S1 File - A Function for the hnRNP A1/A2 Proteins in Transcription Elongation

Fig. A. mycUP1 expression is stimulated by the depletion of nuclear hnRNP A1 and A2 in WI38VA13 cells. Western analysis was used to test the effects of sorbitol and RNAi targeting of A1 and A2 in a WI38VA13 clone stably expressing mycUP1. Fig. B. Examples of sqRT-PCR results for RNAi and sorbitol treatments. Dilution of a control cDNA sample was used to evaluate the fold difference using Quantity One software (Bio-Rad). Fig. C. mycUP1 expression is stimulated by sorbitol, even in the presence of cycloheximide. Quantification of duplicate experiment testing the impact of cycloheximide on the stimulation of mycUP1 elicited by sorbitol. HCT116 cells expressing mycUP1 were treated with 1 or 10 μg/ml of cycloheximide 1 hour prior to sorbitol treatment (1 hour). RNA was collected 24 hours later. Histograms show a log scale quantification of mycUP1 RNA levels (sqRT-PCR) that have been normalized to β-actin RNA levels. Fig. D. Chromatin immunoprecipitation assays using anti-RNA polymerase II antibody. A, ChIP-PCR assays were performed to monitor time-dependent changes of pol II occupancy on the Kitlg gene following sorbitol treatment. B, ChIP assays were performed to monitor time-dependent changes in pol II occupancy on the mycUP1 gene following sorbitol treatment. The numbers indicate the position of the center of the amplicon relative to the transcription start site (see Fig 3). C, The values for specific positions in Kitlg and mycUP1 from panels A and B were plotted relative to time to illustrate that stimulation of mycUP1 transcription is detected after 2 hours while the drop in Kitlg expression is detected after 1 hour. D, Density of ChIP-seq reads for RNA polymerase II (H-224 antibody) on Egr1 (major RefSeq transcript indicated) in mock-treated, DRB-treated and siA1/A2-treated samples. E, Density of Pol II ChIP-seq reads on Kitlg in mock-treated, DRB-treated and siA1/A2-treated samples Only the promoter-proximal region of the gene is shown. The boxed portion represents the region of the Kitlg gene where an increase in pol II occupancy is noted in DRB-treated and A1/A2-depleted samples. Table A. Sequences of siRNAs. Table B. Oligonucleotides used for RT-PCR analysis. Table C. Oligonucleotides used for the qPCR time-course analysis of transcription and for chromatin immunoprecipitation assays. (PDF)