Xbp1s

GRP78, CHOP, PERK, p-PERK, eIF2α, BCL-2, Bax, cytochrome c, caspase-3, p-LKB1, LKB1, p-AMPK, AMPK, PARP and p- eIF2α antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and IRE1, p-IRE1, XBP1s,Fas, FasL, FADD, caspase-8 and caspase-12 antibodies were purchased from Abcam (Cambridge, UK) while β-actin, PGC1-α and ATF6 antibodies were purchased from Santa Cruz (CA, USA). Unless otherwise noted, all chemical reagents were purchased from Sigma (St. Louis, MO, U.S.A).

TMF has been isolated from the leaves of M. exotica and purified by repeated column chromatography and recrystallization in acetoacetate. The purity was found to be more than 98%. Fetal bovine serum (FBS), Dulbecco's modified Eagle's minimum essential medium (DMEM) (low glucose), penicillin, and streptomycin were purchased from Gibco (Life Technologies, NY, USA). GRP78, PERK, p-PERK, eIF2α, p-eIF2α, ATF6, IRE1α, XBP1s, CHOP, JNK, GSK-3β, Bax, Bak, and GAPDH monoclonal antibodies and the peroxidase-conjugated secondary antibody were obtained from Abcam (Cambridge, UK).

Protein extracts were resolved through 8% SDS–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (BioRad), probed with an antibody directed against MHC-I (Cat. No. sc-32235, Santa Cruz, 1:200), IRE1α (Cat. No. 3294, Cell Signaling Technology, 1:1000, pIRE1α (Cat. No. NB100-2323, Novus Biologicals, 1:1000), XBP-1 (Cat. No. ab37151, Abcam, 1:200), XBP-1s (Cat. No. 619502, BioLegend, 1:500), GRP78 (Cat. No. 3177, Cell Signaling Technology, 1:1000), CHOP (Cat. No. 2895, Cell Signaling Technology, 1:1000), DRP-1 (Cat. No. 14647, Cell Signaling Technology, 1:1000), TPP2 (Cat. No. 66017-1-Ig, Proteintech, 1:1000), GAPDH (Cat. No. 60004-1-Ig, Proteintech, 1:20,000), β-actin (Cat. No.20536-1-AP, Proteintech, 1:2000), then with a peroxidase-conjugated secondary antibody (Cat. No. SA00001-1, SA00001-2, Proteintech,1:1000). and visualized by chemiluminescence (GE).

Western blotting was applied to analyze Runx2, osteocalcin, GRP78, CHOP, XBP1s, β-actin, phosphorylated and total IRE1α, PERK. If not mentioned, the antibodies were bought from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) except GRP78, CHOP, and XBP1s (Abcam; Cat. ab212054, ab11419, ab241571). Porcine VICs were dissolved in commercial radio immunoprecipitation assay lysis buffer (Beyotime, Jiangsu, China, Cat.P0013B) in accordance with the manufacturer’s instructions. The protein extracted from cytoplasm was then broke down on 4–20% SDS-PAGE gels and moved to polyvinylidene difluoride membrane [5 (link)]. After blocking with 5% (wt/vol) skim milk for 1 h at indoor temperature, membranes were incubated with primary antibodies at 4℃ overnight, followed by incubation with the appropriate HRP-conjugated secondary antibody for 1 h [13 (link)]. Membranes were exploited with enhanced chemiluminescence system; β-actin was analyzed for normalization of protein loading in cytosol. In phosphorylation assay, general levels of IRE1α and PERK were used for normalization. Band density was analyzed with Quantity One Software (Bio-Rad, Hercules, CA).

The cells were collected, the total protein was prepared in RIPA buffer (medium, Beyotime, China), and the cytoplasmic proteins were obtained using the Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime, China). Western blotting was performed as described previously [20 (link)]. The antibodies used for western blotting were PERK, XBP1s and CLO1A1 (Abcam, USA), GRP78 and MMP13 (Santa Cruz, CA, USA), and β-actin (Beyotime Institute of Biotechnology Jiangsu, China).

After formalin fixation and paraffin embedding, the tissues were deparaffinised in xylene and rehydrated in graded ethanol. For immunohistochemistry, the slides were immersed in 3% hydrogen peroxide and incubated with primary Abs overnight. The Abs included XBP1s (Abcam, ab37152, 1:20 dilution), AREG (Abcam, ab234750, 1:50 dilution), bFGF (Abcam, ab92337, 1:200 dilution), IκBα (Cell Signalling Technology, #4814, 1:100 dilution) and KAT6B (Abcam, ab246879, 1:200 dilution). Subsequently, the slides were incubated with HRP‐conjugated secondary Abs (Santa Cruz Biotechnology, sc‐2357, sc‐516102). Finally, after the application of DAB chromogen, tissue sections were stained with haematoxylin. For in situ hybridisation (ISH) analysis, fluorescence ISH (FISH) was performed in tissue sections using an ISH Detection kit (Boster). lncRNAs XLOC_003973 and XLOC_010383 and the miR‐22‐3p detection probe (Boster) were used, and the total staining processes were performed by following the manufacturer's protocol. Images were captured with Panoramic 250 FLASH (3DHistech) and assessed with Case Viewer software.

Equal amounts of protein were separated via electrophoresis on NuPAGE 4-12% gradient precast polyacrylamide gels (Life Technologies, Carlsbad, CA). Proteins were transferred onto Hybond-P PVDF membranes (Millipore, Billerica, MA) and blocked for 1 h at room temperature in 5% non-fat milk prepared in Tris Buffered Saline with 0.1%Tween-20 (vol/vol). Membranes were incubated with primary antibodies overnight at 4 °C. Primary antibody concentrations were as follows: GRP78 (1:1000; Cat#3177), Cl CASP3 (1:250; Cat#9664), GADD153 (1:500; Cat#5554), Cl PARP (1:1000; Cat#9541), ATF4 (1:500; Cat#11815), p-eIF2α (1:500; Cat#3398), NF-κB (1:1000; Cat#8242), XIAP (1:250; Cat#2042), and PDI (1:5000; Cat#3501) were obtained from Cell Signaling Technologies; XBP1s (1:500; Cat#37152) was obtained from Abcam; ATF6 (1:500; Cat#24169-1-AP) was obtained from Proteintech; MCL1 (1:1000; Cat#sc-819) was obtained from Santa Cruz Biotechnology; and NCOA3 (1:5000; Cat#PA1-845) was obtained from ThermoScientific. Following primary incubation, immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced-chemiluminescence detection system (ThermoScientific, Rockford, IL). Immunoreactive band density was then quantified using ImageJ software.