Geneticin

To generate the P_GIL1-GIL1-GFP fusion construct, the GIL1 gene with its 1.5-Kb upstream promoter sequence were amplified with primer GIL1/F and GIL1/R (Table S1) and cloned into pFL2 [47 (link)] by the yeast in vivo homologous recombination approach [48 (link)]. The P_RP27-GIL1-GFP construct was generated with a similar approach by cloning the PCR product amplified with primers GIL1F/RP27 and GIL1R/RP27 (Table S1) into pFL2. The resulting GIL1-GFP construct was transformed into protoplasts of the wild type PH-1. Geneticin (MP Biochemicals, Santa Ana, CA, USA)-resistant transformants harboring the transforming GFP fusion constructs were identified by PCR and examined for GFP signals using a confocal microscopy with Nikon Tie system (Nikon, Japan).

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)

MACF1 overexpressed preosteoblast was constructed as described previously (Yin et al., 2018 (link)). Briefly, MC3T3-E1 cells (1×10⁷ per well) were electroplated (1800V and 30 ms) with the MACF1 overexpression plasmid pEGFP-C1A-ACF7 or control plasmid pEGFP-C1A using Neon Transfection System (Invitrogen) following the manufacturer's instructions. After the electroporation, cells were seeded into a six-well plate with 10 ml α-MEM and cultured for 6 h. Adherent cells were then washed with 2 ml α-MEM twice and the medium was changed to antibiotic-free growth medium (α-MEM with 10% FBS, and 1% L-glutamine). After being cultured for 48 h, the medium was changed to selective growth medium supplemented with 650 μg/ml Geneticin (MP Biomedicals, Santa Ana, CA, USA) and cells were continuously cultured for 2 weeks. MACF1 overexpression preosteoblast was monoclonalized by limited dilution method.
Stable MACF1-knockdown MC3T3-E1 cell line was established by transfection of lentivirus vector carrying shRNA targeting murine MACF1 (NM_001199136.1) or its scramble control as described previously (Hu et al., 2015 (link), 2018 (link)).