Pvdf membrane

A total of 7 days after transfection, iPSC-MSC-GFP and iPSC-MSC-DLX3 were rinsed with PBS and lysed in 0.1 ml RIPA buffer containing 10 mg/ml proteinase inhibitor PMSF (Invitrogen; Thermo Fisher Scientific, Inc.) on ice for 30 min. The lysed cells were centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was collected. During electrophoresis, 20 µg target total protein/lane was separated via SDS-PAGE on a 10% gel (Beyotime Institute of Biotechnology), which were subsequently transferred to PVDF membranes (Thermo Fisher Scientific, Inc.) at 200 mA for 2 h. PVDF membranes were blocked using 5% non-fat milk with TBS with 0.1% Tween-20 at room temperature for 2 h, and then incubated with the primary antibodies at 4°C overnight. The primary antibodies used were as follows: DLX3 (1:500; cat. no. ab64953; Abcam), ALP (1:500; cat. no. ab83259; Abcam), OPN (1:500; cat. no. ab8448; Abcam), OCN (1:500; cat. no. ab93876; Abcam), COL-1 (1:500; cat. no. ab34710; Abcam) and GAPDH (1:2,500; cat. no. ab9485; Abcam). Then, PVDF membranes were incubated with a secondary antibody (1:2,500; cat. no. ab97051; Abcam) at 37°C for 2 h. GAPDH was used as the control. The blotting results were visualized with chemiluminescent western blotting detection reagents (Pierce; Thermo Fisher Scientific, Inc.) and measured by Image-Pro Plus version 6.0 (Media Cybernetics, Inc.).

Caski cells were lysed in Protein Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer's instructions. Protein concentration was determined using the BCA Protein assay kit (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. For each well, protein (60 µg) was separated using 10% SDS-PAGE and then transferred to a PVDF membrane (Thermo Fisher Scientific, Inc.). Following 3 h incubation at room temperature in TBST containing 5% nonfat dried milk (Yili Group, Beijing, China), the PVDF membrane was incubated with primary antibodies against IGF2BP1 (cat. no. ab82968) and GAPDH (cat. no. ab9485)(1:100; both from Abcam, Cambridge, MA, USA) at room temperature for a further 3 h and then washed with TBST three times. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. ab97051, 1:5,000; Abcam) for 40 min at room temperature. The membrane was then washed three times with TBST and the immune complexes on the PVDF membrane were detected using the ECL Western Blotting kit (Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. ImageJ software v.1.48 (National Institutes of Health, Bethesda, MD, USA) was used to analyze relative protein expression, which was presented as the density ratio versus GAPDH.

OC cells were, respectively, lysed in cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific). Then, the protein concentration was determined using the Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. Proteins were separated using 12% sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) and then shifted to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific). The PVDF membrane was sealed with 5% nonfat dried milk in phosphate-buffered saline (PBS; Thermo Fisher Scientific) for 2 h under room temperature. Subsequently, the membrane was incubated with primary antibodies for 12 h, followed by the incubation with horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, UK) for 1 h. The protein band was revealed using the Enhanced Chemiluminescence Western Blotting Kit (Thermo Fisher Scientific). Protein expression was determined using Image-Pro Plus software 6.0 (Media Cybernetics, Rockville, MD, USA), and GAPDH was used as the internal reference. The primary antibodies were the following: anti-Fus (ab70381), anti-p62 (ab91526), anti-cleaved PARP (ab32561), and anti-LC3B (ab48394) from Abcam (Cambridge, UK) and anti-LC3II (cat. no. 4108) and anti-GAPDH (cat. no. 5174) from Cell Signaling Technology (Danvers, MA, USA).