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. 2012 Feb 15;26(4):356–368. doi: 10.1101/gad.182568.111

Figure 6.

Figure 6.

Bladder carcinomas circumvent Pumilio regulation of E2F3. (A) E2F3 DNA, RNA, and protein levels in bladder cancer cells. RT-qPCR analysis of a panel of bladder carcinoma cell lines (T24, SW780, J82, HT1376, and TCCSUP) showing the expression levels of E2F1, E2F3, Pum 1, Pum 2, and select E2F target genes (cdc6, cdc25, and cycA). Black squares represent 0.5–1.0, yellow squares represent 1.1–2.0, light-red squares represent 2.1–3.0, and dark-red squares represent >3. (B) Western blots of Pumilio 2 (Pum2), E2F3, and Tub from nonamplified T24 cells and in amplified TCCSUP cells depleted of Scrambled controls (Scr) or Pumilio 2 (Pum 2) using specific shRNAs. (C) Western blots of HA, E2F3, and Tub from T24 cells transfected with Pum and Nos overexpression constructs. (D) Western blots of HA, E2F3, and Tub from TCCSUP cells transfected with Pum and Nos overexpression constructs. (E) RT-qPCR results of Pumilio transfections from T24 and TCCSUP cells measuring the effect on E2F target gene expression from e2f3, cdc6, cdc25, and cycA. (F) 3′ RACE results from T24 and TCCSUP cells. Comparing E2F3 transcript levels (E2F3), the PRE 1 region in the E2F3 3′ UTR (PRE 1), the 3′ end of the E2F4 3′ UTR (control), and the genomic PRE 1 sequence for E2F3.