Mb001

The extraction of protein was performed as described^{76 (link)}. Proteins were separated by 12% Bis-Tris Plus gels (Genscript, China), transferred to PVDF membrane (Millipore, USA) by 350 mA for 90 min, and blocked for 1 hour at room temperature. Primary antibodies used to probe blots were mouse anti-TICAM-1 (sc-514384, mAb, Santa, USA), mouse anti-FLIPS/L (sc-5276, mAb, Santa, USA), rabbit anti-cleaved caspase-8 (9496, mAb, CST, USA), rabbit anti-total caspase-8 (ab108333, mAb, Abcam, USA), rabbit anti-RIPK1/RIP1 (NBP1-77077, polyclonal Ab, Novus, USA), rabbit anti-RIP3 (ab152130, polyclonal Ab, Abcam, USA), rabbit anti-MLKL (ab184718, mAb, Abcam, USA), rabbit anti-p-MLKL (phospho S358) (ab187091, mAb, Abcam, USA), rabbit anti-TOP1 (20705-1-AP, polyclonal Ab, Proteintech, USA), rabbit anti-BET (13232, mAb, CST, USA), rabbit anti-CDK9 (2316, mAb, CST, USA) and rabbit anti-GAPDH (MB001, mAb, Bioworld, USA), which were incubated overnight at 4 °C. Subsequently, HRP-conjugated secondary antibodies including anti-rabbit and anti-mouse antibodies (Fcmacs, China) were incubated for 1 hour. Blots were then visualized by a chemiluminescent imaging system (Tanon, Shanghai, China).

Western blot analysis was performed as described previously^{59 (link)}. Briefly, proteins were electrophoresed in 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were blocked in 5% milk and incubated overnight at 4 °C with the primary antibodies, followed by incubation with anti-rabbit Dye 680CW or anti-mouse Dye 800CW (LI-COR, MO, USA) at 1/10,000 dilution for 1 h. The specific signals and the corresponding band intensities were evaluated using Odyssey Infrared Imaging system (Odyssey, Berlin, Germany). The protein level was normalized against GAPDH. The following antibodies were used in this study: rabbit anti-CSF1 (0.2 µg/ml; Abcam, ab9693), rabbit anti-CX3CL1 (1/5,000 dilution; Abcam, ab85034), and mouse anti-GAPDH (1/10,000 dilution; Bioworld, MB001).

SiO₂, which has a diameter of approximately 2–5 μm, was purchased from Sigma (S5631). The silica was sterilized overnight (200 °C for 16 h) [55 (link)] and then dissolved in sterile normal saline (NS) at a concentration of 5 mg/ml. The dose of SiO₂ used in vivo and in vitro was based on previous studies [29 (link)]. Antibodies against α-SMA (14395-1-AP, rabbit), CCR2 (16153-1-AP, rabbit), BECN (11306-1-AP, rabbit), ATG5 (60061-1-lg, mouse) and LC3 (14600-1-AP, rabbit) were obtained from ProteinTech, Inc. Antibodies against collagen I (BS1530, rabbit) and GAPDH (MB001, mouse) were obtained from BioWorld, Inc.

The proteins from the amygdala tissue samples were separated using 10–12% gradient polyacrylamide gel (P0012A, Beyotime Biotechnology), transferred to a polyvinylidene difluoride membrane electrophoretically, and probed with Histone H3 (acetylK9) (ab10812, Abcam, 1:500), HDAC1 (ab19845, Abcam, 1:1,000), HDAC2 (ab32117, Abcam, 1:2,000), HDAC3 (ab32369, Abcam, 1:7,000), and PKM zeta (ab59364, Abcam, 1:500), respectively. β-actin (AC004, ABclonal, 1:7,000), Histone H3 (ab1791, Abcam, 1:500), and GAPDH (MB001, Bioworld, 1:5,000) were used as the controls. Horseradish-peroxidase-conjugated secondary antibody (1:10,000) was used to incubate the membrane for 8 h. Protein bands were detected using an enhanced chemiluminescence kit (WBKLS0500, Immobilon, Millipore). Protein expression indicated by the intensity of protein bands was determined using Image J software.

Western blotting was performed as we described before^{13 (link)}. Briefly, cells were washed with PBS and lysed. The concentration of total proteins was determined using a Nanodrop (Thermo Fisher, USA). Proteins were separated by 12% Bis–Tris Plus gels (Genscript, China), transferred to PVDF membrane (Millipore, USA), and blocked for 1 h at room temperature and incubated with primary antibodies, anti-transferrin receptor (TfR) (1:1000; ab214039, Abcam, USA), anti-NCOA4 (1:1000; A5695, ABclonal, China), anti-ferritin (1:1000; ab65080, Abcam), anti-ferroportin (FPN) (1:1000; ab78066, Abcam), anti-LC3B (1:1000; 83506, CST, USA), anti-HIF-1α (1:1000; ab2185, Abcam), anti-CDK9 (1:1000;2316,CST), anti-BRD4 (1:1000; ab128874, Abcam), anti-p38 (1:1000; 8690, CST), anti-Phospho-p38 (1:1000; 4511, CST), anti-JNK (1:1000; 9252, CST), anti-Phospho-JNK (1:1000; 4668, CST), anti-ERK1/2 (1:1000; 4695, CST), anti-Phospho-ERK1/2 (1:1000; AF1015, Affinity, China), or GAPDH (1:1000; MB001, Bioworld, USA). Subsequently, HRP-conjugated secondary anti-bodies including anti-rabbit and anti-mouse antibodies (Fcmacs, China) were incubated for 1 h. Blots were then visualized by a chemiluminescent imaging system (Tanon, China). The optical density of each lane was read using ImageJ (Bethesda, USA).