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. 2018 Dec 19;9:5378. doi: 10.1038/s41467-018-07620-0

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — The effects of SRPK1 inhibition on global RNA splicing and BRD4 isoform levels. a Frequency and type of significantly altered splicing events (FDR ≤0.001) in THP-1 cells after 24 h of treatment with 3 μM SPHINX31. b Number and distribution of genes with one or more differential exon usage events (FDR <0.001) in THP-1 cells after 24 h of treatment with 3 μM SPHINX31. c Overlap of genes with splicing changes after genetic or pharmacological inhibition of SRPK1 in THP-1 cells (hypergeometric test). d, e Quantification, by isoform-specific qRT-PCR of selected isoform changes identified upon pharmacological vs genetic inhibition of SRPK1 (mean ± s.d., n = 3). f Intron-exon structure of BRD4 long and short isoforms g Western blot of THP-1 cell lysates after SPHINX31 treatment (72 h), showing a marked switch from the BRD4S to the BRD4L protein isoform using both an N-terminal antibody that detects both isoforms (N-Ab) and a C-terminal Ab that detects only BRD4L (C-Ab). h Schematic illustration of the target sites/sequences for two gRNAs designed to specifically disrupt the BRD4 exon 12 splice acceptor site, which defines BRD4S (sgRNA sequences underlined, with PAM sequence underlined in red). i Western blot for BRD4 in THP-1 cells transduced with each of these two gRNA display the same BRD4 isoform switch as seen with SPHINX31. j Competitive co-culture of THP-1 cells transfected with lentiviral gRNAs against BRD4 (BFP positive) vs non-transfected cells normalized to %BFP on day 4 (mean ± s.d., n = 3). gRNAs were designed against known essential BRD4 domains (BD2 or ET) or the splice acceptor of BRD4S exon 12. k Dose-response curves of THP-1 cells to SPHINX31 after overexpression of wild-type BRD4L, BRD4S and bromodomain mutant (Y97A/Y390A) BRD4S (mean ± s.d., n = 3). l Western blot for BRD4 (N-terminal antibody, N-Ab) in THP-1 cells transduced with a gRNA targeting SRPK1 and plasmids expressing a phosphomimic version of SRSF1 cDNA or an empty control, showing block of the BRD4S-to-BRD4L isoform switch upon expression of the former. **P < 0.001 (t-test)