Col1α1

The protein was extracted from fresh tissues and mRTECs by using RIPA lysate. The total protein concentration was measured using a BCA protein quantitative kit and denatured by heating. The protein samples were assessed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), semi-dry transferred to polyvinylidene fluoride (PVDF), and incubated with primary antibody against α-SMA (1:350, Affinity, USA) and Col1α1 (1:500, Abcam, USA) at 4°C overnight. Next, after being washed by TBST three times and incubated with a specific secondary antibody (1:3,000; Boster, China) for 2 h at room temperature. Immunoreactivity was visualized using an ECL detection system.

Total protein was extracted from LX-2 cells, mouse, and human liver tissues by lysing in RIPA buffer (Millipore, Billerica, MA, USA) supplemented with protease inhibitors (1% phenylmethanesulfonyl fluoride (PMSF)). The protein concentration was quantified by the BCA protein assay kit (Beyotime, Nanjing, China) using BSA as standard. Total protein was separated by loading on 6%-10% Trisglycine SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). Primary antibodies incubated on the membranes used in this study were as follows: α-SMA(1:500; Affinity, Changzhou, China), Col1α1 (1:1000; Abcam, Cambridge, MA, USA), Notch2 (1:1000; Cell Signaling Technology, Beverly, MA, USA), Notch3 (1:1000; Abcam), Hes1 (1:1000; Abcam), β-actin (1:1000; Abcam), GAPDH (1:2500; Abcam), and Vinculin (1:1000; Abcam). The expression levels of β-actin, GAPDH, or Vinculin were used as the control for total protein amount. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:1000; Cell Signaling Technology) was used as a secondary antibody at room temperature for 1 h. Protein bands were detected using the chemiluminescence HRP substrate (Millipore). The densitometric analysis of immunoblots was measured by Gel-Pro Analyzer software (Media Cybernetics, Rockville, MD, USA).

The RAW 264.7 cells were cultured with PBS, LPS, KR−1, and KR−2 media for 30 min, and MC3T3-E1 cells were grown in various CM osteogenic media for 14 days. Subsequently, the cells were lysed for 30 min in lysis buffer with protease inhibitors (MedChemExpress), and cell lysates were analyzed by gel electrophoresis after samples were collected for processing and transferred to nitrocellulose membranes. The membrane was incubated with the relevant antibody for 12 h at 4 °C before exposure, after blocking had been performed with a 5% bovine serum albumin solution (Solarbio) for 1 h. The following antibodies were used for Western blotting: p65, phospho-p65, IκBα, phospho-IκBα, GAPDH, β-tubulin, OCN, Runx-2, and COL1α1 (all 1:1000 dilutions, Abcam, Cambridge, UK).

Twenty micrograms of protein from MSC homogenates was separated on 15% or 10% polyacrylamide denaturing gels and electroblotted onto polyvinylidene fluoride membranes. The primary antibodies used were α-SMA (1:1000, Abcam, USA), col1α1 (1:1000, Abcam, USA), Ras (1:1000, Cell Signaling Technology, USA), p-Erk1/2 (1:1000, Cell Signaling Technology, USA), Erk1/2 (1:1000, Cell Signaling Technology, USA), p-Smad2/3 (1:1000, Cell Signaling Technology, USA), and Smad2/3 (1:1000, Cell Signaling Technology, USA). The results were normalized to tubulin (1:1000 dilution, Abcam).