Atf 4

(E)2’-hydroxychalcone (2’-HC; purity ≥ 98%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Hydroxychloroquine (HCQ; purity ≥ 98%), 3-Methyladenine (3-MA; purity ≥ 98%), and N-acetylcysteine (NAC; purity ≥ 99%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin (Rap; purity ≥ 98%) was purchased from Aladdin (Shanghai, China).
The primary antibodies used were as follows: β-actin, Bax, Bcl-2, PARP, cleaved-PARP p25, caspase-9, caspase-3, LC3B, Beclin1, and p-IκB. They were purchased from ABclonal (Wuhan, China). p62/SQSTM1 was obtained from Proteintech (Wuhan, China). ERK, JNK, p-JNK, p38, p-p38, NF-κBp65, IκB, p-IκB, p-eIF2α, and MMP9 were obtained from Abmart (Shanghai, China). P-ERK, p-NF-κBp65, ATF-4, and CHOP were purchased from Wanleibio (Shenyang, China). HRP Goat Anti-Rabbit IgG (Abclonal), Alexa Flour 594-Goat Anti-Rabbit IgG (Abbox, Jiangsu, China), Cy3 Goat Anti-Rabbit IgG (H + L) (Abclonal), and FITC Goat Anti-Rabbit IgG (Servicebio, Wuhan, China) were used as secondary antibodies for a Western blot or immunofluorescence.

Cellular proteins were extracted in RIPA lysis buffer (BioTeke Corporation) on ice. An equal protein content of cell lysates was loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrophoretically resolved, and then transferred onto polyvinylidene difluoride western blot membranes (Roche, Basel, Switzerland). The membranes were blocked for 3 h at 25 °C in 5% skim milk, and then incubated with specific primary and secondary antibodies. Immunoblots were detected using an ECL Western Blotting Substrate (Solarbio Science & Technology) and visualized using a Tanon 5200 digital imaging system (Tanon Science & Technology, Shanghai, China). Primary antibodies were caspase-3, cleaved-caspase-3, Bcl-2, Bax, GRP78, CHOP, IRE1, elF2α, ATF4, ATF6, p-38, p-p38, ERK, p-ERK, JNK, p-JNK, AKT and p-AKT (Wanleibio, Shenyang, China), β-actin, MPST, p-IRE1 (Bioss, Beijing, China), CBS, CSE (Omnimabs, Alhambra, CA, USA), and p-elF2α (Abbkine, Wuhan, China). Secondary antibodies (goat anti-rabbit IgG/HRP antibody, goat anti-mouse IgG/HRP antibody) were purchased from Bioss. Western blotting quantification results were evaluated with Image J software.