Hitransg a reagent

For cell transfection, wild-type and S34F mutant U2AF1 were introduced into the GV492 (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin) lentiviral vectors with the open reading frame (ORF) clone of human U2AF1 (ref. ID NM_006758) and a carboxy-terminal FLAG-tag. Based on direct sequencing, this study identified the constructs overall. Plasmids were transfected with HitransG A reagent (GeneChem, Shanghai, China) at a corresponding MOI reaching 20 in accordance with the vendor’s directions when the cells reached 80% confluence. Cell samples were harvested 72 h after transfection, and in this study, stably transfected clones were transfected with 4 μg/ml puromycin and counted to determine the efficiency of green fluorescence protein (GFP). Normally, more than 80% of the cells were GFP-positive cells. By performing the western blotting assay and the flow cytometry analysis, we confirmed the transgene expression. Untransfected cells acted as negative controls. FoxO3a-shRNA (shRNA-FOXO3a) contained specific sequences targeting the FOXO3a sequence (5’-TTCCAAACTTGTACGCAGTTT-3’; 5’-AAGCTTGTCACTCCTGTTAAT-3’), and a control shRNA acted as a silencing negative control, as supplied by GeneChem Company (Shanghai, China). Cells were transfected following the manufacturer’s directions.

Lentiviruses used in this study were purchased from Genechem (Shanghai, China). Lentiviral infection was performed according to manufacturers’ instructions. In brief, RBE and HUCCT1 cells were seeded in 6-well plates in 2 mL complete medium and transduced by lentivirus at a multiplicity of infection of 15 to 20 in the presence of the HitransG A reagent (Genechem). We then used 5 g/mL puromycin (Selleck, Shanghai, China) for the selection of transduced cells. After continuous selection for approximately two weeks, the surviving cell colonies were expanded. The efficacy of overexpression and knockdown was verified by Western blotting.

After the microglia confluence reached more than 30%, microglia were incubated with shRNA-ARAP3 (10⁷ TU/mL, Genechem, Shanghai, China) loaded in lentivirus (LV) supplemented 1× HitransG A reagent (Genechem) in complete medium for 12 h. The complete medium then was cultured with cells for 60 h.