for 27 days, separately. Tumor volume was measured twice a week. The growth curve was shown. f The fold change of tumor volume at day 27 relative to day 1 was shown as mean ± SEM. g The survival curves were assessed by percent of mice with tumor volume <500 mm³