Differential regulation of MNSFβ gene expression in HCC-94 cells by RNA interference and overexpression. (a) Identification of an effective RNA interference sequence targeting the MNSFβ mRNA by RT-PCR analysis. Total RNA were extracted from the transfected cells for RT-PCR analysis, and P1 was demonstrated to be the most effective RNA interference sequence of MNSFβ mRNA. (b) RT-PCR detection of MNSFβ mRNA expression levels in HCC-94 cells transfected with different plasmids. Lane 1: total RNA extracted from HCC-94 cells without plasmid transfection, Lane 2: total RNA extracted from HCC-94 cells transfected with pDsRed-N1/MNSFβ, Lane 3: total RNA extracted from HCC-94 cells transfected with pDC316-EGFP-U6/MNSFβ-P1, Lane 4: total RNA extracted from HCC-94 cells transfected with pDsRed-N1 (as the overexpression control), Lane 5: total RNA extracted from HCC-94 cells transfected by pDC316-EGFP-U6/MNSFβ-NC (as the siRNA interference control), and Lane 6: total RNA extracted from human fibroblasts (as negative control). (c) Western blotting analysis of MNSFβ protein in culture supernatants of HCC-94 cells transfected with different plasmids. Lane 1: supernatants of human fibroblasts (as negative control), Lane 2: supernatants of HCC-94 cells transfected with pDC316-EGFP-U6/MNSFβ-NC, Lane 3: supernatants of HCC-94 cells transfected with pDC316-EGFP-U6/MNSFβ-P1, Lane 4: supernatants of HCC-94 cells without plasmid transfection, and Lane 5: supernatants of HCC-94 cells transfected with pDsRed-N1/MNSF. β-actin (lower panel of (b, c)) was used as reference both in the RT-PCR and western blotting analysis.