Additional File 1:

IRF1 hnRNA transcripts are enriched in the MEN1-depleted cell line. (a-e) qRT-PCR to quantitate IRF1 total and pre-mRNA expression in shRNA-MEN1 and shRNA-NS cell lines that were uninduced (un) or treated with IFN-γ. IRF1 expression was normalized to β-actin and presented as fold change relative to the uninduced, shRNA-NS condition. Numbers are the base pair location of the primers used on the IRF1 gene; see Figure 1c. Error bars are standard error (n = 4). **P ≤ 0.01, *P ≤ 0.05.

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Additional File 2:

Increased rate of transcription does not account for elevated hnRNA pool in the shRNAmir-MEN1 cell line. (a-b) qRT-PCR to quantitate IRF1 pre-mRNA and total RNA expression in shRNA-MEN1 and shRNA-NS cell lines that were uninduced or treated with IFN-γ and collected at indicated time points. IRF1 expression was normalized to β-actin and presented as fold change relative to the uninduced, non-silencing shRNAmir condition. Error bars are standard error (n = 4). **P ≤ 0.01.

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Additional File 3:

CPE assay MEN1 protein levels. (a) MEN1 western blot of whole cell extracts prepared from shRNA-MEN1 cells treated with doxycycline for 24 hours or not. GAPDH served as the loading control.

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Additional File 4:

Phosphoserine 2 Polymerase II levels are not altered in the MEN1-depleted cell line. (a, b) Chromatin immunoprecipitation of shRNA-MEN1 and shRNA-NS cell lines treated with IFN-γ for 90 minutes or uninduced using the antibody specific for the phosphoserine 2 form of Pol II.

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Additional File 5:

Aberrant TSS transcripts at Pol I, Pol II and Pol II genes. (a) qRT-PCR to quantitate transcripts initiated from a TSS upstream of the canonical TSS for IRF1, GAPDH and RN7SK (a Pol III gene) in shRNA-MEN1 and shRNA-NS cell lines. IRF1, GAPDH and RN7SK mRNA transcripts and aberrant TSS transcripts were normalized to β-actin; the aberrant TSS transcripts were calculated as a fraction of total mRNA, and presented as fold enrichment relative to the non-silencing shRNAmir condition. Error bars are standard error (n = 2). A student's one-tailed t-test determined significance. *P ≤ 0.05. (b) Effective qPCR primers could not be designed at the TSS of the 45S rRNA that is processed to 18S rRNA (RN18S1, a Pol I gene) and so endpoint PCR was used to attempt to detect aberrant TSS transcripts. 25 cycles of PCR did not generate an amplicon, nor did a second round of 25 cycles using an aliquot (10%) of the first reaction as template (arrow), but when genomic DNA was used a template an amplicon of the correct size was produced (positive control). This experiment was done for two biological replicate cDNA preparations, though only one is shown. 18S ORF primers detect the RN18S1 rRNA. RT+ indicates reverse transcriptase was included in the cDNA reaction while RT- indicates it was omitted.

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