Figure 1. Autophagy regulates the timing of MOMP and PUMA levels.

(A) siRNA targeting ATG12 in HeLa cells inhibits trehalose-induced (Tre) autophagy shown by reduced accumulation of LC3-II after treatment with lysosomal protease inhibitors Pepstatin and E64D.

(B) An example of complete and rapid MOMP visualized in HeLa cells stably expressing IMS-RP. Loss of punctate IMS-RP staining was used to determine when MOMP took place after TRAIL treatment.

(C) Trehalose treatment delays MOMP after TRAIL treatment (25ng/ml) and this delay is blocked by autophagy inhibition using ATG12 siRNA. Cells were imaged individually after treatment with or without trehalose and siRNA as indicated. Bars indicate the mean time to MOMP, p values by Student’s t- test.

(D) Western blot analysis of time course of TRAIL-treated HeLa cells −/+ Trehalose to analyze cleavage of initiator caspase-8 or BID. Autophagy induction by trehalose did not delay caspase-8 or Bid cleavage.

(E) Autophagy inhibition by shRNA knockdown of ATG5, ATG7, ATG12, Vps34 or p62/SQSTM1 all cause an increase in PUMA levels.

(F) PUMA knockdown is sufficient to delay MOMP in response to TRAIL treatment. Cells were infected with shRNA targeting PUMA in the presence or absence of trehalose as indicated and time to MOMP determined by imaging individual cells. PUMA knockdown is sufficient delay MOMP similarly to trehalose. Bars indicate mean time to MOMP, p values were calculated by t-test, ns indicates p>0.05.

See also Supplementary Figures 1 and 2.