MACF1 Overexpression and Knockdown Cell Lines

MACF1 overexpression cells were constructed by transfection of plasmid PEGFP‐C1A‐ACF7 as previously described,²¹ (link) with PEGFP‐C1–transfected cells as control. MC3T3‐E1 cells (1 × 10⁷ per well) or primary BMSCs (5 × 10⁶ per well) were electroplated (1800V, 30ms) with the MACF1 overexpression plasmid PEGFP‐C1A‐ACF7 or normal plasmid PEGFP‐C1A by using Neon Transfection System (Invitrogen) according to manufacturer's instructions. After the electroporation, cells were seeded into a 6‐well plate with 10ml α‐MEM and cultured for 6h. Adherent cells were then washed by 2ml α‐MEM two times, and medium was changed to antibiotic‐free culture medium. After culture for 48 hours, medium was changed to the selective growth medium supplemented with 650 μg/mL geneticin (MP Biomedicals, 0215878291) and cells were cultured for two weeks. Screened cells were used as MACF1 overexpression MC3T3‐E1 cells and BMSCs.
Stable MACF1 knockdown MC3T3‐E1 cell line was made by transfection of lentivirus vector carrying shRNA targeting murine MACF1 (NM_001199136.1) or its scramble control as described previously.²³ (link)