Periostin

Western blot was performed as the previously described [26 (link)]. Briefly, total protein was extracted using RIPA buffer added with PMSF and phosphatase inhibitors. Protein concentration was determined using BCA protein assay (Millipore, Switzerland). Then, 20 μg of protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, USA). After blocked with 5% non-fat milk, the membrane was incubated with primary antibody against PCNA (1:2000), Snail (1:1000), Vimentin (1:1000), E-cadherin (1:1000), N-cadherin (1:1000), integrin β1 (1:1000), Shc (1:1000), phosopho-Shc (1:2000), ERK1/2 (1:1000) or phosopho-ERK1/2 (Thr202/Tyr204) (1:2000) (Cell Signaling Technology, USA), periostin (1:1000), collagen I (1:5000), α-SMA (1:300) or anti-vitamin D receptor antibody (1:1000) (Abcam, USA), GAPDH (1:1000), tubulin (1:1000) or β-actin (1:1000) (Beyotime, China) at 4 °C overnight and the corresponding HRP-conjugated secondary antibody for 1 h at room temperature on the next day. The membrane was developed with enhanced chemiluminescence (ECL; New Cell & Molecular Biotech Co., China).

The cells seeded in the 6-well plates were collected, lysed in 2.5×sodium dodecyl sulfate (SDS) gel loading buffer (30 mM Tris-HCl, pH 6.8, 1% SDS, 0.05% bromphenol blue, 12.5% glycerol, and 2.5% mercaptoethanol) and boiled for 30 min; then, 20 μL of the protein was loaded onto 8% concentrated gel for protein electrophoresis separation. After that, the protein was transferred to the PVDF membrane (Millipore, Boston, MA), which was then blocked using 5% skim milk powder for 1 h at room temperature. Then, the membranes were incubated with primary antibodies specific for Cleaved-caspase 3, p-SMAD2, SAMD2/3(Cell Signaling Technology), EDA-Fibronectin, Periostin, Vimentin, α-SMA, p-Smad3 (Abcam), gapdh (#KC-5G5), and β-actin (#KC-5A08) (KangChen, Shanghai) at 4°C overnight, washed with PBST, and further incubated with a secondary antibody for 1 h. Finally, the immunoreactive protein was detected by a chemiluminescence assay (AI680RGB, GE HealthCare, United States) using the FDbio-Dura ECL kit (FDbio Science Biological Technology Co., LTD, China).

Western blotting was performed as described previously^{46 (link)}. Blots were incubated overnight at 4 °C with polyclonal antibodies to periostin (Abcam, Cambridge, UK), fibronectin (Dako, Glostrup, Denmark), type I collagen (Southern Biotech, Birmingham, AL, USA), or β-actin (Sigma Chemical Co., Perth, Australia). The membranes were washed three times for 10 min in 1 × PBS with 0.1% Tween-20 and incubated in buffer A containing a 1:1000 dilution of horseradish peroxidase-linked goat anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). TINA image software (Raytest, Straubenhardt, Germany) was used to measure the band densities, and the changes in the optical densities of bands from the treated groups relative to control cells or tissues were used for analysis.

To analyze the CLC and HERS cell differentiation in vivo after induction with conditioned medium, cells were seeded on the top surfaces of iTDM and then transplanted in SD rat great omentum. Briefly, iTDMs were first placed in 24-well plate, then seeded with an initial amount of 5 × 10⁵ CLC and HERS cells respectively, and cultured in vitro for 7 days according to Table 1 with changing of medium every 2 days. Afterwards, SEM examination was performed to detect the growth and morphology of cells on iTDMs, and implantation of iTDMs with cells in rat great omentum was carried out under deep anesthesia. iTDM without cell seeding was used as blank control. For each sample, at least three replicates were made. 4 weeks later, implants were harvested from the omentum under deep anesthesia. Samples were then fixed with 4% paraformaldehyde overnight at 4 °C, demineralized with 10% EDTA (pH 8.0) and embedded in paraffin. Sections were made and stained with hematoxylin and eosin (H&E). Immunohistochemical staining was also performed using antibodies for AMBN (Millipore, USA), AMGN (Millipore, USA), BSP (Abcam, UK), COL I (Abcam, UK) and periostin (Abcam, UK) at a dilution of 1:200 according to the manufacturers’ protocol. Histological images were taken under microscope (Olympus, Japan).

For the paraffin section, the Matrigel was broken with a cold KBM (Lonza). The hAOs were fixed in 4% PFA for 30 min and dehydrated with graded ethanol solutions for 30 min. Then, the blocks were embedded into paraffin and made into 4-μm sections. For hematoxylin and eosin (H&E) staining, the sections were rehydrated and then were stained. For immunofluorescence, antigen retrieval was achieved by citrate buffer, pH 6.0 at 121°C or 10 μg/mL proteinase K at 37°C. After cooling down, slides were treated with blocking solution, and the following first antibodies were used: OCT3/4 (Santa Cruz, TX, United States), OTX2 (R&D systems), Brachyury (R&D systems), SOX17 (R&D systems), PCNA (Abcam, CAM, United Kingdom), cleaved caspase-3 (Cell Signaling Technology, MA, United States), E-cad (BD biosciences, CA, United States), cytokeratin 14 (Abcam), p63 (GeneTex, CA, United States), collagen IV (Abcam), PITX2 (Abnova, Taiwan), DLX3 (Invitrogen, OR, United States), amelogenin (AMELX; Santa Cruz), and ameloblastin (AMBN; Biorbyt, UK, dentin sialoprotein (DSP; Santa Cruz), periostin (Abcam), cementum protein (CEMP1; Abcam), and human leukocyte antigen (hLA; Abcam). For visualization, anti-mouse or rabbit IgG conjugated with Alexa Fluor 488 or 555 dye (Invitrogen) was applied and observed under a confocal laser microscope (DMi8; Leica, Germany).