Tcf 4

We extracted total proteins from cells, animal tissues and human tissue specimens, respectively, and determined protein concentrations using a BCA kit (Beyoncé, Shanghai, China). Total proteins were solubilized using RIPA lysis buffer (Beyoncé, Shanghai, China) containing various protease inhibitors. The total protein was separated by SDS-PAGE and was transferred to PVDF membranes (Millipore, Burlington, MA, USA) by electroblotting. The membranes were blocked with 5% skimmed milk for 1 h at room temperature. The membranes were incubated overnight at 4 °C with specific primary antibodies. The membrane then was incubated with the secondary antibody at room temperature for 2 h and was detected with an ultrasensitive ECL chemiluminescence kit (New Saimei, Suzhou, China) and imaged with a chemiluminescence imaging system (Shanghai Tennant, Shanghai, China). The primary antibodies were as follows: Cyclin A2 (#18202-1-AP; Proteintech, Wuhan, China), Cyclin D1 (#60186-1-Ig; Proteintech, Wuhan, China), β-catenin (#51067-2-AP; Proteintech, Wuhan, China), TCF4 (#GR3295722-2; Proteintech, Kunming, China), E-cadherin (#20874-1-AP; Proteintech, Wuhan, China), Anti-mouse IgG (H+L) (#14709; CST, Parsons, KS, USA), Anti-rabbit IgG (H+L) (#14708; CST, Parsons, KS, USA), GAPDH mouse-derived (#60004-1-Ig; Proteintech, Wuhan, China), and GAPDH rabbit source (#10494-1-AP; Proteintech, Wuhan, China).

Tissues dissected from the mouse brains and cultured cells were lysed in RIPA buffer [20 mM Tris-HCl (pH = 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 1 mM PMSF, 10 mg/ml aprotinin, 1 mg/ml pepstatin A and 1 mg/ml leupeptin]. The equivalent denatured samples were subjected to SDS-PAGE, transferred and probed with antibodies against Tcf4 (1:1,000, proteintech), GAPDH (1:4,000, Abcam). Bmp7 (1:1000, Proteintech), Smad1 (1:1000, Proteintech), phosphorylated Smad1 (1:1000, Abcam), Neurogenin2 (1:1000, Abcam), and FLAG (1:1000, sigma).

Bicinchoninic acid method was used to measure the protein concentration of each sample. An amount of protein lysate (20 μg) was separated on 10% SDS‐PAGE gels and transferred to PVDF membranes (Merck, USA). The membranes were then incubated with skimmed milk at 25°C for 1 h and then incubated with CD9 (20597‐1‐AP; Proteintech, China), TSG101(28283‐1‐AP; Proteintech, China), ALIX (12422‐1‐AP; Proteintech, China), β‐catenin (51067‐2‐AP; Proteintech, China), AXIN2 (20540‐1‐AP; Proteintech, China), TCF4 (22337‐1‐AP; Proteintech, China), LEF1 (14972‐1‐AP; Proteintech, China) and β‐actin (20536‐1‐AP; Proteintech, China) overnight at 4°C. The membranes were washed by TBST and incubated with secondary antibodies at 25°C for 2 h. An enhanced chemiluminescence reagent (Boster, Wuhan, China) was used to detect the protein bands. Relative expression of proteins in cells were calculated using the internal control as β‐actin. The experiments were performed at least in triplicate and repeated at least three times, and the data are representative of replicate experiments.

Oridonin (C₂₀H₂₈O₆, CAS No. 28957–04-2, purity: 98%) was purchased from Yuanye Bio-Technology Co. (Shanghai, China). Oridonin was dissolved in dimethyl sulfoxide (DMSO, Sigma, D2650) and kept at 4 °C. In Fig. 1A, the chemical structure is depicted. LF3 (CAS: 664969–54-4) was purchased from Selleck (https://www.selleck.cn/). Z-VAD-FMK (Z-VAD, HY-16658B), Chloroquine (CQ, HY-17589A), and 4-Phenylbutyric acid (4-PBA, HY-A0281) were purchased from MCE (https://www.medchemexpress.cn/). Primary antibodies: ATF4 (Proteintech, 10,835–1-AP), CHOP (Proteintech, 15,204–1-AP), TCF4 (Proteintech, 22,337–1-AP), CFTR (Proteintech, 20,738–1-AP), FYN (Proteintech, 66,606–1-Ig), YOD1 (ABclonal, A13270), IRE1α (Cell Signaling Technology, Cat# 3294), p-IRE1α (Novus Biologicals, NB100-2323), PERK (Proteintech, 24,390–1-AP), p-PERK (ABclonal, AP0886), TP53 (Proteintech, 60,283–2-Ig), Wnt (Abcam, Ab15251), β-catenin (Abcam, Ab16051), β-actin (Proteintech, 66,009–1-Ig). Secondary antibodies: HRP enzyme-labeled goat anti-rabbit IgG (Servicebio, GB23303) and HRP enzyme-labeled goat anti-mouse IgG (Servicebio, GB23301).