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. 2021 Jul 21;12(10):769–787. doi: 10.1007/s13238-021-00858-3

Figure 4.

Figure 4

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.