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. Author manuscript; available in PMC: 2009 Oct 9.
Published in final edited form as: Nature. 2009 Feb 15;458(7239):762–765. doi: 10.1038/nature07823

Figure 3.

Figure 3

3a. P493-6 cells were treated with tetracycline (Tet) for different lengths of time to inhibit Myc expression or were treated first with tetracycline for 48h and then washed (Wash) to remove tetracycline with the times after wash out indicated. RNA was then harvested for real-time PCR for GLS1 as described in Methods section. Data are shown as mean ± SD, n = 3 PCR reactions.

3b. Northern analysis of miR-23a and miR-23b expression in P493 cells treated with or without tetracycline for 24h and then transfected with anti-sense miR-23a and miR-23b LNAs or Scrambled control LNA. 48h after transfection, cells were harvested and northern blot assays were performed as described in the Methods section. U6 snRNA probe was used as the loading control.

3c. Chromatin immunoprecipitation (ChIP) assay with P493 cells documents Myc binding to the promoter region of C9orf3, whose transcript is processed to miR-23b. The positions of the amplicons are depicted in the cartoon of the C9orf3 gene below the bar graphs (mean ± SD, n=3) demonstrating the binding of Myc in the amplicon 2 region in a tetracycline dependent manner. Anti-HGF serves as a non-specific antibody control.

3d. Inhibition of GLS-3′-UTR luciferase reporter by miR-23. Upper panel. Glutaminase reporter (wild-type GLS-3′UTR or mutant Mut-GLS-3′-UTR) or control (PGL3) luciferase constructs were transfected into MCF-7 cells with the following oligonucleotides: scrambled control LNA (cont-LNA) nucleotide or antisense (miR-23-LNA). The ratio of normalized reporter to control luciferase activity is shown. Cells were co-transfected either with 100ng reporter vectors and 4ng pSV-Renilla, and further co-transfected with 10nM LNA antisense for miR-23 or control LNA. After 24h, luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). Data shown are luciferase activity (RLU = relative light unit) normalized to control group (mean ± SD, n=4). (*) denotes mean (± SD) that is significantly different (P < 0.05 by t test). Lower panel illustrates miR-23a, miR-23b, GLS-3′UTR and MutGLS-3′UTR sequences.

3e. Analysis of GLS protein levels in P493 and PC3 cells treated with control (Cont-LNA) or antisense miR-23 LNAs (miR-23-LNA). Left panel, P493 cells were treated with or without tetracycline for 24h and then transfected with antisense miR-23a and miR-23b LNAs or Scramble control probe. After 72h, cells were harvested for immunoblot assay with anti-Myc and anti-GLS antibodies. Tubulin serves as a loading control. Right panel, PC3 cells were transfected with siRNA for MYC (siMyc) or control siRNA (siCont). After 24h, cells were transfected with LNA knockdown probes for miR-23a and miR-23b or Scramble control probe. Cells were cultured for 72h and then were harvested for immunoblot assay. Each experiment was repeated twice with a representative experiment shown.