Sictl

siRNA for REST (siREST) (EMU029201), siRNA for Sp1 (siSp1) (EMU061231) and siRNA for KMT2A (siKMT2A) (EMU180041) were purchased from Sigma-Aldrich, whereas the negative control (siCTL) (001910-10-05) was purchased from Dharmacon. NSC34 at 60% confluence were transfected with siRNAs (25 nM) by using Lipofectamine 2000 in Optimem medium, according to the protocol by the manufacturer (Invitrogen Srl, S. Giuliano Milanese Italy). For constructs transfection in NSC34 cells have been used the following plasmids: (1) pcDNA3.1, (2) pF151 pcDNA3.1(+)SOD1WT and (3) pF155 pcDNA3.1(+)SOD1-G93A, that were a gift from Elizabeth Fisher (Addgene plasmid # 26397 and # 26401) (Stevens et al., 2010 (link)). The amount of DNA constructs used for transient transfection (60% confluence) was: 500 ng DNA in 24-multiwell plates for MTT and LDH assays, 10 μg of DNA in 100 mm dishes for quantitative real time PCR (Q-PCR), Western blot and ChIP analysis. 24 h after transfection low serum medium containing vehicle or 100 nM MeHg was added. Transfection efficiency was evaluated by western blot analysis to demonstrate the presence of the human SOD1 protein and by Q-PCR to assess the levels of REST, Sp1 and KMT2A mRNAs after siRNAs treatment (Chong et al., 2021 (link)).

SMCs were transfected with small interfering RNA targeting rat TLR2 (siTlr2; final concentration, 25 nM; siGenome SmartPool; Dharmacon, Lafayette, CO) or non-targeting control small interfering RNA (siCtl; Dharmacon). Cells at 70% confluence were washed twice with HBSS, DMEM without antibiotics containing 10% fetal bovine serum was added and transfection was performed using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After 24 hours, media were removed, cells were washed twice with HBSS and DMEM with 10% fetal bovine serum was added for an additional 24 hours, at which time, media were removed and the cells were cultured under experimental conditions as indicated.