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. Author manuscript; available in PMC: 2013 Aug 22.
Published in final edited form as: Cancer Cell. 2012 Jul 10;22(1):51–65. doi: 10.1016/j.ccr.2012.05.019

Figure 2. Reducing Pol I Transcription Rates in Eμ-MycLymphoma Cells Using shRNA-mirs Against UBF or RRN3 Strongly Activates Programmed Cell Death.

Figure 2

(A) Western blot analysis of UBF in cultured in Eμ-Myc lymphoma cells transduced with one of two independent shRNA-mirs targeting UBF (shUBF-1 and -2) or empty vector (LMS), and in B220+ splenic B cells isolated from 4- to 6-week-old Eμ-Myc mice (+TG) and their respective wild-type littermates (WT). Relative Ubf mRNA expression determined by qRT-PCR (LMS versus shUBF-1, p < 0.001; LMS versus shUBF-2, p < 0.001; n = 3).

(B) Western blot analysis of RRN3 as for UBF in (a) utilizing two independent shRNA-mirs targeting RRN3 (shRRN3-1 and -2). Relative Rrn3 mRNA expression determined by qRT-PCR (LMS versus shRRN3-1, p < 0.001; LMS versus shRRN3-2, p < 0.001; n = 4).

(C) 47S pre-rRNA synthesis determined by [32P] orthophosphate labeling in cells transduced with shRNA-mirs targeting UBF or RRN3 as compared to LMS alone (representative of n = 2).

(D) Pre-rRNA expression determined by qRT-PCR of the ITS1 in cells transduced with select shRNA-mirs and B cells described in (a) (LMS versus shUBF-2, p < 0.01; LMS versus shRRN3-1, p < 0.01; n = 4).

(E) GFP competition assay of GFP+, transduced Eμ-Myc lymphoma cells (LMS, shUBF, shRRN3) cocultured with GFP–, mock transduced Eμ-Myc lymphoma cells. Data expressed as fold change in percentage of GFP+ cells compared to Day 2 posttransduction (n = 3).

(F) Percentage of apoptotic cells determined by subG1 DNA content analysis expressed as fold change over LMS (6.3% ± 0.61 SEM) (LMS versus shUBF-1, p < 0.01; LMS versus shUBF-2, p < 0.001; n = 3) (LMS versus shRRN3-1, p < 0.05; n = 6). See also Figures S2A and S2B.

(G) GFP competition assay in Eμ-Myc lymphoma cells with BCL2 overexpression, expressed as fold change in percentage of GFP+ cells on Day 4 compared to Day 2 posttransduction (shRRN3-1 p < 0.01; n = 3). One-way ANOVA with Tukey's multiple comparison posttest (a,b,d,f). Student's two-tailed t test (g). Error bars represent SEM. See also Figures S2C and S2D.