Mitochondrial marker antibody sampler kit

For primary antibodies, we used mouse antibody against PARP1 (cat# 556494, 1 : 1,000, BD Bioscience), mitochondrial marker antibody sampler kit (cat# 8674, 1 : 3,000, Cell Signaling, rabbit antibody to CoxIV, Cytochrome C, HSP60, PHB1, pyruvate dehydrogenase, SDHA, and mouse antibody to SOD1), mouse antibody against GAPDH (GT239, 1 : 5,000, GeneTex), mouse antibody against FLAG (clone M2, Sigma), rabbit antibody against PINK1 (cat# 100-494, 1 : 2,000, Novus Biologicals), and mouse antibody against UQCRC2 (cat# ab14745, 1 : 3,000, Abcam). For secondary antibodies, we used horseradish peroxidase- (HRP-) conjugated mouse antibody against FLAG (cat# A8592, 1 : 5,000, Sigma-Aldrich), HRP-conjugated antibody against HA (1 : 3,000, Roche), HRP-conjugated mouse antibody against β-actin (AC15, 1 : 10,000, Sigma-Aldrich), HRP-conjugated sheep antibody against mouse IgG (cat# RPN4301, 1 : 5,000, GE Healthcare), HRP-conjugated donkey antibody against rabbit IgG (cat# RPN4101, 1 : 5,000, GE Healthcare), Alexa Fluor 488-conjugated donkey antibody against mouse IgG (H+L) (cat# A21202, 1 : 1,000, Invitrogen), and Alexa Fluor 568-conjugated donkey antibody against rabbit IgG (cat# A10042, 1 : 1,000, Invitrogen). LentiCRISPR and lentiCRISPR-sgRNA-eGFP#2 constructs were purchased from Addgene. Oligonucleotides were ordered from and synthesized by Cosmo Genetech.

To quantify specific gene expression, samples were lysed in lysis buffer (Sigma-Aldrich). Samples containing equal quantities of protein were run on 10% to 12% SDS polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked in 5% BSA and incubated overnight at 4°C with anti-catalase and anti-human PRL-1 (1:1,000, both from Abcam, Cambridge, UK); anti-GPX3, anti-HLAG (4h84), and anti-PGC1A (1:1,000, all from NovusBio, Littleton, CO, USA); anti-POU5F1 (1:1,000, AbFrontier, Seoul, Republic of Korea); antibodies from a Mitochondrial Marker Antibody Sampler Kit (1:1,000, Cell Signaling Technology, Danvers, MA, US); anti-HO-1 (1:500, Cell Signaling Technology); anti-ATP5B (1:500, Santa Cruz Biotechnology, Dallas, TX, USA); anti-NRF2 (1:1,000, Bioss, Woburn, MA, USA); anti-TFAM (1:1,000, Thermo Fisher Scientific, Waltham, MA, USA); and anti-GAPDH (1:3,000, AbFrontier). The membranes were then incubated with peroxidase-conjugated secondary anti-rabbit IgG and anti-mouse IgG (1:8,000, Bio-Rad). Bands were detected using enhanced chemiluminescence reagent (Bio-Rad Laboratories).

To assess the specific gene expression of PD-MSCs^PRL-1 and cirrhotic liver tissues, samples were lysed in protein lysis buffer (Sigma-Aldrich). The protein lysates were loaded on sodium dodecyl sulfate polyacrylamide gels, and separated proteins were transferred to PVDF membranes. The following primary antibodies were used: anti-human PRL-1, anti-Oct4 (1:1000; all from Abcam, Cambridge, UK), anti-albumin, anti-HLA-G (4 h84) (1:1000; all from Novus Biologicals, Littleton, CO, USA), anti-ATP5B (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-ROCK1, anti-RhoA, anti-CDK4, anti-cyclin D1, and mitochondrial marker antibody sampler kit (1:1000; all from Cell Signaling Technology, Denvers, MA, USA). The loading control was anti-GAPDH (1:3000; AbFrontier, Seoul, Korea). The following secondary antibodies were used: anti-mouse IgG (1:5000; Bio-Rad, Hercules, CA, USA) and anti-rabbit IgG (1:10,000; Bio-Rad). The bands were detected using ECL reagent (Bio-Rad).