Combination treatment of AC220 and spautins induces CMA. (A) WB quantification of mutant p53 levels in ES2 (top) for the indicated time points and in MDA-MB-231, Sum159, and MDA-MB-435 (bottom) cells treated with AC220 and/or C43 for 16 h. (B) Mutant p53 levels in scramble (SCR) or FLT3 siRNA–transfected ES2 cells treated with C43 for 24 h. This experiment used the same blot as that in Fig. 1 E with additional antibodies. (C) p53 levels in ES2 cells treated with C43 and AC220 for 24 h in the absence or presence of proteasome (MG132) or lysosomal inhibitor (ClQ). (D) p53, IκB, and GAPDH levels in ES2 and Sum159 cells treated with AC220 and/or C43 for the indicated time points. (E) WB of p53, Lamp2A, and Hsc70 levels in nontargeting (N.T.), Hsc70, or Lamp2A siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. (F) The cellular ATP levels (fold) in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 16 h. (G) Cell death indicated as Annexin V/PI positivity (fold) and WB of PARP-1 and caspase-3 cleavage in ES2 cells treated with AC220 and/or C43 in the presence or absence of zVAD or 7N-1 for 24 h. STS was used as a positive cell death inducer. (H) Cell death (fold) of nontargeting (N.T.), Lamp2A, or Hsc70 siRNA–transfected ES2 cells treated with AC220 and/or C43 for 24 h. Anti–α-tubulin was used as a loading control. Cells were treated with 0.1% DMSO (control: vehicle) or 1 µM AC220 and 10 µM C43, unless otherwise stated. In all the experiments, treatment groups were compared with the control group, unless otherwise indicated. Error bars indicate ±SD. *, P < 0.05; **, P < 0.01.