Anti alas1

Tissue or cells were placed on glass slides, fixed with 4% paraformaldehyde for 30 min and rinsed twice with PBS. Subsequently, the cells were incubated with anti-ALAS1 (Proteintech, Rosemont, IL, USA, Cat# NBP1-91656) at 4°C for 12 h and then with the secondary antibody at 25°C for 2 h. After washing three times in PBS, the nuclei were labelled with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (1 μg/mL, Sigma) for 5 min. ALAS1 and DAPI fluorescence images were captured using a multichannel fluorescence microscope.

Succinylacetone was purchased from MedChemExpress (Cat# HY-W010184). Antibodies were used to determine protein expression including anti-ALAS1 (Proteintech, Rosemont, IL, USA Cat# NBP1-91656), anti-MMP2 (Wanleibio, Shenyang, China Cat# WL03224), anti-MMP9 (Wanleibio, Shenyang, China, Cat# WL03206), anti-PCNA (Cell Signaling Technology, USA, Cat# 13110), anti-c-Myc (Abcam, Cambridge, MA, USA, Cat# ab32072), and anti-β-actin (Cell Signaling Technology, USA, Cat# 4967).
The small interfering RNA (siRNA) oligonucleotides [siRNA-NC: 5'-UUCUCCGAACGUGUCACGUTT-3' (sense) and 5'-ACGUGACACGUUCGGAGAATT-3' (anti-sense); siRNA-ALAS1#1: 5'-GGUGCAGUAAU GACUACCUTT-3' (sense) and 5'-AGG UAG UCAUUACUGCACCTT-3' (anti-sense); siRNA-ALAS1#2: 5'-GCAGACAUAAC AUCUACGUTT-3' (sense) and 5'-ACGUAGAUG UUAUGUCUGCTT-3' (anti-sense)] were constructed and provided by GenePharma (Shanghai, China).