Phosphorylated p65

Lipopolysaccharides (LPS) (Escherichia coli O111: B4) were purchased from Sigma-Aldrich (United States). An adipogenic differentiation medium of human mesenchymal stem cells was obtained from Procell Life Science &Technology Co., Ltd., (Wuhan, China). Antibodies for flow cytometry—CD73, CD90, CD105, and CD45—were purchased from Elabscience (Wuhan, China). Antibodies for Western blot were procured from suppliers as follows: CD9, CD81, and GAPDH were acquired from Elabscience (Wuhan, China); ALIX and HSP70 were obtained from Abcam (Cambridge, United Kingdom); Wnt5a, p65, and phosphorylated-p65 were bought from Cell Signaling Technology (United States); anti-rabbit HRP-conjugated antibody was from Absin (China); PKH67 was from Fluorescence (China); and 4′,6-diamidino-2-phenylindole (DAPI) were from Solarbio (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-1β were obtained from Absin (China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-10 were obtained from Elabscience (China).

Total proteins were extracted using RIPA lysis buffer, and samples containing equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. The following antibodies were used for Western blot analysis: IκBα (Cell Signaling Technology, Danvers, MA, USA), phosphorylated IκBα (Cell Signaling Technology), p65 (Cell Signaling Technology), phosphorylated p65 (Cell Signaling Technology), phosphorylated p38 (Cell Signaling Technology), p38 (Cell Signaling Technology), phosphorylated Akt (Cell Signaling Technology), Akt (Cell Signaling Technology), phosphorylated ERK1/2 (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), FHL2 (Abcam, Cambridge, UK), LC3B (Cell Signaling Technology), Beclin-1 (Cell Signaling Technology), β-actin (CWBiotech, Shanghai, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; CWBiotech, Shanghai, China).

Cells were lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) and SDS-PAGE was performed. Antibodies used in this study are listed as follows: Human, CREB (9197, Cell Signaling Technology, Danvers, MA), phosphorylated CREB (9198, Cell Signaling Technology), JNK (9252, Cell Signaling Technology), phosphorylated JNK (9255, Cell Signaling Technology), Caspase-12 (2202, Cell Signaling Technology), GAPDH (AP0060, Bioworld, St. Louis Park, MN), PKAcα (4782s, Cell Signaling Technology), Ki-67 (9449s, Cell Signaling Technology), Cleaved-Caspase-3 (9664s, Cell Signaling Technology), p65 (8242, Cell Signaling Technology), phosphorylated p65 (3033, Cell Signaling Technology), M1 E1 and NS3 (produced by Beijing Protein Innovation).

Cells were lysed in RIPA buffer (Sigma) supplemented with 1% protease and phosphatase inhibitors (Roche) for 30 min on ice. The lysates were then centrifuged at 12,000 rpm for 30 min at 4 °C, and the proteins in the supernatant were quantified using a BCA assay kit (Sigma) according to the manufacturer’s instructions. Equal amounts of protein diluted in sodium dodecyl sulfate (SDS) loading buffer, separated by 10% SDS–polyacrylamide gel electrophoresis, and electrotransferred onto polyvinylidene fluoride membranes (EMD Millipore Billerica). The membranes were blocked with 5% skim milk in TBST at room temperature for 60 min and incubated overnight at 4 °C with primary antibodies against GAPDH, TRAP, CTSK, NFATc1, p65, phosphorylated p65, phosphorylated p38, p38, ERK-1/2, phosphorylated ERK-1/2, JNK, and phosphorylated JNK (each diluted 1:1000; Cell Signaling Technology). Afterwards, the membranes were washed three times with TBST and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted 1:3000; Santa Cruz Biotechnology) at room temperature for 60 min. The membranes were washed three times with TBST, and signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore).

Cells were collected and lysed with lysis buffer (Cell Signaling Technology, Cat# 9803S). Cell lysates were subjected to SDS-PAGE assay, transferred to a Polyvinylidene fluoride (PVDF) membrane (Millipore, Cat# IPVH00010), and immunoblotted with antibodies against GAPDH (Cat# 2118), RUNX2 (Cat# 12556), p65 (Cat# 8242), phosphorylated p65 (Cat# 3033), phosphorylated smad1/5/9 (Cat# 13820) and NFATc1 (Cat# 8032) (Cell Signaling Technology).

Nuclear protein was extracted from cells and mouse lung tissue using a Nuclear Cytoplasm Extraction Kit (KeyGen Biotech, Nanjing, China). Aliquots of 30 μg of protein lysate were separated using SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Bedford, MA). After blocking with 5% skim milk at room temperature for 1 h the membrane was incubated overnight with primary antibody at 4°C, and then incubated with secondary antibody at room temperature for 1 h. Finally, ECL reagent (Beyotime, Nanjing, China) was used for chemiluminescent detection. Primary antibodies used for immunoblot analysis included phosphorylated p65 (Cell Signaling Technology, Danvers, United States) and histone H3 (Proteintech, Rosemont, United States).

Total protein was extracted with the RIPA lysis buffer (Beyotime Biotechnology) containing 1% phenylmethane sulfonyl fluoride (PMSF). Proteins from the cytoplasm and nuclear fraction were separately extracted using a nuclear protein extraction kit (R0050; Solarbio). A BCA Protein Assay Kit (23227; Solarbio) was used to quantify protein concentrations. Subsequently, proteins were denatured by boiling, separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene fluoride (PVDF) membrane. This was immersed in non-fat milk powder (5%) for 2 h at room temperature to block non-specific binding sites on the membrane. Next, the PVDF membrane was incubated with primary antibodies against p65 (1:1000; #8242; Cell Signaling Technology), phosphorylated-p65 (1:1000; #3033; Cell Signaling Technology), IKBα (1:1000; #4812; Cell Signaling Technology), p-NF-KB inhibitor alpha (p-IKBα; 1:1000; #2859; Cell Signaling Technology), β-actin (1:1000; 20536-1-AP; Proteintech), and Histone H3 (1:1000; #9715; Cell Signaling Technology) at 4°C overnight, followed by the secondary goat anti-mouse or rabbit HRP–linked antibodies. Target bands were visualized by enhanced chemiluminescence reagent (Thermo Fisher Scientific, United States) and analyzed using Image J.