Journal: Nature Communications
Article Title: KAP1 negatively regulates RNA polymerase II elongation kinetics to activate signal-induced transcription
doi: 10.1038/s41467-024-49905-7
Figure Lengend Snippet: a Scheme showing dTAG strategy targeting the C-terminus of KAP1 with the dTAG cassette. FKBP-V: degron; 2xHA: two tandem HA tags; P2A: 2A peptide sequence; Puro: Puromycin resistance cassette; BSD: Blasticidin resistance cassette. b Western blot showing dTAG-mediated KAP1 degradation kinetics in HCT116 KAP1 dTAG cells. Blots are representative of two independent experiments. c Scheme showing the experimental design alongside the cell treatments. d RT-qPCR assay highlighting gene expression of one representative IEG ( FOS ) in DMSO- and dTAG-treated cells during a serum stimulation time course. Data represent mean ± SEM ( n = 3 biological replicates, two-sided Student’s t -test comparing DMSO to dTAG at each of the indicated time points). P -values are indicated. Black: DMSO, red: dTAG. e RNA-Seq volcano plot identifying IEGs as upregulated genes upon 30 min of serum stimulation ( n = 3 biological replicates, two-sided Likelihood Ratio Test using the Benjamini–Hochberg (BH) multiple comparisons adjustment, false discovery rate [FDR] <0.05). Dots marked red were identified as differentially expressed. f Violin plot showing upregulation of IEGs organized by induction of expression: 2-fold IEGs ( n = 236), 4-fold IEGs ( n = 69), 8-fold IEGs ( n = 34), and 16-fold IEGs ( n = 16). Random ( n = 236) denotes random genes selected from non-DE genes ( n = 10,361). Data represents the Log 2 FC value computed from RNA-Seq analysis between the DMSO-S0 and DMSO-S30 condition (generated from 3 biological replicates). The n number represents the number of genes in each cluster plotted. The median is denoted as a dashed black line. g Violin plot showing downregulation of IEGs upon acute KAP1 depletion organized by induction of expression: 2-fold IEGs ( n = 236), 4-fold IEGs ( n = 69), 8-fold IEGs ( n = 34), and 16-fold IEGs ( n = 16). Random ( n = 236) denotes random genes selected from non-DE genes ( n = 10,361). Data represents the Log 2 FC value computed from RNA-Seq analysis between the DMSO-S30 and dTAG-S30 conditions (generated from 3 biological replicates). The n number represents the number of genes in each cluster plotted. Median % changes in expression are listed above the violin in each IEGs cluster and Random non-DE genes. The median is also denoted as a dashed black line. h Western blot showing protein expression of two representative IEGs (c-Fos and ATF3) during a serum time course ± dTAG treatment. Blots are representative of three independent experiments. Source data are provided as a Source Data file.
Article Snippet: The original HCT116 parental cell line was purchased from ATCC (catalog CCL-247).
Techniques: Sequencing, Western Blot, Quantitative RT-PCR, Gene Expression, RNA Sequencing, Expressing, Generated
ATCC is a verified supplier 
ATCC manufactures this product 
![a Scheme showing dTAG strategy targeting the C-terminus of KAP1 with the dTAG cassette. FKBP-V: degron; 2xHA: two tandem HA tags; P2A: 2A peptide sequence; Puro: Puromycin resistance cassette; BSD: Blasticidin resistance cassette. b Western blot showing dTAG-mediated KAP1 degradation kinetics in <t>HCT116</t> KAP1 dTAG cells. Blots are representative of two independent experiments. c Scheme showing the experimental design alongside the cell treatments. d RT-qPCR assay highlighting gene expression of one representative IEG ( FOS ) in DMSO- and dTAG-treated cells during a serum stimulation time course. Data represent mean ± SEM ( n = 3 biological replicates, two-sided Student’s t -test comparing DMSO to dTAG at each of the indicated time points). P -values are indicated. Black: DMSO, red: dTAG. e RNA-Seq volcano plot identifying IEGs as upregulated genes upon 30 min of serum stimulation ( n = 3 biological replicates, two-sided Likelihood Ratio Test using the Benjamini–Hochberg (BH) multiple comparisons adjustment, false discovery rate [FDR] <0.05). Dots marked red were identified as differentially expressed. f Violin plot showing upregulation of IEGs organized by induction of expression: 2-fold IEGs ( n = 236), 4-fold IEGs ( n = 69), 8-fold IEGs ( n = 34), and 16-fold IEGs ( n = 16). Random ( n = 236) denotes random genes selected from non-DE genes ( n = 10,361). Data represents the Log 2 FC value computed from RNA-Seq analysis between the DMSO-S0 and DMSO-S30 condition (generated from 3 biological replicates). The n number represents the number of genes in each cluster plotted. The median is denoted as a dashed black line. g Violin plot showing downregulation of IEGs upon acute KAP1 depletion organized by induction of expression: 2-fold IEGs ( n = 236), 4-fold IEGs ( n = 69), 8-fold IEGs ( n = 34), and 16-fold IEGs ( n = 16). Random ( n = 236) denotes random genes selected from non-DE genes ( n = 10,361). Data represents the Log 2 FC value computed from RNA-Seq analysis between the DMSO-S30 and dTAG-S30 conditions (generated from 3 biological replicates). The n number represents the number of genes in each cluster plotted. Median % changes in expression are listed above the violin in each IEGs cluster and Random non-DE genes. The median is also denoted as a dashed black line. h Western blot showing protein expression of two representative IEGs (c-Fos and ATF3) during a serum time course ± dTAG treatment. Blots are representative of three independent experiments. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5487/pmc11245487/pmc11245487__41467_2024_49905_Fig1_HTML.jpg)