Figure 3.

Metformin activates chaperone-mediated autophagy by inducing Hsc70 phosphorylation at Ser85. (A) AMPK wild-type (WT) and α1/α2 double knockout (DKO) MEF cells were treated with 20 mmol/L Metformin for 2, 4, 8, 12, and 24 h. Cell lysates were immunoblotted with indicated antibodies. (B) AMPK WT and DKO MEF cells were treated with 20 mmol/L Metformin with or without MG132 (10 μmol/L), Bafilomycin A1 (100 nmol/L), NH₄Cl (20 mmol/L), Leupeptin (100 nmol/L) for 12 h. Cell lysates were immunoblotted with indicated antibodies. (C) 293THK cells were pretreated with or without 1 μg/mL DOX, transfected with Flag or PP-Flag (λ-PPase) plasmids for 24 h, treated with or without 20 mmol/L Metformin for another 12 h. The fluorescence of HK2-GFP was analyzed by flow cytometry. (D) 293THK cells were treated as shown in (C). Cell lysates were immunoblotted with indicated antibodies. (E) HEK293T cells were transfected with Hsc70-Flag for 24 h, treated with or without Metformin (20 mmol/L) for another 8 h, Hsc70 was immunoprecipitated using Flag-agarose beads and analyzed by MS-MS. (F) HEK293T cells were transfected with indicated Hsc70 plasmids for 24 h, treated with or without 20 mmol/L Metformin for another 6 h, the interaction between Hsc70 and HK2 or PKM2 was analyzed by co-immunoprecipitation. (G) HEK293T cells were transfected with Hsc70 WT or Hsc70 S85A plasmids for 24 h, PLA assay for Hsc70 and PKM2 was performed using fluorescence microscopy. Scale bar, 100 μm. (H) Quantification of the mean fluorescence intensity of Texas Red from (G) (data represents mean ± SD; ****P < 0.0001, one-way ANOVA).