Human hepatocytes were purchased from Invitrogen. Sodium oleate was obtained from Sigma-Aldrich and was dissolved in DMEM medium with 1% fatty acid free bovine serum albumin (BSA) (Sigma). Oleate treatment of HepG2 cells was carried out as previously described with minor revision [13 (link),7 (link)]. Specifically, HepG2 cells or Human hepatocytes were plated in 4-well chamber slides with DMEM medium supplemented with 10% FBS (Invitrogen). After 24 h, cells were treated with either control medium (DMEM supplemented with 1% fatty acid free BSA), or medium containing oleate (0.5 mM). The cells were cultured for another 24 h, after which lipid accumulation and miR-206 expression were determined by Oil-Red O staining (Sigma-Aldrich) and qRT-PCR, respectively.
To determine whether PTPN1 mediates the effect of miR-206 on lipogenesis, HepG2 cells cultured in the DMEM containing 0.5 mM oleate were transfected with MC-TTR-miR-206 (200 ng in 4-well chamber slides), or a combination of MC-TTR-miR-206 and PTPN1 Target Protector (TP) (20 nM in 4-well chamber slides). The PTPN1 TP and control TP were designed and generated by Exiqon. Lipofectamine 2000 was used for transfection of MC-TTR-miR-206 and PTPN1 TP. After another 24 h of culture, lipid accumulation was assessed by Oil-Red O staining, microfluorimetry or imaging.
To determine whether PTPN1 mediates the effect of miR-206 on lipogenesis, HepG2 cells cultured in the DMEM containing 0.5 mM oleate were transfected with MC-TTR-miR-206 (200 ng in 4-well chamber slides), or a combination of MC-TTR-miR-206 and PTPN1 Target Protector (TP) (20 nM in 4-well chamber slides). The PTPN1 TP and control TP were designed and generated by Exiqon. Lipofectamine 2000 was used for transfection of MC-TTR-miR-206 and PTPN1 TP. After another 24 h of culture, lipid accumulation was assessed by Oil-Red O staining, microfluorimetry or imaging.