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. 2020 Dec 4;10:573127. doi: 10.3389/fonc.2020.573127

Figure 4.

Figure 4

Involvement of the AMPK-ULK1-LC3 signaling cascade in TRPM8-stimulated autophagy. (A, B) The construct for Flag-AMPK expression was transiently transfected into MCF7 cells. After 48 h of transfection, the AMPK-ULK1-LC3 signaling cascade-related proteins were detected by WB (N = 3). (C, D) MCF7 cells were transiently transfected with wild type TRPM8, mutant V976W, or control vector. After 48 h of transfection, protein lysates were used for WB analysis (N = 3). (E, F) MDA-MB-231 cells treated with 2 μM icilin, 0.5 μM AMTB, or their combination for 48 h were extracted for WB analysis (N = 3). (G, H) MCF7 cells were transfected with siRNA against human TRPM8; siRNA against TRPM8 (siTRPM8-1 and siTRPM8-2) successfully knocked down TRPM8 expression compared with that in the control scramble siRNA samples according to WB analysis using an anti-TRPM8 antibody. The AMPK-ULK1-LC3 signaling cascade-related proteins detected by WB analysis using the indicated antibodies (N = 3). N represents the number of replicate experiments. *P < 0.05; NS, not significant.