Cisd1 16 006 1 ap

Paraffin-section containing aortic tissue and PVAT were deparaffinized, dehydrated, and boiled for epitope retrieval using an antigen retrieval buffer at pH = 6.0 (Opal 4-color IHC Kit, PerkinElmer). Tissue sections were blocked (StartingBlock™ T20 Blocking Buffer, 37539; Thermo-Fisher Scientific) for 1 h and incubated with primary antibodies (cathepsin S: ab18822, Abcam; MMP-12: PA5-13181, Thermo-Fisher Scientific; F4/80: ab6640, Abcam; CISD1: 16,006–1-AP, Proteintech; and NF-κB p65: SC-372, Santa Cruz) overnight at 4℃. The sections were incubated with HRP-conjugated secondary antibodies for 1 h and administrated with 50 × diluted Opal working solution for 10 min at room temperature. The following primary antibodies were stained with a repeat procedure including antigen stripping and blocking steps. DAPI was used to stain cell nuclei via adding mounting media containing fluoroshield with DAPI. The images were visualized by confocal microscopy (C1-Si, Nikon).

Human HCC cell lines PLC/PRF/5 and Huh7 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. HCC-LM3 cells were taken from our laboratory stocks. All the HCC cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Corning, Inc., Corning, NY, USA) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and were maintained in an incubator with 5% CO₂ at 37 °C. Sorafenib and Bafilomycin A1 were obtained from Selleck Chemicals (Houston, TX, USA). Primary antibodies used in this study were listed below: MTX1 (15529-1-AP, Proteintech, Wuhan, China), CISD1 (16006-1-AP, Proteintech, Wuhan, China), Beclin1 (11306-1-AP, Proteintech, Wuhan, China), LC3 (14600-1-AP, Proteintech, Wuhan, China), GAPDH (60004-1-Ig, Proteintech, Wuhan, China), β-Actin (sc-8432, Santa Cruz Biotechnology, USA) and FLAG (abs830005, Absin, China).