Cyclin d1 antibody

The samples were fixed, embedded, cut into 4 μm sections and stained using a conventional immunohistochemistry procedure. Sections were incubated with FASN antibody (dilution 1:50; Proteintech), PRRX1 antibody (dilution 1:80; Novus Biologicals), cyclin D1 antibody (dilution 1:150; Proteintech) and c‐Myc antibody (dilution 1:150; Proteintech) for 2 hours, before incubated with secondary antibodies at room temperature for 30 minutes. The semiquantification of IHC results was observed and analysed by two independent pathologists. The percentage of positive cells was counted in five areas at 200× magnification per slide and was categorized into following groups: 0, <5% (−); 1, 5%‐25% (+); 2, 25%‐50% (++); 3, >50% (+++). The 0 and 1 were low expression groups, and 2 and 3 belonged to high expression groups.

Forty-eight hours after siRNA transfection, RIPA lysis buffer (Servicebio Technology, China) containing Proteinase Inhibitor Cocktail (MedChemExpress, NJ, USA) was used to lyse Huh7 and MHCC97H cells. The BCA kit (Servicebio) was applied to quantify concentration of protein. After electrophoresis, 30 μg of total proteins were separated by SDS-PAGE, then, the proteins were transferred to PVDF membranes (Millipore, USA), and the membranes were immersed in 5% skimmed milk for blocking, and then incubated individually with specific primary antibodies [the internal control α-tubulin polyclonal antibody (Proteintech, China; 1:5000), SFXN4 antibody (Affinity, USA; 1:1000), cyclin D1 antibody (Proteintech, China; 1:2000), MMP2 (Boster, China; 1:200)]. After incubation at 4 ℃ overnight, the PVDF membranes were washed and then immersed in HRP-conjugated secondary antibody for 1 h. An ECL kit (NCMBIO, China) was applied for visualization of protein bands. The density of the bands was quantified by ImageJ software (NIH, USA).