Figure 3

The essential role of GSK3β-TIP60-ULK1 pathway in ER stress-induced autophagy. (a) HeLa cells transfected with control (NC) or TIP60 small interfering RNA (siRNA) for 48 h were treated with TM (10 μg/ml)and bafilomycin A1 (400 μM) for 24 h. The lysates were blotted for LC3B, total and phosphorylated TIP60. The relative amounts of LC3BII were calculated from densitometry performed on immunoblots and normalized to the amount of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data represent the mean±S.E.M. of three independent experiments (**P<0.01; NS, not significant (two-way analysis of variance (ANOVA) followed by Tukey’s test)). (b) The effect of modulating TIP60 on TM-induced change in LC3BII. HeLa cells transfected with Vector, Myc-TIP60 (wild type (WT)) or Myc-TIP60 (S86A) for 20 h were treated with TM (10 μg/ml) and bafilomycin A1 (400 μM) for another 24 h. The lysates were immunoblotted as indicated. Data represent the mean±S.E.M. of three independent experiments (*P<0.05; **P<0.01; NS, not significant (two-way ANOVA followed by Tukey’s test)). (c) The effect of knockdown TIP60 on TM-induced LC3B puncta formation. HeLa cells co-transfected with GFP-LC3 (green) and TIP60 small interfering RNA (siRNA) for 48 h were treated with TM (10 μg/ml) for 24 h and scored for the number of puncta. Quantification shown above represents the mean GFP puncta per cell (n=12) from three independent experiments±S.E.M. (**P<0.01; NS, not significant (two-way ANOVA followed by Tukey’s test); scale bar, 20 μm). (d) The effect of modulating TIP60 on TM-induced LC3B puncta formation. HeLa cells were transfected as indicated and then treated with TM (10 μg/ml) for another 24 h. LC3B puncta formation was evaluated as described in (c) (*P<0.05; **P<0.01, NS, not significant (two-way ANOVA followed by Tukey’s test); scale bar, 20 μm). (e) The effect of ULK1 knockdown on TM-induced change in LC3BII. HeLa cells were transfected with control (NC) or ULK1 siRNA for 48 h were treated with TM (10 μg/ml) and bafilomycin A1 (400 μM) for another 24 h. The lysates were immunoblotted as indicated. Data represent the mean±S.E.M. of three independent experiments (**P<0.01; NS, not significant (two-way ANOVA followed by Tukey’s test)). (f) The effect of GSK3β inhibitor on TM-induced LC3BII change. HeLa cells were treated with TM (10 μg/ml) with or without SB216763 (10 μM) and bafilomycin A1 (400 μM) for 24 h. LC3BII level was determined. Data represent the mean±S.E.M. of three independent experiments (**P<0.01; NS, not significant (two-way ANOVA followed by Tukey’s test))