Figure 7.

Autophagy is essential for the regulatory effect of TRPM8 on the proliferation and migration of breast cancer cells. (A, B) Cell proliferation experiments. MCF7 cells were transfected with TRPM8 constructs. After 24 h of transfection, cells were treated with 10 M CQ. (A) The in vitro colony formation assay. Cells were further cultured in growth media for 7-10 days to form the colonies. The colonies were washed with ice-cold PBS three times, stained with trypan blue, and counted (N = 3). (B) Ki67 expression was detected by immunostaining (N = 3). (C) MDA-MB-231 cells were transiently transfected with siRNA against human ATG7 (siATG7) and a Flag-TRPM8 construct. After 48 h of transfection, protein lysates were extracted for WB analysis to determine the effect of TRPM8 overexpression on basal autophagy in the presence of ATG7 knockdown (N =3). (D, E) Similar experiment was performed in MDA-MB-231 cells transfected with siATG7 and a Flag-TRPM8 construct. (N = 3). (F, G) Cell migration determined by wound healing assay. (F) MCF7 cells were transfected with the TRPM8 constructs for 24 h and scratched with a sterile 10 μl tip. After three washes with 1× PBS, the cells were cultured for 24–72 h in serum-free medium in the presence of 10 μM CQ (N = 3). (G) Similar wound healing assay as in (F) but with MDA-MB-231 cells transfected with siATG7 and a Flag-TRPM8 construct (N = 3). N represents the number of replicate experiments. *P < 0.05; NS, not significant.