EMBO/soda-nel huggingface dataset:
PP2A Phosphatase Is Required for Dendrite Pruning via Actin Regulation in Drosophila (2020)
Figure 3

Fig. 3. Effects of PP2A on ecdysone target gene expression during dendrite pruning.

C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Sox14 or Mical was assessed by immunofluorescence.

A - C Sox14 stainings at 2 h APF. (A) Control c4da neurons. (B) C4da neurons expressing PP2A-29B dsRNA. (C) C4da neuron expressing dominant-negative Mts.

D Quantification of Sox14 expression in (A - C). C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons (identified by characteristic cell body shape). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, *** P<0.0005, Wilcoxon's test.

E - H Mical stainings at the indicated time points. (E) Mical expression in control c4da neurons at 2 h APF. (F) Mical expression in c4da neurons expressing dominant-negative Mts at 2 h APF. (G) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 2 h APF. (H) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF. (I) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 0 h APF.

J Quantification of Mical expression in (E - I). C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons (marked by asterisks). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, ** P<0.005, *** P<0.0005, Wilcoxon's test. Data information: Scale bars in A and E are 10 μm.
PP2A Phosphatase Is Required for Dendrite Pruning via Actin Regulation in Drosophila (2020)
Figure 4

Fig. 4. Interactions between PP2A and the actin regulator cofilin during c4da neuron dendrite pruning.

A - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control.

H Penetrance of pruning defects in panels A - G. * P<0.05, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure.

I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.

J - L GFP-tagged Drosophila cofilin (GFP::tsr) was expressed in c4da neurons under ppk-GAL4, and its localization was assessed at the indicated developmental stages by live imaging. Panels J - L show GFP::tsr localization in c4da neurons at the third instar stage, panels J' - L' show GFP::tsr localization in c4da neurons at 6 h APF. (J, J') Control c4da neurons expressing Orco dsRNA. (K, K') C4da neurons expressing Mical dsRNA. (L, L') C4da neurons expressing PP2A-29B dsRNA.

M Quantification of GFP::tsr punctae in samples J - L'. Data are mean +/- s. d., the numbers of neurons (animals) analyzed for each genotype are given in the figure (data were not derived from independent experiments). ** P<0.005, *** P<0.0005, n. s., not significant, Wilcoxon's test.

Data information: Scale bars are 50 μm in A and 10 μm in J.
PP2A Phosphatase Is Required for Dendrite Pruning via Actin Regulation in Drosophila (2020)
Figure 5

Fig. 5. PP2A interacts with actin regulators during dendrite pruning.

A - H Genetic interactions between PP2A-29B and actin regulators during dendrite pruning. The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. (A) C4da neuron coexpressing PP2A-29B dsRNA and tdtomato as suppression control. (B) C4da neuron coexpressing PP2A-29B dsRNA and GFP::tsr. (C) C4da neuron coexpressing PP2A-29B dsRNA and RacN17. (D) C4da neuron coexpressing PP2A-29B dsRNA and RhoN19. (E) C4da neuron expressing PP2A-29B dsRNA in ena210/+ background. (F) C4da neuron coexpressing PP2A-29B dsRNA and Orco dsRNA as suppression control. (G) C4da neuron coexpressing PP2A-29B dsRNA and Chicadee dsRNA. (H) C4da neuron coexpressing PP2A-29B dsRNA and moesin dsRNA.

I Penetrance of pruning defects in panels E - L. n. s., not significant, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure.

J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.

Data information: Scale bars in A and E are 50 μm.
A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A (2022)
Figure 1

Figure 1: CentSIRT6 missense variant identified in AJ centenarians demonstrates lower deacetylation activity

A Age, gender and BMI information of the Ashkenazi Jewish Centenarian cohort. Table showing the age, gender and BMI information in control and centenarian groups. Pie chart showing the percentage of male/female individuals in control and centenarian groups. Right panel showing the age and BMI distribution of all individuals recruited in targeted capture-seq. Data were presented as mean and SD, Students t-test, two tailed, *** p<0.001.

B, C Histograms of normalized allele counts in individuals 75+ years old compared to all age groups (N=125,748) across (B) all chromosome 19 SNPs and small insertions/deletions, and (C) missense SNPs and indels only. Dotted vertical lines show centSIRT6.

D The structure of the SIRT6 gene. centSIRT6 contains two missense mutations in the C-terminus N308K and A313S. White boxes represent UTRs, colored boxes represent protein coding regions, and blue boxes represent enzymatic domains.

E Michaelis-Menten kinetic parameters as calculated by saturation curves using differential concentrations of myristoylated peptide or NAD+. n=3 technical replicates (TR); error bars=SD.
A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A (2022)
Figure 2

Figure 2: CentSIRT6 possesses reduced deacetylase activity

A, B Deacetylase activity on H3K9 (A) and H3K18 (B) residues shows reduced activity in centSIRT6 allele. Designer histones were incubated with purified SIRT6 and 5 mM NAD+, then resolved by SDS-PAGE and analyzed by immunoblotting with acetyl-specific histone antibodies. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisks indicate p<0.05 compared to WT, color corresponds to allele. H3K9ac, H3K18ac and H3 are blots, SIRT6 loading Coomassie stain HeLa histone preps were also probed as shown in Figure EV3A.

C Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels. Relative fraction represents the portion of the peptide encompassing H3K9-17 compared to the summed total of all of the peptide quantitation values for the same region. Acetylation of other H3 peptides is shown in Supplementary Data 1. Histone preparation is shown in Figure EV3F. n=3 biological replicates (BR); error bars=SD. Students t-test, two tailed

D Whole cell histone H3 acetylation levels in cumate-inducible SIRT6 human fibroblasts, assessed by Western blot. Cu, cumate; PQ, paraquat. Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot, and administered accordingly to respective cell lines (Figure EV3B, C). Cells were incubated with appropriate cumate dose for 48hrs prior to harvest, with PQ-treated cells receiving PQ 24hrs after initial cumate induction.

E-H Relative abundance of H3K9ac at SIRT6-dependent NF-κB target genes. Anti-H3K9ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 BR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition. Red asterisk indicated p<0.05 compared to +Cu WT.

I, J Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity. Anti-H3K18ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 TR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition.
A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A (2022)
Figure 3

Figure 3: CentSIRT6 demonstrates enhanced mADPr activity

A Self-ribosylation of SIRT6 using biotin-labeled NAD+. Recombinant SIRT6 was incubated with NAD+ conjugated with a biotin residue and then run on an SDS-PAGE. Each allele was assessed relative to its 0 hr time point and normalized to SIRT6 total protein loading controls. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05. Black asterisks indicate significance over 0 hr control. Red asterisks indicated significance over time-matched WT SIRT6.

B Self-ribosylation of SIRT6 with titration of NAD+. mADPr-specific antibody (Bonfiglio et al., 2020) was used to detect ribosylated wild type SIRT6 and centSIRT6 proteins. Plot was fit the Michaelis-Menten best-fit with GraphPad software. Km NAD was 142+29 for centSIRT6 and 106+45 for wild type SIRT6 and maximal signal was ~2x greater for centSIRT6 compared to the wild type SIRT6. Two separate batches of recombinant protein of each SIRT6 allele were prepared and assayed. n=3 TR; error bars=SD.

C Activation of PARP1 by SIRT6 variants. SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly-ADPr antibody. Poly-ADPr activity of PARP1 results in a wide range of product size. Activity was assessed by quantifying poly-ADPr signal in whole lanes for each sample. n=3 TR; error bars=SD. Two-way ANOVA. Asterisk indicate p<0.05. Black asterisks indicate significance over HPRT control. Red asterisks indicated significance over wild type SIRT6.
A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A (2022)
Figure 4

Figure 4: CentSIRT6 enhances LINE1 retrotransposon suppression and DNA repair

A qRT-PCR analysis of LINE1 expression in cumate-inducible SIRT6 fibroblasts. Primers assessed both 5' (ORF1) and 3' (ORF2) LINE1 sequences from the L1HS family of evolutionarily active LINE1 retrotransposons. Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes. Asterisk indicate p<0.05. n=3 TR; error bars=SD. Students t-test, two tailed.

B LINE1 EGFP retrotransposition assay. Three independent transfections with LINE1 EGFP reporter plasmid were conducted and quantified by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicated significance over SIRT6 KO control. Black asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.

C In vitro KAP1 ribosylation by SIRT6. Asterisk indicate p<0.05. Red asterisk indicate significance over 0 hr, black asterisk indicate significance over compared to WT SIRT6. n=3 BR; error bars=SD. Students t-test, two tailed.

D, E Stimulation of NHEJ and HR by SIRT6 variants. Reporter cell lines were co-transfected with SIRT6-expressing plasmid, I-Sce1 plasmid, and DsRed transfection control. After 72 hr recovery, reactivation of the GFP reporter was measured by flow cytometry. Stimulation of NHEJ or HR was calculated as radio of GFP+/DsRed+ positive cells. Representative FACS traces are shown in Figure EV4A. Blots demonstrate equivalent SIRT6 abundance in transfected cells. Asterisk indicate p<0.05 Black asterisk indicate significance over control, red asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA

F Basal γH2AX foci in cumate-inducible SIRT6 fibroblasts. Foci were scored in at least 80 cells per condition. Representative images of the foci are shown in Figure EV4B. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.

G DNA repair kinetics in cumate-inducible SIRT6 fibroblasts. Cells were grown on slides and irradiated with 2 Gy gamma radiation, followed by immunostaining for γH2AX. Irradiation was conducted when the cells were at 75% confluency on slides. Cells were fixed and foci scored at t=0.5 hr, 2 hr, 4 hr, 6 hr, and 24 hr post-irradiation. Foci were scored in at least 80 cells per genotype per time point. Representative images of the foci are shown in Figure EV4C. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.

H, I Oxidative stress resistance. Cumate-inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 hours. Resistance was determined by apoptosis staining 48 hours after exposure. Representative FACS traces are shown Figure EV4D. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.
A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A (2022)
Figure 5

Figure 5: CentSIRT6 induces increase cell death in cancer cells

A Number of adherent cells after transfection with SIRT6 variants. Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 hours. HCA2 are normal human foreskin fibroblasts. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.

B, C Apoptosis staining of cancer cell lines 48 hours after transfection. Cells were stained with Annexin V/PI and analyzed by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicates significance over control, black asterisk indicates significance over WT SIRT6.

n=3 BR; error bars=SD. Two-way ANOVA.
A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A (2022)
Figure 6

Figure 6: CentSIRT6 allele shows enhanced interaction with LMNA

A Proteins showing stronger interaction with centSIRT6 compared to the wild type SIRT6. Proteins were prepared from cumate-induced SIRT6 cell lines and immuno-precipitated with SIRT6 antibody; rabbit pre-immune serum was used as a control. Prior to analysis by mass spectrometry, samples were labeled with tandem-mass tags.

B IP experiments on lysates from cumate-induced fibroblasts expressing wild type or centSIRT6 alleles with antibodies to SIRT6, LMNA, and mADPr. SIRT6 expression was induced 48 hours prior to IP. n=3 BR. One representative set of IPs is shown.

C CentSIRT6 shows stronger interaction with LMNA compared to the wild type SIRT6. Quantification of the IP experiment shown in (B). SIRT6 IP followed by Western blot with antibodies to LMNA.

D LMNA shows enhanced interaction with centSIRT6 compared to the wild type. Quantification of the IP experiment shown in (B). LMNA IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6.

E centSIRT6 shows enhanced mADPr. Quantification of the IP experiment shown in (B). SIRT6 IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues (Bonfiglio et al., 2020)

F LMNA shows enhanced mADPr signal in cells expressing centSIRT6. Quantification of the IP experiment shown in (B). IP with mADPr antibody (Bonfiglio et al., 2020) using extract from cumate-induced SIRT6 fibroblasts, followed by Western blot with antibodies to LMNA.

G SIRT6 and LMNA are colored red and featured as central points in two opposing nodes of interactions. A third node (upper middle) shows interaction partners that are shared by SIRT6 and LMNA. Proteins whose interactions are enhanced by the centSIRT6 allele are colored yellow. Proteins that interacted equally with wild type and centSIRT6 alleles are uncolored. H1.5 is a special case and colored orange because it showed increased interaction with the centSIRT6 and decreased interaction with LMNA in the presence of the centSIRT6 allele. Proteins known to be ribosylated in previous reports are shown as hexagons.

Data information: For C-F, n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05
hnRNP R negatively regulates transcription by modulating the association of P-TEFb with 7SK and BRD4 (2022)
Figure 1

Figure 1. hnRNP R depletion increases transcription

A. Schematic of the strategy for the generation of hnRNP R knockout cells by prime editing. The PAM sequence is underlined in red, the protospacer sequence recognized by the pegRNA is marked in black, the inserted nucleotides are marked in purple and the TGA stop codon is indicated by asterisks.

B. Western blot analysis of hnRNP R in three independent HNRNPR wildtype (+/+), heterozygous (+/-) and homozygous (-/-) HeLa cell lines.

C. Confocal microscopy images of nuclei stained with DAPI. Scale bar: 10 µm.

D. Quantification of the DAPI area in (C). Data are mean with standard deviation (SD); ***P ≤ 0.001; unpaired two-tailed t-test (n = 3 biological replicates).

E. RNA FISH with a Cy3-labeled oligo-dT probe. DAPI was used for nuclear staining. Scale bar: 10 µm.

F. Quantification of mean fluorescence intensity of the Cy3 oligo-dT signal in (E). Data are mean with SD; ****P ≤ 0.0001; Mann-Whitney test (n = 129 cells for HNRNPR+/+ and n = 176 cells for HNRNPR-/-).

G. Schematic of the mRNA quantification procedure (Picelli et al, 2014). TSO, template-switching oligonucleotide.

H. Fluorescence detection of tRNA amplification by qPCR.

I. Quantification of relative mRNA levels detected by qPCR from cDNA at different dilutions. Data are mean with SD; **P ≤ 0.01, ***P ≤ 0.001; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).

J. Fluorescence detection of mRNA amplification by qPCR using ISPCR primers.
hnRNP R negatively regulates transcription by modulating the association of P-TEFb with 7SK and BRD4 (2022)
Figure 2

Figure 2. Increased Ser2 phosphorylation of RNA pol II in hnRNP R-depleted cells

A. Western blot analysis of Ser2-phosphorylated and total RNA pol II, Cyclin T1, CDK9 and GAPDH protein levels.

B. Quantification of relative expression of Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9 in (A). Data are mean with SD; **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates).

C. Immunostaining with antibodies against Ser2-phosphorylated and total RNA pol II in. Scale bar: 20 μm.

D. Immunostaining with antibodies against CDK9. Scale bar: 10 μm.

E. Immunostaining with antibodies against Cyclin T1. Scale bar: 10 μm.

F. Western blot analysis of subcellular fractions for Calnexin, GAPDH, histone H3, Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9. Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.

G. Quantification of relative expression of Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9 in subcellular fractions in (F). Data are mean with SD; *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).
hnRNP R negatively regulates transcription by modulating the association of P-TEFb with 7SK and BRD4 (2022)
Figure 3

Figure 3. P-TEFb is released from 7SK in hnRNP R-deficient cells

A. Western blot analysis of the indicated proteins in the input (In) lysates used for co-immunoprecipitation.

B. Western blot analysis of Cyclin T1, HEXIM1, CDK9 and hnRNP A1 co-immunoprecipitated by an anti-LARP7 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.

C. Western blot analysis of Cyclin T1, HEXIM1, CDK9, hnRNP A1 and LARP7 co-immunoprecipitated by an anti-MePCE antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.

D. Quantification of CDK9, Cyclin T1 and HEXIM1 co-immunoprecipitating with LARP7 in (B). Data are mean with SD; **P ≤ 0.01, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates).

E. Quantification of CDK9, Cyclin T1 and HEXIM1 co-immunoprecipitating with MePCE in (C). Data are mean with SD; *P ≤ 0.05, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates).

F. Quantification of hnRNP A1 co-immunoprecipitating with LARP7 and MePCE in (B) and (C). Data are mean with SD; *P ≤ 0.05; unpaired two-tailed t-tests (n = 3 biological replicates).

G. Western blot analysis of subcellular fractions Calnexin, GAPDH, histone H3, hnRNP R and hnRNP A1. Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.

H. Western blot analysis of hnRNP R co-immunoprecipitated by an anti-hnRNP A1 antibody from subcellular fractions. Immunoprecipitation with mouse-IgG antibody was used as control.

I. Quantification of 7SK RNA in subcellular fractions by qPCR. Data are mean with SD; **P ≤ 0.01, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).

J. Quantification of 7SK RNA co-immunoprecipitating with hnRNP A1 from subcellular fractions. Data are mean with SD; *P ≤ 0.05, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).
hnRNP R negatively regulates transcription by modulating the association of P-TEFb with 7SK and BRD4 (2022)
Figure 4

Figure 4. hnRNP R depletion enhances P-TEFb binding to BRD4

A. Western blot analysis of BRD4, hnRNP R, Cyclin T1, CDK9, α-Tubulin and histone H3 in the input lysates used for co-immunoprecipitation.

B. Western blot analysis of hnRNP R, Cyclin T1, CDK9 and histone H3 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.

C. Quantification of CDK9 co-immunoprecipitating with BRD4 in (B). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).

D. Western blot analysis of BRD4 (long and short exposure) co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with mouse-IgG antibody was used as control.

E. Quantification of BRD4 co-immunoprecipitating with CDK9 in (D). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).

F. Quantification of histone H3 co-immunoprecipitating with BRD4 in (B). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).

G. Western blot analysis of BRD4, hnRNP R, Cyclin T1, CDK9, α-Tubulin and histone H3 in the input lysates used for co-immunoprecipitation. Lysates were pre-treated with RNase as indicated.

H. Western blot analysis of hnRNP R, Cyclin T1, CDK9 and histone H3 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.

I. Western blot analysis of BRD4, hnRNP R, CDK9 and α-Tubulin in the input lysates used for co-immunoprecipitation.

J. Western blot analysis of hnRNP R and CDK9 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.

K. Quantification of CDK9 co-immunoprecipitating with BRD4 in (J). Data are mean with SD; ****P ≤ 0.0001; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).

L. Western blot analysis of phospho-CDK9(Thr186) co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.

M. Quantification of phospho-CDK9(Thr186) co-immunoprecipitating with BRD4 in (L). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).