Fig 1: The regulation of miRNA target genes. (A - F). The protein expression of PDHA2 (A), GPT (B), LDHA (C), OGDH (D), HK2 (E) and PCK1 (F) were analyzed in OIP5-AS1 knocking out (KO) PA-17, AD-23 and CA-34 cells or control wild type (WT) cells by western blotting. The relative protein levels were quantified and normalized to GAPDH. The original gel data were showed in supplementary Fig. 8. (G - O). The antagomirs (Anta) or controls (Ctr) of miR-873 (G, H and I), miR-335 (J, K and L) and miR-323a (M, N and O) were individually transfected into PA-17, AD-23 or CA-34 cells. The RNA expression of PDHA2 (G, J and M), GPT (H, K and N) and LDHA (I, L and O) were measured by qRT-PCR. The relative RNA levels were quantified and normalized to U6 snRNA. Data are means + SD from three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, N.S, no significance
Fig 2: The selective sponging of miRNAs by OIP5-AS1. (A). The scheme for selective sponging of miR-873, miR-335 and miR-323a by OIP5-AS1. (B, C and D). The PDHA2 shRNA (ShRNA) or shRNA control (Sh-Ctr) plasmids were transfected into PA-17 cells and selected by puromycin. The stable clone cells with PDHA2 shRNA were transiently transfected with PDHA2 cDNA (OE) or vector control (V-Ctr) plasmids. The production of pyruvate (B), alanine (C) or lactic acid (D) were measured by Colorimetric Assay kits. (E, F and G). The GPT shRNA (ShRNA) or shRNA control (Sh-Ctr) plasmids were transfected into AD-23 cells and selected by puromycin. The stable clone cells with GPT shRNA were transiently transfected with GPT cDNA (OE) or vector control (V-Ctr) plasmids. The production of pyruvate (E), alanine (F) or lactic acid (G) were measured by Colorimetric Assay kits. (H, I and J). The LDHA shRNA (ShRNA) or shRNA control (Sh-Ctr) plasmids were transfected into CA-34 cells and selected by puromycin. The stable clone cells with LDHA shRNA were transiently transfected with LDHA cDNA (OE) or vector control (V-Ctr) plasmids. The production of pyruvate (H), alanine (I) or lactic acid (J) were measured by Colorimetric Assay kits. Data are means + SD from three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, N.S, no significance
Supplier Page from Sino Biological, Inc. for Human GPT Gene ORF cDNA clone expression plasmid