EMBO/SourceData huggingface dataset:
Figure 6(A-C) Ciliary length measured by fluorescence microscopic analysis at 1 year of age (1y). (A) Representative immunostaining of the ciliary marker Gt335 (green) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas. Counterstaining by DAPI, scale bar 10µm. (B) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining. Scale bar 500nm. (C) Length of connecting cilia (CC) appear elongated in USH1C when applying a Fiji script to immunostained histological sections (2 pigs, 1y, 1 retina each, number of examined connecting cilia is indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).(D, E) Measuring CC length in TEM sections. (D) Representative images of longitudinal cross sections and principle of determining CC length. Scale bar 500nm. (E) Elongation of CC length is confirmed in TEM sections of USH1C PRC. (1 pig, 1y, 1 retina each, number of examined connecting cilia indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).Length of primary cilia in dermal fibroblasts. (F) Staining of ciliary structures in fibroblasts stained with ARL13B (ciliary shaft, green) and PCNT2 (ciliary base, red), DAPI (nucleus, blue). Scale bar upper panel 5 µm, lower panel 2.5 µm. (H) Similarly, ciliary length of fibroblasts from a human USH1CR31X/R80fs patient are elongated, compared to a healthy control (7 TR, number of cells examined indicated, KS-test, ****p<0.0001).(I-M) Outer segment (OS) PRC architecture by TEM of longitudinal retinal sections. (I) At 3w, WT show normal sub-cellular organization, including parallel stacking of photosensitive discs and calyceal processes (black arrows, representative image from 2 pigs, 2 retinas, scale bar 600nm). (J) In 3w USH1C rods, membrane discs appear also in vertical orientation (white arrows, representative image from 2 pigs, 2 retinas, scale bar 750nm). At 1y, interstitial gaps appeared in disc stacks of the OS in USH1C pigs (K, arrow, scale bar 550nm) and vesicles are found at the OS base (L, asterisk, scale bar 500nm). (M) The disc architecture in 1y animals was substantially distorted (arrows, representative image from 2 pigs, 2 retinas, scale bar 850nm).
Figure 7(A) AAV transduction of retinal layers. AAV8, AAV9 and Anc80 encoding eGFP under the ubiquitous CMV promoter were injected subretinally into the eyes of mature WT pigs in vivo. Upper Panel: Epifluorescence of dissected eyes demonstrates eGFP fluorescence in the bleb region of whole mount preparations of all three AAV samples. Dashed circles indicate bleb size. Lower panel: Epifluorescence images of longitudinal retina sections revealed eGFP expression in the PRC layer (OS, IS and ONL) for all AAV. Increased exposure (4x) documented transduction of MGC (arrows) by AAV9 and Anc80 but not by AAV8 (representative images from 1 pig, 1 retina each, 2 TR, scale bars 25 µm).(D) Efficacy of AAV gene therapy was examined in vivo by sub-retinal injection of Anc80 virus capsid expressing harmonin_a1 under a ubiquitous promoter into a USH1C pig (see also Figure EV5). Left panel: Sub-retinal injection reconstituted harmonin expression in the AAV-treated eye but not in the sham-treated eye (3 TR). Protein extract from a WT retina served as positive control, actin staining as loading control. The 72kD band correlates with harmonin_a isoforms while higher MW indicates putative dimer formation. Middle panel: Immunofluorescence reveals absence of harmonin in the retina of the PBS-injected control eye and harmonin abundance (green) in the retina of the AAV-treated eye. (Representative image from a single animal. Blue: DAPI, scale bar 25 µm). Right panel: Photopic gfERG reveal increased a- and b-wave response to single flash and flicker stimulation in the AAV-treated (red), compared to the sham-treated (black) eyes of two USH1C pigs.
Figure 1A. Pull-down assays of yTBP (61-240) with Kap95p, Kap114p and Kap121p. GST-fused yTBP (61-240) was incubated with recombinant Kap95p, Kap114p and Kap121p. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. B. Band intensities of Kap -βs from the SDS-PAGE gels in (A) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.C-E. ITC titration curves (upper) and binding isotherms (lower) of (C) Kap114p, (D) Kap95p, and (E) Kap121p with yTBP (61-240). Salt concentration and Kd values are indicated.
Figure 2A, B. Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC. SEC (Superdex 200) elution profiles of the (A) Kap114pRanQ69L and (B) Kap114pyTBP complexes. Peak fractions were analyzed by SDS-PAGE and stained with Coomassie blue. The void volume (Vo) of the peak fraction and absorbance (a.u.) at 280 nm for each complex is indicated.SV-AUC analysis of (C) yTBP The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).SV-AUC (D) Kap114p and (E) the Kap114pyTBP complex. The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).F. SE-AUC analysis of the Kap114pyTBP complex. The average molecular mass determined by the Sedfit program was ~133.5  kDa. The lower panel shows the residual difference (standard deviation) between experimental and fitted values.Plots of the distance distribution function P(r) for the (G) Kap114pRanQ69L complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pRanQ69L (H) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.The ab initio envelopes determined by GASBOR for the (I) Kap114pRanQ69L complexes. Models of the (J) Kap114pRanQ69L complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR.Plots of the distance distribution function P(r) for the (K) Kap114pyTBP complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pyTBP (L) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.
Figure 3B. Pull-down assays of yTBP with Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). GST-fused yTBP (61-240) was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. C. Band intensities of Kap-βs from the SDS-PAGE gels in (B) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.D, E. ITC titration curves (upper) and binding isotherms (lower) of (D) Kap114p (Δ347-371) and (E) Kap114p (Δ899-956) with yTBP (61-240). Salt concentration and Kd values are indicated.F. Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type, KAP114 knockout strains, and KAP114 knockout strains rescued by KAP114 deletion mutants.G. His-tagged RanQ69L was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. H. Band intensities of Kap-βs from the SDS-PAGE gels in (G) were quantified and normalized by His-Ran intensity. Quantitative plots show percent Kap-βs binding on His-Ran. I. GST pull-down assays of yTBP (61-240) with Kap114p or Kap114p (Δ347-371) in the presence or absence of RanQ69L. GST-fused yTBP (61-240) was incubated with recombinant Kap114p and His-tagged RanQ69L at a molar ratio of 1:4:8 Kap114p:yTBP:RanQ69L. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. J. Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L. Band intensities of Kap114p and Kap114p (Δ347-371) from the SDS-PAGE gels were used to determine the average fraction of bound protein.
Figure 4B. EMSA analysis of yTBP binding to DNA. We incubated 5 nM FAM-labeled oligonucleotide (TGTATGTATATAAAAC) with indicated concentrations of purified yTBP (0.125 to 8 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).C. Indicated concentrations (0.25 to 16 μM) of purified recombinant Kap114p were incubated with yTBP (4 μM) and FAM-labeled DNA (5 nM), followed by native PAGE analysis. Kap114p (16 μM) was incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).D. Super-shift EMSA analysis of TFIIA binding to yTBPDNA complex. We incubated 5 nM FAM-labeled DNA and yTBP (0.5 μM) with indicated concentrations of purified TFIIA (0.08 to 5 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).E. Indicated concentrations (0.06 to 4 μM) of purified Kap114p were added to the reaction containing 0.5 μM of TFIIA, yTBP and FAM-labeled DNA (TGTATGTATATAAAAC), before conducting native PAGE analysis. TFIIA and Kap114p (4 μM) were incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). Electrophoretic bands containing protein-DNA complex and free DNA are indicated.
Figure 5A. Wild type and KAP114 knockout strains were diluted serially and spotted onto YPD plates in the presence of 1.5 M NaCl.B. The KAP114 knockout strain was transformed with vector control (lane 2) and a plasmid containing wild type KAP114, KAP114 (Δ347-371), or KAP114 (Δ899-956) (lanes 3-5). Wild type and KAP114 knockout strains containing plasmids were serially diluted and grown in minimal medium plates supplemented with 1.5 M NaCl. Growth temperatures are indicated.C. A heatmap generated by three independent biological replicates shows gene expression profiles of wild type and KAP114 knockout strains grown in the presence of 1.5 M NaCl. The Log2 (fold-change) values and the color scale are shown.E, F. (E) qPCR analysis of gene expression in the KAP114 knockout strains (green bars) compared to wild type (black bars) under regular conditions. (F) qPCR analysis of gene expression in the KAP114 knockout strain (green bars) compared to wild type (black bars) grown in high salt conditions. qPCR fold-changes in mRNA expression are relative to control (YPD media, black bars). Actin was used as the internal control.G. ChIP-qPCR assays showing yTBP-GFP binding to the promoter of indicated genes in wild type and KAP114 knockout strains grown in high salt conditions (light green bars) compared to under regular conditions (black bars). Chip-qPCR fold-enrichment is relative to control (YPD media, black bars).H. Endogenous Ran, GFP-yTBP and proliferating cell nuclear antigen (PCNA; loading control) in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions were analyzed by Western blot using antibodies for Ran, GFP and PCNA. I, J. Analysis of Ran and GFP-yTBP in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions. The combined bar and scatter plot shows the band intensity ratios of Ran (I) and GFP-yTBP (J) over PCNA. Each dot represents an individual data point.
Figure 1B WB showing depletion of PERK in Perk KO astrocytes and neurons.D-I eIF2α phosphorylation in WT and Perk KO astrocytes (N=3) and neurons (N=6) ± 20-24 hours of TM-induced ER stress. Representative WB (D, G), quantification of absolute phosphorylated eIF2α level (p-eIF2α/eIF2α) (E, H) and the ER stress-induced p-eIf2α response (F, I). Data information: (E, H, Data are normalised to untreated WT. (F, I Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: ** P<0.01, *** P<0.001, **** P<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, H, Students t-test (F, I)J-O Protein synthesis in WT and Perk KO astrocytes (N=5) and neurons (N=5) ± 20-24 hours of TM-induced ER stress. Representative images obtained by high-content microscopy showing puromycinilated proteins detected by immunofluorescence (J, M), quantification of puromycin intensity as measure for de novo protein synthesis (K, N) and the ER stress-induced protein synthesis response (L, O). Astrocytic soma (GFAP, white), dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green). Scale bar: 25 μm. Data information: K, N) Data are normalised to untreated WT. , L, O) Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: ** P<0.01, *** P<0.001, **** P<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test K , N); Nested t-test (L, O).
Figure 2A-F ATF4 expression in WT and Perk KO astrocytes (N=3) and neurons (N=9) ± 20-24 hours of TM-induced ER stress. (A, D) Representative confocal images of ATF4 immunofluorescence. Astrocytic soma (GFAP, cyan), dendrites (MAP2, cyan), nuclei ((Δ)Cre, green), ATF4 (magenta). Scale bar: 10 μm. (B, E) Quantification of absolute nuclear ATF4 intensity. Data are normalised to untreated WT. (C, F) Quantification of the ER stress-induced ATF4 response. Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant p-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F).G-J (G, I) Venn diagram showing the number of significantly upregulated ATF4 target genes in WT (grey and blue) and Perk KO (green and blue) astrocytes (G, N=3) and neurons (I, N=3) upon 20-24 hours of TM-induced ER stress determined by mRNA-seq. Percentage: fraction of upregulated ATF4 target genes in WT cells that are also upregulated in Perk KO cells. (H, N=3; J, N=3) Log2 fold change of ATF4 target genes that are significantly upregulated in both WT and Perk KO cells (blue in Venn diagram). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant p-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant.
Figure 3Protein synthesis and ATF4 expression in mouse (A-F) astrocytes (A-C and neurons (D-F ± 20-24 hours of TM-induced ER stress ± PERK-i (Pi). (A, D, Representative images obtained by high-content microscopy. Astrocytic soma (GFAP dendrites (MAP2, white), nuclei (ΔCre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. (B, N=5; E, N=5; Quantification of puromycin intensity as measure for de novo protein synthesis. (C, N=5; F, N=7; Quantification of absolute nuclear ATF4 intensity. Data information: Data are normalised to untreated WT and presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test.Protein synthesis and ATF4 expression in human (G-L) astrocytes G-I, ) and neurons J-L) ± 20-24 hours of TM-induced ER stress ± PERK-i (Pi). G, J) Representative images obtained by high-content microscopy. Astrocytic soma Phalloidin, white), dendrites (MAP2, white), nuclei (ΔCre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. ; H, N=3; K, N=3) Quantification of puromycin intensity as measure for de novo protein synthesis. I, N=4; L, N=3) Quantification of absolute nuclear ATF4 intensity. Data information: Data are normalised to untreated WT and presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test.
Figure 5A-F Protein synthesis (A-C, N=3) and ATF4 expression (D-F, N=5) in WT and Perk KO neurons overexpressing eIF2αWT or phospho-deficient eIF2αS51A ± 20-24 hours of TM-induced ER stress. Representative images obtained by (A) confocal and (D) high-content microscopy. Dendrites (MAP2, white/cyan), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (B) and ATF4 response (E). Data are normalised to WT eIF2αWT. Quantification showing the percentage change in the ER stress-induced protein synthesis (C) and ATF4 (F) response induced by eIF2αS51A compared to eIF2αWT. (C) Calculated from data in (B): [(eIF2αS51A-eIF2αWT)/eIF2αWT] *100. (F) Calculated from data in (E): [(eIF2αS51A-eIF2αWT)/eIF2αWT]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E, Nested t-test (C, F).Expression of p-eIF2α sensitive (5'ATF4-uORFWT) and p-eIf2α insensitive (5'ATF4-uORFMut) EYFP translational reporters in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=3). (I, K) Representative confocal images. Dendrites (MAP2, cyan), nuclei ((Δ)Cre, red), endogenous ATF4 (magenta), ATF4 reporter (green). Scale bar: 10 μm. (J, L) Quantification of EYFP intensity, normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test J, L)
Figure 6A-F Protein synthesis and ATF4 expression in ISRIB-treated WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=4). (A, D) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (B) and ATF4 (E) response. Data are normalised to WT without ISRIB. Baseline level (without ER stress) is depicted by dashed line. Quantification showing the percentage change in the ER stress-induced protein synthesis (C) and ATF4 (F) response induced by ISRIB. (C) Calculated from data in (B): [(ISRIB+-ISRIB-)/ISRIB-]*100. (F) Calculated from data in (E): [(ISRIB+-ISRIB-)/ISRIB-]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F)G-M Protein synthesis and ATF4 expression in 2b5he and 2b5ho astrocytes and neurons ± 20-24 hours of TM-induced ER stress in the presence or absence of PERK-i (Pi) (N=3). (G, I, K, M) Representative images obtained by high-content microscopy. Astrocytic soma (Phalloidin, white), dendrites (MAP2, white), nuclei (DAPI, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. (H, L) Quantification of puromycin intensity as measure for de novo protein synthesis and (J, N) absolute nuclear ATF4 intensity. Data are normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Three-way ANOVA with Tukey's post hoc test (H, J, L, N).
Figure 7A Differentially expressed genes (DEGs) involved in oxidative stress related processes comparing TM-treated Perk KO and WT neurons. Blue arrow: Ang.B, C Ang mRNA level in WT and Perk KO neurons and astrocytes ± 20-24 hours of TM-induced ER stress, determined by mRNA-seq (N=3). FPMK: Fragments Per Kilobase of transcript per Million. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, C,D, E Representative WB and quantification of ANG protein level (N=3). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test E,F, G tRNA unfolding in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=4). (F) Representative images obtained by high-content microscopy of 1-methyladenosine (m1A) immunofluorescence to determine tRNA unfolding. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), unfolded tRNA (m1A, green). Scale bar: 25 μm. (G) Quantification of m1A intensity, normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test G,H-M Abundance of small RNAs in WT and Perk KO astrocytes (N=3) and neurons (N=6) ± 20-24 hours of TM-induced ER stress, determined by high-resolution automated electrophoresis (H, K) Representative gel image. Electropherogram (I, L) and zoom (J, M) showing quantified fluorescence units (FU) of small RNAs, size ranges of tRNAs and tsncRNAs are indicated. Shown is the mean of the biological replicates.N-R Protein synthesis and ATF4 expression in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress in the presence or absence of ANG-I (N=3). (N) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesis of proteins (Puromycin, green). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (O) and ATF4 response (Q). Data are normalised to WT without ANG-i. Baseline level (without ER stress) is depicted by dashed line. Quantification showing the percentage change in the ER stress-induced protein synthesis (P) and ATF4 response (R) induced by ANG-i. (P) Calculated from data in (O): [(ANG-i+-ANG-i-)/ANG-i-]*100. (R) Calculated from data in (Q): [(ANG-i+-ANG-i-)/ANG-i-]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test O, Q); Nested t-test (P, R).
Figure 1 (B) U2OS stably expressing FLAG-TIP60 or empty vector (vec) were subjected to FLAG- immunoprecipitation from nuclear lysates and analysed by Western blotting  (C) HEK293T cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG- TIP60S90A or empty vector (vec). The samples were subjected to FLAG affinity-purification, dephosphorylated with shrimp alkaline phosphatase (rSAP) and incubated as indicated with CDK9/cyclinT1 in presence of ATP. Phosphorylation of TIP60 was analysed by a phosphoS90-specific TIP60 antibody, and the blot was further probed with antibodies specific for FLAG and CDK9  (D) p53-/- hRasG12V MEF were treated with DMSO or 1µM SNS-032 (SNS) for 1h. Nuclear lysates were analysed by Western blotting  (E) p53-/- hRasG12V MEF were treated with DMSO, 1µM SNS-032 (SNS) or 50µM DRB followed by addition of 100nM Calyculin A as indicated. Nuclear lysates were analysed by Western blotting  (F) U2OS cells stably expressing FLAG-TIP60WT were transfected with a pool of four different CDK9-targeting siRNAs or a control siRNA targeting Luciferase. Nuclear lysates were analysed by Western blotting