Wl02385

A549 cells were incubated with 10 μM HA15 for 48 h. The cells were then harvested and whole proteins extracted with 100 μL RIPA buffer containing 5 μL protease inhibitor (20×), 2 μL phosphatase inhibitor, and 1 μL 100 mM phenylmethanesulfonyl fluoride. Proteins were extracted using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, CA, USA). The primary antibodies used in the study were: GRP78 (1:10,000, a kind gift from Professor Deqiang Wang, Chongqing Medical University), B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and Beclin-1 (50599 and 11306, both 1:1000, Protein tech, Wuhan, China), Bcl-2 and p53 (BM0200 and BM0101, both 1:200, BOSTER, China), p62 (WL02385, 1:1000, Wanleibio, China), and actin (TA-09, 1:1000, ZSGB-BIO, China), LC3B (A5202, 1:500,bimake,USA), LaminA/C (4777, 1:2000, Cell Signaling Technology, MA, USA), Phospho-p53(ser15) (9284, 1:500, Cell Signaling Technology, MA, USA).

Primary anti-VEGFA (ab52917, Abcam, USA), anti-vWF (PB0273, Boster, Wuhan, China), anti-PCNA (BM0104, Boster, Wuhan, China), anti-LC3 (WL01506, Wanlei, Shenyang, China), anti-p62 (WL02385, Wanlei, Shenyang, China), anti-ERα (ab32063, Abcam, USA), and anti-PR (sc-810, Santa Cruz, TX, USA) antibodies were diluted in 0.5% goat serum in PBS. The sections were heated in a microwave in sodium citrate solution for antigen recovery and pretreated with 0.3% H₂O₂ in methanol to quench endogenous peroxidase activity. Then, the samples were incubated with 3% goat serum to block nonspecific antibody binding sites. Subsequently, the samples were incubated with primary antibodies at 4 °C overnight. Immunoreactivity was visualized using a Mouse and Rabbit Specific HRP/DAB Detection IHC kit (ab64264, Abcam) according to the manufacturer’s instructions.