ATM‐CHK2‐Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress

A

Intracellular ROS levels detected in H1299 cells treated with HBSS starvation.

B

Western blot detection of p62 in H1299 cells transfected with the indicated shRNA in normal medium or after HBSS starvation.

C

Western blot detection of p62 in H1299 cells cotransfected with the indicated shRNA and plasmid and cultured in normal or HBSS starvation medium. CHK2 NTm, shRNA nontargetable mutant CHK2 rescue plasmid.

D, E

Quantification of p62 and LC3‐II/I protein levels in H1299 cells transfected with the indicated shRNA in normal medium or after H₂O₂ (500 μM) treatment. Results are presented as mean ± s.e.m. from three independent experiments, *P < 0.05, **P < 0.01 (Student's t‐test).

F, G

Quantification of p62 and LC3‐II/I protein levels in H1299 cells cotransfected with the indicated shRNA and plasmid and cultured in normal medium or after H₂O₂ (500 μM) treatment. Results are presented as mean ± s.e.m. from three independent experiments, *P < 0.05 (Student's t‐test).

H

Quantification of p62 protein levels in H1299 cells transfected with the indicated shRNA in normal medium or after HBSS starvation. Results are presented as mean ± s.e.m. from three independent experiments, *P < 0.05 (Student's t‐test).

I

Quantification of p62 protein levels in H1299 cells cotransfected with the indicated shRNA and plasmid and cultured in normal medium or after HBSS starvation. Results are presented as mean ± s.e.m. from three independent experiments, *P < 0.05 (Student's t‐test).

J

Quantification of p62 protein levels in MEFs of indicated genotype during growth in normal medium or after HBSS starvation. Results are presented as mean ± s.e.m. from three independent experiments, **P < 0.01 (Student's t‐test).

K

Quantification of p62 protein levels in MEFs of the indicated genotype during growth in normal medium or after H₂O₂ (500 μM) treatment. Results are presented as mean ± s.e.m. from three independent experiments, **P < 0.01 (Student's t‐test).

L

Western blot detection of p62 in MEFs of the indicated genotype during growth in normal medium or after HBSS starvation.

M

Western blot detection of p62 in MEFs of the indicated genotype during growth in normal medium or after H₂O₂ (500 μM) treatment.

N

Representative images of GFP‐LC3 puncta in HeLa/GFP‐LC3 cells transfected with the indicated shRNA, pretreated with 60 nM CQ or vehicle (DMSO), and cultured for 1 h in normal, HBSS starvation medium or treated with H₂O₂ (500 μM). Scale bar, 50 μm.

O

Quantification of GFP‐LC3 puncta in HeLa/GFP‐LC3 cells transfected with the indicated shRNA, pretreated with 60 nM CQ or vehicle (DMSO), and cultured for 1 h in normal, HBSS starvation medium or treated with H₂O₂ (500 μM; n = 30). Results are presented as mean ± s.e.m. from three independent experiments, ***P < 0.001 (Student's t‐test).

P

Western blot detection of LC3 in H1299 cells transfected with the indicated shRNA, pretreated with 60 nM CQ or vehicle (DMSO), and cultured for 3 h in normal or treated with H₂O₂ (500 μM).

Figure EV1. CHK2 is involved in oxidative stress‐induced autophagy.