CARS senses cysteine deprivation to activate AMPK for cell survival

A, B

WB analysis of p‐AMPK and total AMPK protein expression in 293T cells transfected with shRNAs targeting GCN2 (A) or Sirt3 (B) and further treated with cystine‐deficient medium for 8 h. Actin served as the loading control.

C

293T cells were transfected with HA‐CaMKK2 alone or with Flag‐AHCY for 48 h and then cultured with cystine‐deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti‐Flag, and a WB analysis was performed. The red arrow indicates the target band.

D

WB analysis of p‐AMPK and total AMPK protein expression in 293T cells transfected with shRNAs targeting AHCY and further treated with cystine‐deficient medium for 8 h. Actin served as the loading control.

E

WB analysis of p‐AMPK and total AMPK protein expression in 293T cells transfected with shRNAs targeting CARS and further treated with glucose‐deficient medium for 2 h. Actin served as the loading control.

F

WB analysis of p‐AMPK and total AMPK protein expression in CARS‐overexpressing 293T cells treated with DMSO, pim1, or Takinib for 8 h. Actin served as the loading control.

G

WB analysis of CARS protein expression in 293T, RCC4 and SK‐MES cells treated with cystine‐deficient medium for 0, 6, 12 or 24 h. Actin served as the loading control.

Figure EV2. CARS is critical for AMPK activation under cystine starvation condition.