Figure 6
Effect of Atf4 knockdown on membrane transporter expression under ER stress preconditioning. NS, non-silencing. (A) RT-qPCR analysis of Atf4 mRNA. Total RNA was extracted from untreated cells or following treatment with 0.3 μg/ml TPG for 16 h. The histogram depicts Atf4 mRNA normalized to Actb mRNA, represented as the fold increase over non-pretreated controls. Values shown are the means ± SE of 3 separate experiments. ***Significantly different from TPG-untreated cells by a one-way ANOVA (here p < 0.001). ###Significantly different from TPG-treated NS siRNA-transfectants by a one-way Welch’s t-test (here p < 0.001). (B) Synthetic siRNA-mediated knockdown of Atf4. Western blots of C2C12-DMPK160 cells transfected with NS siRNA or Atf4 siRNA were analyzed using the indicated antibody probes. Cropped blots are shown; all gels were run under the same experimental conditions. (C,E) Effect of Atf4 knockdown on the expression of membrane transporter (C) or Snhg1 (E) mRNA was analyzed by RT-qPCR. Total RNA was extracted from untreated cells or following treatment with 0.3 μg/ml TPG for 16 h. The histogram depicts the indicated mRNA normalized to Actb mRNA. Values shown are the means ± SE of 3 separate experiments. *, ***Significantly different from TPG-untreated cells by a one-way ANOVA (*p < 0.05, ***p < 0.001). ###Significantly different from TPG-treated NS siRNA-transfectants by a one-way Welch’s t-test (here p < 0.001). (D) Western blots of C2C12-DMPK160 cells transfected with NS siRNA or Atf4 siRNA were analyzed using the indicated antibody probes. Cropped blots are shown; all gels were run under the same experimental conditions.
