P smad1 5

Mouse tissue frozen sections were fixed with 4% paraformaldehyde (10 minutes) and permeabilized with 0.5% Triton X‐100 (10‐15 minutes). To reduce non‐specific background staining, sections were blocked with 5% bovine serum albumin for 1 hour at room temperature. Then, sections were incubated overnight at 4°C with the following primary antibodies: Runx2 (1:100; Proteintech, Cat No. 20700‐1‐AP), P‐Smad1/5 (1:100; Cell Signaling, Cat No. 13820), OPN (1:100; Bioworld Technology, Cat No. BS1264), vimentin (1:100; Abcam, Cat No. 92547) and α‐SMA (1:600; Sigma, c6198). Next, Alexa Fluor 488 goat anti‐rabbit or CyTM3 (Jackson ImmunoResearch) was incubated for 1 hour at room temperature. DAPI staining was used to observe the nucleus. Then, fluorescent images were obtained using confocal laser scanning microscope (Carl Zeiss).

Protein extracts were prepared with RIPA buffer, and equal amounts of protein were resolved by SDS-PAGE and then transferred to nitrocellulose membranes that were probed with antibodies against pSmad2 (1/200 dilution, Cell Signaling), total Smad (1/200 dilution, Cell Signaling), pSmad 1/5 (1/200 dilution, Cell Signaling), total Smad 1/5 (1/200 dilution, Cell Signaling) and a β-actin-HRP antibody (1:50,000 dilution; Sigma) used as a loading control. Densitometry was performed using ImageJ software and at least three independent experiments were performed for each condition.

Liver tissue samples were prepared with RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, Heidelberg, Germany). Extracted proteins (40 μg/lane) were separated by electrophoresis using 4%–10% bis-tris gels and nitrocellulose membranes (GE Healthcare, Freiburg, Germany). Membranes were incubated with antibodies directed against phosphorylated STAT3 (at tyrosine705 (P-STAT3, Cat. No. 9145 L, Cell Signalling Technology)), STAT3 (Cat. No. 4904, Cell Signalling Technology), phosphorylated SMAD 1/5/8 (P-SMAD 1/5, Cat. No. 9516S, Cell Signalling Technology), SMAD1 (Cat. No. 6944, Cell Signalling Technology), Ferroportin (Cat. No. MTP11-A, Alpha Diagnostics) and α-tubulin (Cat. No. T6074, Sigma-Aldrich). Washed membranes were incubated with horseradish peroxidase–linked anti-rabbit or anti-mouse IgG (New England Biolabs, Frankfurt, Germany). Membranes were incubated with ECL-Plus (Bio-Rad, Munich, Germany), and chemiluminescence was detected with a ChemiDoc™ XRS+ (Bio-Rad, Munich, Germany). Densitometrical analysis was performed with Image Lab™ (Bio-Rad, Munich Germany).

The cells were washed two times with cold PBS, and the total protein was extracted using an RIPA lysis buffer. The protein concentration was quantified using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), and proteins were separated via 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The transfer membranes were blocked with 5% fat-free milk at room temperature for 1 h and then incubated with primary antibodies against BMP2 (1 : 1000; Abcam, Cambridge, UK), ERK1/2, p-ERK1/2, JNK, p-JNK, p38, p-p38, IκBα, p-IκBα, p65, p-p65, p-Smad1/5, Smad1/5 and Smad4 (1 : 1000; Cell Signaling Technology) at 4 °C overnight. After three washes, the membranes were incubated with appropriate secondary antibodies that were conjugated with IRDye 800CW at room temperature for 1 h. Immunoreactive bands were detected using the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). The GAPDH antibody (1 : 1000; Cell Signaling Technology) was used as a control. The intensity of each band was analyzed using the ImageJ software (Bethesda, MD, USA).

DPSCs were lysed and assayed with a BCA protein assay kit (Beyotime). Samples containing 15–30 μg of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins were then transferred to polyvinylidene fluoride membranes (Millipore). The membranes containing the transferred proteins were blocked with 5% bovine serum albumin and reacted with the primary antibody overnight at 4℃. The membranes were then labeled with corresponding secondary antibody of horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG for 1 h at room temperature before visualization with SuperSignal enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, US). Primary and secondary antibodies against the following proteins were used in this study: METTL3 (96391, 1:1000); METTL14 (51104, 1:1000); WTAP (56501, 1:1000); p-Smad1/5 (9516, 1:1000) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). NOG (sc-293439, 1:1000); RUNX Family Transcription Factor 2 (RUNX2, sc-390351, 1:1000); Dentin Sialophosphoprotein (DSPP, sc-73632, 1:1000); Smad1/2/3 (sc-7960, 1:1000); p-Smad3 (sc-517575, 1:1000) were obtained from Santa Cruz Biotechnology. GAPDH (60004-1-Ig, 1:3000); goat anti-mouse IgG (SA00001-1, 1:3000) and goat anti-rabbit (SA00001-2, 1:3000) were purchased from ProteinTech Group (ProteinTech, Wuhan, China).