Overexpression lentivirus

UNC5B(399‐945aa)‐3Flag overexpression lentivirus and UNC5B(412‐945aa)‐3Flag overexpression lentivirus were synthesized by Obio Technology Co. Ltd. Tables 1 and 2 indicated the composition reports. Full‐length UNC5B‐Flag overexpression lentivirus was purchased from GenePharm Co. Ltd. The Lipofectamine 3000 Reagent (Life Technologies Co. Ltd.) was used for these plasmids and related transfection.
Antibodies against UNC5B (ab104871, ab54430), caspase‐3 (ab179517), mutant P53 (ab32049) and BCL‐2 (ab32124) were purchased from Abcam. Antibodies against cleaved PARP (D64E10), S6 Ribosomal Protein (5G10), UNC5B (D9M7Z) and beta‐actin (3700S) were obtained from Cell Signaling Technology (CST). Antibodies against Fibronectin, beta‐catenin, vimentin, N‐CA and E‐CA were also obtained from the Epithelial‐Mesenchymal Transition (EMT) Antibody Sampler Kit (9782T, CST). Antibody against FLAG (F1804) was obtained from Sigma‐Aldrich Co. Ltd. Antibody against P53 (21891‐1) was purchased from Proteintech Co. Ltd. UNC5B (ab54430, Abcam) was used for immunofluorescence and flow cytometry. UNC5B (D9M7Z) was used as a mixture reagent in Co‐IP. Universal Magnetic Co‐IP Kit (54002, Active Motif) was purchased from Active Motif Co. Ltd. Fast silver staining kit (P0017S, Beyotime) was purchased from Beyotime Co. Ltd.

The siRNAs were designed and synthesized by GenePharma (Shanghai, China). Three interference sequences were designed for the target gene. The interference efficiency was verified by qPCR and western blotting methods. Two sequences with a knockdown efficiency of >70% were selected for further experiments. Details of the sequences are shown in Supplementary Table 2. Transfection was carried out according to the instructions. After transfection, osteogenic induction medium was added to induce the induction of MSCs into osteoblasts. On the seventh day of osteoinduction, siRNA was transfected again for a second knockdown. The overexpression lentivirus was constructed by OBiO Technology (Shanghai, China). Mut-ALKBH5 contains a mutation of histidine to alanine at position 204. MSCs were incubated with the lentiviruses for 24 h at a multiplicity of infection of 30.
LY294002 and SC79 were purchased from Calbiochem and dissolved into dimethyl sulfoxide. MSCs were treated with 10 μM LY294002 and 4 µg/ml SC79 after siRNA or lentivirus transfection.