Image lab software version 5

Protein lysates were prepared by incubating cells in RIPA buffer on ice for 30 min, followed by sonication using Q700 Sonicator (QSonica, Newtown, CT, USA). Proteins were resolved by SDS-PAGE and transferred to PVDF membrane using standard protocols. Following primary antibodies were used at a 1:1000 dilution (except Lamin B, which was used at 1:2000 dilution) in this study: UBF (sc-13125, Santa Cruz Biotechnology, Dallas, TX, USA); RUNX1 (4334S, Cell Signaling Technologies, Danvers, MA, USA); Cyclin B (4138S, Cell Signaling Technologies, Danvers, MA, USA); Beta-Actin (3700S, Cell Signaling Technologies, Danvers, MA, USA), and CDT1 (ab70829, AbCam, Cambridge, UK); Lamin B1 (ab16048, AbCam, Cambridge, UK). Horseradish peroxidase conjugated secondary antibodies used in this studies were: goat anti-mouse IgG at 1:5000 dilution (31460, Invitrogen, Carlsbad, CA, USA), goat anti-rabbit IgG HRP conjugated (31430, Thermo Fisher Scientific, Asheville, NC, USA) at 1:1000, 1:2000, or 1:5000 dilutions. Blots were developed using Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and imaged using Molecular Imager^® Chemi doc™ XRS+ Imaging System (Bio-Rad, Hercules, CA, USA) aided by Image Lab Software Version 5.1 (Bio-Rad, Hercules, CA, USA).

The total DNA from the PAM, PK15, ST, and PIEC cells was extracted with a cell DNA Isolation Mini Kit (Vazyme, Nanjing, China). DNA fragments containing antigenic peptides and inserted or deleted regions were amplified by PCR using the extracted DNA samples above. All PCR products were sequenced with gene-specific primers. All the images of agarose gel electrophoresis were captured with the Image Lab Software version 5.1 (Bio-Rad Laboratories, Hercules, CA, USA). The specific primers are shown in Table S1.

To obtain nuclear protein extracts from MSCs, 24 h after culture on substrates with distinct stiffness, cells were washed with PBS and then scraped in Laemmli buffer. The subcellular fractionation was done following the protocol described in^{75 (link)}, and nuclear fractions were collected into microtubes and then heated at 95 °C for 5 min. Next, the samples were spun down and passed ten times through a 25 G needle and further analyzed by Western blot. Proteins were separated by SDS-PAGE [12.5% (w/v) acrylamide–bisacrylamide (Bio-Rad) gels] and transferred onto PVDF membranes that were subsequently probed with specific antibodies against H4K16ac (Abcam) and H4 (Cell Signaling) followed by the incubation with the respective alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch). The membranes were incubated for 5 minutes with enhanced chemifluorescence substrates (ECF – GE Healthcare) and imaged in a Molecular Imager FX Pro Plus system (BioRad) using the Quantity One software (BioRad). The acquired images were analyzed with Image Lab software, version 5.1 (BioRad).

Proteins were extracted from snap-frozen kidney cortexes and were quantified with a Bio-Rad protein assay. Equal amounts of protein lysates were loaded and separated on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes or polyvinylidene difluoride membranes (Millipore, USA). Nonspecific proteins were blocked by incubating the membranes in 5% non-fat milk for 1 hour at room temperature. The membranes were then incubated with primary antibodies at 4°C overnight for specific proteins, followed by incubation with HRP-conjugated secondary antibodies for 1 hour at room temperature. HRP activity was visualized using Clarity Western ECL Substrate and a ChemiDoc MP Imaging System (Bio-Rad Laboratories, USA). Image Lab software version 5.1 was applied for densitometric analysis (Bio-Rad Laboratories, USA). The following primary antibodies were used in this study: polyclonal anti-HIF-2α from rabbit (Abcam; ab199; 1:200 dilution), polyclonal anti-HIF-3α from rabbit (Abcam; ab176464; 1:1000 dilution), and monoclonal anti-β-actin from mouse (Sigma; A5441; 1:5000 dilution).

The levels of p-STAT3, p-STAT5, p-SMAD3, and TGFβR2 proteins were assessed in liver tissue as previously described [34 (link)]. The liver tissue was homogenized in RIPA buffer containing protease and phosphatase inhibitor cocktail (BioBasic, Ontario, Canada). The total protein levels were quantified using the Biorad assay, 20–30 μg of total protein from the cell lysate were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). Membranes were blotted overnight at 4°C with the primary antibodies, anti p-STAT3, anti p-STAT5, anti p-SMAD3 or anti TGFβR2 (ThermoFisher Scientific, Rockford, IL, USA) at concentration (1:1000), and then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit IgG (ThermoFisher Scientific, Rockford, IL, USA) for 1 hr at room temperature. β-actin was used as loading control. Band intensity was analyzed by ChemiDoc imaging system with Image Lab software version 5.1 (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Cells were lysed in NP40 buffer (Beyotime, Shanghai, China) for 10 min on ice and then centrifuged at 10,000 g at 4°C to remove cell debris. Equal amounts (30 μg) of cell extract were resolved by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA), followed by incubation with primary rabbit monoclonal antibodies against human STAT3 and pSTAT3 (1:1000 dilution; R&D Systems) and rabbit monoclonal antibodies against human MMP2/9, E-cadherin and α-tubulin (1:1000 dilution; ProteinTech, Chicago, IL). Then, the cells were incubated with peroxidase-conjugated affinipure secondary IgG antibodies (H+L) (1:2000; ProteinTech, Chicago, IL). Proteins were detected using a chemiluminescence detection system, and bands were quantitated by Image Lab™ Software, Version 5.1 (both from Bio-Rad, Hercules, CA).

Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Samples containing 20 μg of total protein were prepared in SDS sample buffer (Invitrogen, Carlsbad, CA, USA) using standard methods. Samples were resolved by SDS-PAGE in 10% polyacrylamide gels and blotted to PVDF membranes by semi-dry transfer. Membranes were blocked in 3% milk/TBST for 1 h at room temperature prior to application of primary antibody overnight at 4°C. Chemiluminescent protein detection occurred by application of HRP-conjugated secondary antibody for 1 h at room temperature followed by treatment with Clarity Western ECL (Bio-Rad) peroxidase substrate. Blot luminescence was digitally imaged using a ChemiDoc MP Imaging System with Image Lab Software Version 5.1 (Bio-Rad). Protein levels were quantified by densitometry using ImageJ software (NCBI). A mild or harsh stripping protocol (Abcam) was performed prior to blot re-probing.