Fig 1: GPC5 expression affects proliferation in U3-DT cells.(A) Western blot analysis. Cells were treated with siRNA for 3 days. Cells were lysed and the levels of GPC5 were examined by Western blotting with anti-GPC5 Rabbit mAb. Lane 1, control siRNA-treated cells; Lane 2, GPC5 siRNA-treated cells; Lane 3, markers. (B) Growth curve. siRNA-treated cells (2,000 cells/well) were then seeded in 96-well culture plates and proliferation was assessed by CCK-8 assay at the indicated time points. An exponential approximation line of control (blue) or siRNA-treated (red) dots was drown between for 14 to 86hr. The results are expressed as the mean ± S.D. of quintuplicate wells. P < 0.05. (C) Cell-cycle distribution of U3-DT cells. Cell cycle distribution (%) of U3-DT cells incubated in medium containing siRNA (red) or not (blue) is shown. (D) Immunofluorescence profile of U3-DT cells. Double immunofluorescence labeling with anti-human GPC5 (red) and Ptc1 (green) antibodies in U3-DT cells. Nuclei are labeled with DAPI (merged image). Arrows: GPC5 and Ptc1 positive-merged spots (yellow) are clearly visible in U3-DT cells. Scale bar = 50 µm.
Fig 2: Microprocessor dependent termination prevents transcriptional interferencea. Chromatin RNA-seq profiles across MIR17HG-GPC5 tandem gene locus showing readthrough transcription following Microprocessor depletion. Black arrow and red box highlights reduction in GPC5 exon 1 peak following Microprocessor depletion. b. RT-qPCR analysis of chimeric transcripts versus GPC5 exon 1. Gene specific RT primer (arrowhead) in exon 1 and PCR amplicons are indicated by black bars. c. Chromatin RNA-seq profiles across convergent OGFRL1-LINC00472 gene locus. d. Read quantification for protein coding genes subject to transcriptional interference following Microprocessor depletion. e. mRNA levels of GPC5 and OGFRL1 were determined by RT-qPCR using exon specific primers. RNA levels are expressed relative to control siRNA which were set to 1. All values are normalized to GAPDH mRNA. f. Protein levels of GPC5 and OGFRL1 are reduced by transcriptional interference. Direction of transcription is indicated by green arrow and miRNA by red vertical lines. All experiments used HeLa cells. Error bars represent s.d. of an average (n=3 independent experiments).
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