Figure 2.

Effect of ATF6α silencing on sensitivity to cisplatin treatment. (A) OS and hFOB cells were treated with increasing concentrations of cisplatin or irinotecan. Twenty-four hours post-treatment, cell viability was measured as described in methods. Data were normalized to an untreated control well and graphed, and half maximal inhibitory concentration (IC50) values were calculated from the dose-response curve as the concentration of the drug that produced a 50% decrease in the mean luminescence relative to untreated control wells. Data are presented as means ± standard errors of the means from three to 4 separate experiments. Curves were fit by non-linear regression and the significance of differences between IC₅₀ were calculated using one-way ANOVA and post hoc Tukey’s multiple comparison tests The significance scores of all treatments versus hFOB groups are indicated; IC50 cisplatin, hFOB vs 143b, ****P < .0001, hFOB vs U2OS, ****P < .0001and IC50 irinotecan hFOB vs 143b, ****P < .0001, hFOB vs U2OS, ****P < .0001 (B) Total RNA from siControl and siATF6α expressing OS cells was extracted and expression of ATF6α levels was quantitated by qPCR, normalizing against 18s rRNA expression. Data represent mean ± SE of 3 independent experiments. **P < .01; ***P < .001. (C) Protein lysates from OS cells transfected with sicontrol and siATF6α siRNAs were analyzed by WB for the expression of ATF6α as described in methods (upper panel). GAPDH was used as a loading control and quantified using ImageJ (lower panel). Columns represent mean± SE of three independent experiments. **P < .01, *P < .05 siATF6α versus siControl (D) siControl and siATF6α expressing OS cell lines were treated with cisplatin (12.5 μM)(left panel) or irinotecan (20 μM-143b, 50 μM for U2OS) (right panel) overnight, and then fixed and stained with anti-cleaved caspase-3 antibody and analyzed for caspase-3 activation by immunofluorescence. The percent of cleaved caspase-3 positive cells was analyzed using Zen software and expressed as a fold-change after normalization to untreated siControl cells. Approximately one thousand DAPI positive cells were scored for caspase-3 activation. Columns represent mean± SE of five independent experiments performed in triplicate. **P < .01, *P < .05 siATF6α+drug versus siControl + drug. (E) Representative immunofluorescence images of cleaved caspase-3 positive cells in siControl and siATF6α expressing OS cells following treatment with cisplatin (upper panel) or irinotecan (lower panel).