Fibronectin

Total protein was obtained from cells using RIPA lysis buffer (Beyotime, Haimen, China) containing 1% protease inhibitor Cocktail (Sigma‐Aldrich, St. Louis, MO). Equal quantities of protein were separated on a sodium dodecyl sulfate‐polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (Bio‐Rad, Hercules, CA). The membrane was blocked at room temperature for 2 h with 5% skimmed milk in Tris‐buffered saline with Tween 20, and then probed overnight at 4°C with primary antibodies against Bcl‐2, caspase‐3, cleaved caspase‐3, GAPDH (dilution 1: 1000; Cell Signaling Technology, Danvers, MA), fibronectin, vimentin (1: 200; Santa Cruz Biotechnology, Santa Cruz, CA), or cytokeratin (1: 1000; Dako, Carpinteria, CA). Subsequently, the membranes were exposed to horseradish peroxidase‐labeled IgG for 2 h, and the bands were visualized using a Bio‐Rad imaging system.

The obtained native submandibular glands and dental pulp tissue were fixed in 4% paraformaldehyde overnight, dehydrated with graded ethanol, embedded in paraffin, and then sectioned at 5 μm. Hematoxylin and eosin (H&E) staining was performed to characterize the ECM histology and immunofluorescence staining was conducted to label some vital ECM proteins of both tissues, which included collagen type I alpha (COL1, mouse monoclonal to collagen I; Santa Cruz, United States), collagen type III alpha 1 (COL3, mouse monoclonal to collagen 3; Santa Cruz), and fibronectin (mouse monoclonal to fibronectin; Santa Cruz). For immunofluorescence, deparaffinized sections were blocked against non-specific binding using goat serum and then incubated with diluted primary antibodies (anti-COL1, 1:200; anti-COL3, 1:200; anti-fibronectin, 1:200) at 4°C overnight. Alexa Fluor 488 goat anti-mouse IgG (ABclonal, United States) was used as a secondary antibody to visualize components specific to the ECM and DAPI was used to label nuclei. The stained sections were observed under a fluorescence microscope (Olympus BX61, cellSens Standard software).

Cells were lysed on ice for 20 min using a lysis buffer and centrifuged at 14,000 ×g for 10 min at 4°C. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were transferred onto nitrocellulose membranes and the membranes were blotted with appropriate primary antibodies at a dilution of 1:1000. The membranes were then treated with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Bound antibodies were detected with Super Signal West Pico PLUS Chemiluminescent Substrate (ECL; Amersham, Arlington Heights, IL, USA) and developed with Kodak X-OMAT films (Kodak, New Haven, CT, USA). Primary antibodies used in this study were COL-1A (ab34710; Abcam, Cambridge, UK), α-SMA (ab5694; Abcam, Cambridge, UK), fibronectin (sc-69681; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), TGF-β (sc-130348; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and GAPDH (ATGEN, Seongnam-si, Gyeonggi-do, South Korea) antibodies.

Recombinant human TGF-β1 and LiCl were purchased from Sigma-Aldrich (St Louis, Mo, USA). The DMEM-F12 medium and fetal bovine serum (FBS), were supplied by Gibco (BRL Grand Island, NY, USA). Akt1, Akt2, Akt3, p-Akt (Thr308), p-Akt (Ser473), GSK3β (glycogen synthase kinase-3β), p-GSK3β, p-Smad3 and E-cadherin were purchased from Cell Signaling Technology (Beverly, MA). Collagen 1, α-SMA and Snail were obtained from Abcam (Cambridge, UK). TGF-β1, β-catenin, fibronectin, Vimentin and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cells were washed with cold PBS, scraped into RIPA buffer and centrifuged. The cell lysates were subjected to 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) hybridization transfer membrane. The primary antibodies used were as follows: HA (Sc-7392, Santa Cruze), CD44 (15675-1-AP, ProteinTech group), Fibronectin (sc-8422, Santa Cruze), Vimentin (5741, Cell Signaling Technology), p21cip1 (sc-6246, Santa Cruze), p27kip2 (sc-1641, Santa Cruze) and β-actin (Sc-47778, Santa Cruze). Secondary staining and detection were carried out in accordance with standard protocols^{16 (link), 64 (link)}.