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. 2022 Jun 2;31:101289. doi: 10.1016/j.bbrep.2022.101289

Fig. 2.

Fig. 2

ZIC5 positively regulates survival of PDAC and CCA cells. PANC-1 (PDAC cell line) and RBE (CCA cell line) were transfected with a negative control (siNeg) or ZIC5 (siZIC5) siRNA. The target sequence of the siRNA differed between #1 and #2. (A) At 2 days after transfection, ZIC5 mRNA expression was assessed by qPCR. The relative expression level was normalized to that of the housekeeping gene ACTB as an internal control. Statistical analysis was performed using Dunnett's multiple comparison of means test. (B) Cell proliferation was assessed at the indicated times. Relative number of viable cells at Day 4 or 5 (compared to that at Day 0) from three independent experiments is indicated in the bar graphs. Statistical analysis was performed using Dunnett's multiple comparison of means test (***P < 0.001, **P < 0.01, *P < 0.05). (C) PANC-1 and RBE cells were transfected with a negative control (siNeg) or ZIC5 (siZIC5) siRNA. After 24 h, cells were treated with gemcitabine (Gem) (0.2 μM) as indicated. After 2 days, the cells were incubated with Caspase-3/7 Green Detection Reagent (green) and Hoechst33342 (blue). The percentage of caspase-positive cells from three independent experiments was determined. Statistical analysis was performed using Tukey's multiple comparison test (***P < 0.001, **P < 0.01, *P < 0.05). (D) After 14 days of gemcitabine treatment (0.1 μM), cells were stained with Giemsa solution. Stained area was quantified using Image J. Statistical analysis was performed using Tukey's multiple comparison test (***P < 0.001). (E) MiaPaca-2 cells stably overexpressed ZIC5 and control cells (vector) were treated with gemcitabine (Gem) (0.2 μM) for 2 days, and then apoptosis assays were performed. Statistical analysis was performed using the two-tailed non-paired Student's t-test (*P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)