Opi mem

For transfection of siRNA, cells were plated at a density of 1×10⁵ cells per well in 6-well plates, cell density was ~60–80% per well. Then cells were transfected with negative control (NC), or specific siRNAs for HDAC7, NudCD1 and GGH (100 nM; Shanghai GenePharma Co., Ltd.), respectively, using Lipofectamine^® RNAiMAX transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). For transfection, 125 µl opi-MEM (Thermo Fisher Scientific) was mixed with 25 pmol siRNA and another 125 µl opi-MEM was mixed with 7.5 µl RNAiMAX and the mixture was incubated at room temperature for 15 min respectively. Then the iMAX solution was added to the siRNA solution, mixed well and incubated at room temperature for another 15 min. Finally, the 250 µl mixture was added into indicated wells. After transfection of 24 h, the culture medium was aspirated and replaced with new complete medium for another 24 h.
The siRNA sequences were: HDAC7: 5′-ACUUCUUGGGCUUAUAGCGCA-3′, 5′-CGCUAUAAGCCCAAGAAGUCC-3′; NUDCD1: 5′-AGUGUAUAUUGAUCAUCUCGA-3′, 5′-GAGAUGAUCAAUAUACACUGG-3′; GGH: 5′-UUUUUGCAUUAAUAUUCCGAU-3′, 5′-CGGAAUAUUAAUGCAAAAAUG-3′; NC: 5′-UUCUCCGAACGUGUGACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′.