Bovine serum albumin (bsa)

Total protein of tissues was extracted through the ice-cold radioimmunoprecipitation lysis buffer (RIPA, Thermo Fisher Scientific, 89,900), which contained the protease inhibitor cocktail (Merk, P8340). Subsequently, the extracted proteins were quantified through the BCA Assay Kit (Beyotime, P0010) and boiled for degeneration. Then, we separated the proteins in SDS-PAGE (Yeasen, 20315ES05) and transferred them into the PVDF membrane (Merk, 3,010,040,001). After being blocked into 5% Bovine serum albumin (BSA, Yeasen, 36104ES25), the PVDF membrane was then incubated with primary antibodies: Anti-beta-actin (Proteintech, 20,536, 1:1000), Anti- ZBTB16 antibody (ABclonal, A5863, 1:1000), Anti-SERPINA10 antibody (ABclonal, A7106, 1:1000), and Anti-CD38 antibody (ABclonal, A1680, 1:1000). Stepwise, the membranes were incubated in secondary antibodies: Goat Anti-Mouse IgG (Proteintech, SA00001-1, 1:1000) and Goat Anti-Rabbit IgG (ABclonal, AS014, 1:1000), followed by enhanced chemiluminescence to display bands.

Undifferentiated human ESC HUES8 and iPSC PGP1 were maintained in mTeSR1 (StemCell Technologies, Cat# 85850) on Matrigel-coated cell culture plates at 37 °C with 5% CO₂. For directed differentiation of DE, human PSCs were cultured in DMEM-F12 or DMEM medium supplemented with 0.2% bovine serum albumin (Yeasen Biotechnology, Cat# 36101ES76), 100 ng/mL Activin A (PeproTech, Cat# 120-14P), 2.5 μM CHIR99021 (Selleck, Cat# S2924) for 24 h, then the CHIR99021 was removed from a medium for the following 3–4 days. Next, the differentiation of the pancreatic progenitor cells was performed according to a previous report [47 (link)]. Small molecules used in this study are listed as follows: XCT790 (XCT), 2–5 μM (MCE, Cat# HY-10426); ATN-224 (ATN), 5–10 μM (MCE, Cat# HY-16074); Mdivi-1, 10 μM (MCE, Cat# HY-15886); IACS-010759 (IACS), 0.1–20 nM (Selleck, Cat# S8731); Dynasore (DYNA), 1–3 μM (Selleck, Cat# S8047); FCCP, 1–2 μM (Selleck, Cat# S8276); Oligomycin A (Oligo), 0.1–10 μM (Apexbio, Cat# A5588); Doxycycline (DOX), 100 ng/ml (Sigma, Cat# 324385). Of note, the concentrations of the chemicals we used in this study showed little effect on the survival of cells.

Cells were fixed with 4% paraformaldehyde for 10–15 min at room temperature (RT) followed by washing with 1× PBST (1× PBS + 0.3% Triton X-100 (Vetec)) for three times with 5 min each time at RT. Then, cells were blocked in blocking buffer (1× PBST + 5% bovine serum albumin (Yeasen)) for 0.5–1 h at RT followed by the incubation with the primary antibody (anti-Nanog antibody, 1:500, ab80892, Abcam; anti-Oct4 antibody, 1:500, sc-8629, SANTA CRUZ) overnight at 4 °C. After three-time washing by PBST with 15 min each time at RT, cells were stained with the appropriated secondary antibodies for 1 h at RT. Finally, 10-min incubation with Hoechst (1:5000) was used to stain nuclei.

Tissues and cells were lysed with RIPA buffer containing protease inhibitor (Bimake, #B14002) and phosphatase inhibitor (Bimake, #B15003). Bicinchoninic acid (BCA) protein assay kit (Yeasen, #20201ES90) was used to detect protein concentration. Equal quantity of proteins was separated by SDS‐PAGE and transferred to PVDF membrane. The membrane was blocked with 5% bovine serum albumin (Yeasen, #36101ES80) for 1–3 h at room temperature, and subsequently incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1–3 h at room temperature. Next, the electrochemiluminescence reagents (Yeasen, #36208ES76) were used to detect the antibody signals, and quantitative analysis of the developed results was performed by ImageJ software using vinculin as an internal reference to calculate the relative expression levels of target protein. Primary antibodies of IMPA1(Abcam, #ab184165), Vinculin (Sigma, #V9131), and HA (CST, #3724S) were used in this study. Other antibody information is shown in Table S5.

Dissociated hPSC-derived cardiomyocytes at 20–21 days post differentiation were plated onto Matrigel-coated six-well plates in mature medium, which consists of RPMI 1640 without glucose (Gibco), 500 μg/ml bovine serum albumin (Yeasen), 213 μg/ml l-ascorbic acid 2-phosphate (Sigma-Aldrich) supplemented with 10 mM d-galactose (Sigma-Aldrich), 4 mM l-lactic acid (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 20 μg/ml insulin (Sigma-Aldrich), 1× chemically defined lipid concentrate (Sigma-Aldrich), and 200 ng/ml triiodo-l-thyronine (Sigma-Aldrich). At day 2, the medium was supplemented with 100 ng/ml G418 for another 7 days. From day 9, cells were cultured in mature medium with a medium change every other day.

Frozen skin samples were cut into pieces and lysed with ice-cold radioimmunoprecipitation (RIPA) lysis buffer containing protease inhibitors (Beyotime, China) to obtain tissue homogenates. The protein concentration of each sample was detected using a bicinchoninic acid (BCA) kit (Beyotime, China). After normalization to PBS, the lysates were mixed with loading buffer and incubated for 10 min at 95°C. Approximately 20 µg of protein from each sample was separated by 10% SDS-PAGE and transferred to PVDF membranes (Merck Millipore, USA). After blocking in 5% bovine serum albumin (Yeasen, China) for 1 h, the membranes were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 h. GAPDH and β-tubulin were used as loading controls. Bound antibodies were detected using the ECL Western blot detection system (Merck Millipore, USA) and quantified by ImageJ (National Institutes of Health, USA). The information for the primary antibodies used in the experiment is listed in Supplementary Table S4.