Fig 2: Identification of CMA-inducing drugs by high-throughput screening. (A) 293THK cells were pretreated with or without 1 μg/mL DOX and transfected with siRNA targeting Hsc70 for 48 h, treated with Spautin-1 (10 μmol/L) and AC220 (2 μmol/L) for another 12 h, fluorescence of HK2-GFP was imaged by fluorescence microscopy. Scale bar, 1000 μm. (B) The fluorescence intensity of (A) were quantified from four independent experiments, Mean FITC-A% represents the average fluorescence intensity of cells (data represents mean ± SD; n = 4, ****P < 0.0001, t-test). (C) 293THK cells were treated as in a, the substrate protein (HK2-GFP) was detected by Western blot. (D) 293THK cells were treated as in (A). Total RNA was extracted by FastPure® Cell/Tissue Total RNA Isolation Kit, mRNA levels of GFP and Hsc70 were analyzed by qPCR (data represents mean ± SD; n = 3, ****P < 0.0001, t-test). (E) 293THK cells were pretreated with or without 1 μg/mL DOX for 24 h, incubated with 2,197 FDA-approved drugs or drug candidates for 24 h, and the HK2-GFP fluorescence was analyzed by flow cytometry and compared with the fluorescence of cells treated with DMSO. The results were presented in the form of a heatmap. Red and blue colors represent the degree of increase and decrease of HK2-GFP levels, respectively, following drug treatments. (F) 293THK cells were pretreated with or without 1 μg/mL DOX, transfected with siRNA (two different sequences #1 and #2) of Hsc70 for 48 h, treated with or without Metformin for another 12 h, cell lysates were analyzed by Western blot using anti-Hsc70, anti-GFP, and anti-Tubulin antibodies

Fig 3: Metformin activates IKKα/β kinases to promote Hsc70 phosphorylation at Ser85 in a TAK1-dependent and AMPK-independent manner. (A) HEK293T cells were transfected with indicated siRNA for 48 h, treated with or without 20 mmol/L Metformin for another 12 h. Cell lysates were immunoblotted with indicated antibodies. (B) In vitro kinase reactions using purified IKKα, IKKβ, and Hsc70. The reactions were immunoblotted with indicated antibodies. (C) HEK293T cells were transfected with Hsc70-Flag for 24 h, treated with or without Metformin (20 mmol/L) for another 6 h, and the interaction between IKKβ and Hsc70 was detected by co-immunoprecipitation. (D) 293THK cells were pretreated with or without 1 μg/mL DOX, transfected with siRNA (#1 and #2 represent two different sequences) of IKKβ for 48 h, treated with or without 20 mmol/L Metformin for another 12 h, fluorescence of HK2-GFP was analyzed by flow cytometry (data represents mean ± SD; ****P < 0.0001, one-way ANOVA). (E) 293THK cells were pretreated with or without 1 μg/mL DOX, treated with 20 mmol/L Metformin with or without 5 μmol/L TPCA1 for 12 h, fluorescence of HK2-GFP was analyzed by flow cytometry. (F) 293THK cells were treated as shown in (E). Cell lysates were immunoblotted with indicated antibodies. (G) H4 cells were transfected with siRNA (#1 and #2 represent two different sequences) of IKKβ for 48 h, treated with or without 20 mmol/L Metformin for another 12 h. Cell lysates were immunoblotted with indicated antibodies. (H) 293THK cells were pretreated with or without 1 μg/mL DOX, transfected with siRNA (#1 and #2 represent two different sequences) of TAK1 for 12 h, treated with or without 20 μmol/L Metformin for another 48 h, the fluorescence of HK2-GFP was analyzed by flow cytometry (data represents mean ± SD; ****P < 0.0001, one-way ANOVA). (I) H4 cells were transfected with siRNA (#1 and #2 represent two different sequences) of TAK1 for 12 h, treated with or without 20 μmol/L Metformin for another 48 h. Cell lysates were immunoblotted with indicated antibodies.

Fig 4: Metformin induces APP degradation through activation of chaperone-mediated autophagy. (A) SH-SY5Y cells were treated with Metformin (20 μmol/L) for 8, 12, 24, 36 and 48 h. Cell lysates were immunoblotted with indicated antibodies. (B) SH-SY5Y cells were treated with or without Metformin (20 μmol/L) for 36 h, with MG132 (10 μmol/L), Bafilomycin A1 (100 nmol/L), NH4Cl (20 mmol/L), Leupeptin (100 nmol/L) for another 12 h. Cell lysates were immunoblotted with indicated antibodies. (C and D) SH-SY5Y cells were transfected with siRNA (#1 and #2 represent two different sequences) of Hsc70 (C) or Lamp2a (D) for 12 h, treated with or without Metformin (20 μmol/L) for another 48 h, cell lysates were immunoblotted with indicated antibodies. si-Control: scrambled siRNA. (E and F) SH-SY5Y cells were transfected with Hsc70-Flag (E) or Lamp2a-Flag (F) for 24 h, treated with or without Metformin (20 μmol/L) for another 12 h, the interaction between APP and Hsc70 was analyzed by immunoprecipitation. (G) SH-SY5Y cells were transfected with Hsc70 WT-Flag or Hsc70 S85A-Flag for 24 h, treated with or without Metformin (20 μmol/L) for another 12 h, the interaction between APP and Hsc70 was analyzed by immunoprecipitation. (H) SH-SY5Y cells were transfected with siRNA (#1 and #2 represent two different sequences) of IKKβ for 12 h, treated with or without Metformin (20 μmol/L) for another 48 h. Cell lysates were immunoblotted with indicated antibodies. si-Control: scrambled siRNA. (I) SH-SY5Y cells were treated with Metformin (20 μmol/L) for 36 h, with or without TPCA1 (5 μmol/L) for another 12 h. Cell lysates were immunoblotted with indicated antibodies. (J) SH-SY5Y cells were transfected with siRNA (#1 and #2 represent two different sequences) of TAK1 for 12 h, treated with or without Metformin (20 μmol/L) for another 48 h. Cell lysates were immunoblotted with indicated antibodies. si-Control: scrambled siRNA.

Fig 5: 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).