Figure 2

Effect of altered Myc levels on Uhg and snoRNA expression. A) Gene set enrichment analysis (GSEA) of snoRNAs. The 8,019 detectable genes are ordered by relative expression (log2 of the ratio of Myc-knockdown over GFP-knockdown) from left to right, and all detectable snoRNAs are mapped onto these genes (black vertical bars). NES indicates the normalized enrichment score, p the nominal p-value and q the false discovery rate. B and C) RNA levels were assayed 24 hours after addition of Myc-dsRNA to S2 cells by quantitative PCR (B) and Northern blotting (C); of ten investigated snoRNAs, five gave no signal or such a weak signal that a reduction in response to Myc-knockdown could not be reliably quantified, four were clearly reduced upon Myc-knockdown and one was unaffected (see Additional file 1: Table S5). D) qPCR assays carried out six hours after addition of 125 μM CuSO₄ to S2-Myc cells to induce Myc overexpression. Error bars show standard deviations of biologically independent duplicates. Selected snoRNAs are grouped with the transcripts of their host Uhg genes. Panel C, arrowheads point to mature snoRNAs; the identity of the cross-reactive slower migrating band in the Me28S-G2703c blot (asterisk) is unclear. The locations of molecular weight DNA markers are indicated. The same RNA samples were analyzed by reverse transcription and quantitative PCR for the reference genes snm158 and rab6, as well as for the snoRNAs. C’, qPCR results and quantification of the Northern blot bands. C”, part of each sample that was not used for RNA isolation (shown in panels C and C’) was analyzed by Western blotting with mouse anti-Myc antibody (top) and mouse anti-α-Tub84B (bottom). The experiment shown in panels C, C’ and C” is representative of biologically independent duplicates. snoRNA, small nucleolar RNA; Uhg, U-snoRNA host genes.