EMBO/soda-nel huggingface dataset:
FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner (2019)
Figure 8

FIGURE 8. HOXB8 is a FOXA2 partner in the poorly differentiated PANC1 cells.

A) Distribution of the positions of the best HOXB motif relative to the summit of the PANC1-specific FOXA2 peaks. The best PWM identified in the enrichment motif analysis and used to perform the CentriMo analysis is indicated. The graph indicates the average density in 20 bp bins in a window of 600 bp.

B) Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP-seq peaks.

C) Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP-seq peaks in PANC1 cells. The FOXA2 ChIP-seq track in CFPAC1 cells is also shown for comparison.

D) FOXA2 ChIP-seq in CFPAC1 cells transduced with a HOXB8-expressing lentivirus and in their matched mock-infected control. Gained (brown) and lost (yellow) FOXA2 peaks are shown. Pie-charts on the right show the fraction of gained, unchanged and lost peaks overlapping HOXB8 ChIP-seq peaks in transduced CFPAC1 cells.

E) TF motif over-represented in gained or lost FOXA2-bound genomic regions.

F) Expression of HNF1β protein in wild-type and HOXB8-expressing PANC1 cells.

G) Representative snapshots from CFPAC1 cells transduced with the HOXB8-expressing lentivirus.

EXPANDED VIEW FIGURE LEGENDS
Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides (2021)
Figure 2

Fig. 2. (PR)20 peptides impair reactions using RNA substrates in vitro and in vivo.

(A) Reverse transcription of RNA (500 ng) with oligo-dT in the presence of increasing doses of (PR)20. Data represent the fluorometric quantification of the resultant cDNA (n=3), mean values ± SD.

(B) Percentage of RNA (1 μg) remaining after a 15´ digestion with increasing doses of RNase A in the presence or absence of (PR)20 (5 μM) (n=3). Data represent mean values ± SEM.

(C, D) High-throughput microscopy (HTM)-mediated analysis of 5-ethynyl-uridine (EU) (C) and polyA+ mRNA (D) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 16 h. EU was added 30´ prior to fixation (n=3). Black lines indicate median values in both panels. In D, boxes define upper and lower quartiles and whiskers mark the highest and lowest observations.

(E) Effect of (PR)20 (10 μM, 16 h) in the number of Cajal Bodies (CB) identified by immunofluorescence with anti-COILIN and anti-SMN antibodies (n=3). Data represent mean values ± SD.

(F) Accumulation of Sindibis virus (SV) genomic RNA in BHK-21 cells in the absence or presence of increasing doses of (PR)20. The amount of viral and cellular RNA was quantified from total RNA isolated 4 h after infection by quantitative real-time PCR (qRT-PCR) with specific primers (n=3). Data represent mean values ± SEM.

Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses (C-E) are provided in each case. Scale bar (white) indicates 2-5 μm. See also Appendix Fig. S1.
Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides (2021)
Figure 3

Fig. 3. Effects of (PR)20 on DNA-based reactions.

(A) Quantification of EMSA assays evaluating the binding of (PR)20 to 19 nt-long ssDNA, ssRNA, dsDNA and dsRNA molecules. Each probe (0.2 μM) was incubated with increasing concentrations of (PR)20 for 10´. Curve-fitting was performed using non-linear regression with the Hill equation.

(B, C) Percentage of GAPDH or 5.8 rRNA levels quantified by qPCR in reactions containing increasing doses of (PR)20 (n=3).Data represent mean values ± SEM.

(D) HTM-mediated analysis of 5-ethynyl-deoxyuridine (EdU) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 4 h. EdU was added 30´ prior to fixation. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.

(E) HTM-mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM (PR)20 for the indicated times. Note the accumulation of cells with persistent DNA damage (γH2AX) 10 h after IR in the presence of the DPR. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.

(F) Efficiency of CRISPR-mediated gene deletion quantified in mouse embryonic stem cells stably expressing EGFP, a doxycycline-inducible Cas9 and an EGFP-targeting sgRNA. The percentage of EGFP-positive cells was quantified by flow cytometry 72 h after inducing Cas9 expression with doxycycline in the presence or absence of 10 μM (PR)20 (n=3). Data represent mean values ± SEM.

(G,H) Turbidity of solutions containing (PR)20 mixed with the different dNTPs measured by the optical density at 600 nm (O.D. 600) (G) (n=3). Data represent mean values ± SEM. A representative image of these assays is provided in (H).

Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. See also Appendix Fig. S2.
Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides (2021)
Figure 4

Fig. 4. Protamine inhibits reactions using nucleic acid templates.

(A) Quantification of EMSA assays evaluating the binding of protamine to 19 nt-long ssDNA and ssRNA molecules. Each probe (0.2 μM) was incubated with increasing concentrations of (PR)20 for 10´. Curve-fitting was performed using non-linear regression with the Hill equation.

(B) Percentage of GAPDH levels quantified by qPCR in reactions containing increasing doses of protamine (n=3). Data represent mean values ± SEM.

(C) Percentage of RNA (1 μg) remaining after a 15´ digestion with increasing doses of RNase A in the presence or absence of protamine (5 μM). Data derive from one representative experiment that was repeated twice.

(D) Immunofluorescence of Cy3-labelled salmon protamine (red) and the nucleolar factor UBF1 (green) in U2OS cells treated with 30 μM Cy3-protamine for 4 h. Scale bar (white) indicates 2.5 μm.

(E, F) HTM-mediated quantification of the EU levels per nucleus (E) and O-propargyl-puromycin (OPP) levels per cell (F) in U2OS cells exposed to 30 μM protamine for 16 (E) or 24 (F) h. EU and OPP were added 30´ prior to fixation. Black lines indicate median values (****, p<0.0001; t-test).

(G) RT-PCR of NACA and GADD45A mRNAs in U2OS cells treated with protamine (30 μM) or (PR)20 (20 μM) using primers in non-consecutive exons to monitor alternative splicing events. A scheme of the primers used is provided on the right side of the panel.

(H) RT-PCR of GADD45A mRNAs in U2OS cells treated with HA-tagged (PR)20, (GR)20, (PK)20 and (GA)20 peptides (20 μM) using the pipeline defined in (G).
Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides (2021)
Figure 6

Fig. 6. Non-coding oligonucleotides rescue the effects of arginine rich CPPs.

(A) In vitro translation of a luciferase mRNA with or without of 0.5 μM (PR)20 and in the presence of increasing amounts of a 646 nt non-coding RNA (n=3). Data represent mean values ± SEM.

(B) HTM-mediated quantification of OPP levels per cell (PR)20 (10 µM, 16h) alone or together with a 38 nt ssDNA oligonucleotide at the indicated doses. Black lines indicate median values.

(C) Percentage of viable cells as evaluated with a CellTiter-Glo luminescent assay in U2OS cells treated with increasing doses of (PR)20 alone or together with 2 μM of ssDNA oligonucleotides (19 nt or 38 nt) (n=3). Data represent mean values ± SEM.

(D) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h.

(E) Immunofluorescence of HA-(PR)20 (green) and Cy5 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 4 μM of Cy5-labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h.

(F) Clonogenic survival assay of U2OS cells exposed to 7.5 μM with the indicated peptides or protamine (PROT.) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0.5 μM Heparin.

(G) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with heparin (0.5 μM).

Data information: See also Appendix Fig. S3. Scale bar (white) in (D), (E) and (G) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test. See Fig. S3 for an HTM-mediated quantification of the image analyses.
Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides (2021)
Figure 7

Fig. 7. Rescue of the toxicity of (PR)20 in mouse motor neurons.

(A) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without 2 μM of a non-coding 38nt oligonucleotide. Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. Note that besides its impact on overall survival, the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide.

(B,C) Cell viability quantified CellTiter-Glo luminescence assays mMN (B) and NSC34DIFF (C) treated as in (A) (n=3). Data represent mean values ± SEM.

(D) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without heparin (0.5 μM). Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm.

(E, F) Cell viability quantified CellTiter-Glo luminescence assays mMN (E) and NSC34DIFF (F) treated as in (D) (n=3). Data represent mean values ± SEM.

Data information: **, p<0.01; ***, p<0.001. See also Appendix Fig. S4.
Targeting netrin-1/DCC interaction in Diffuse Large-B and Mantle Cell Lymphoma (2015)
Figure 1

Figure 1. Inactivation of DCC-induced apoptosis favors lymphomagenesis in mice.

A. In absence of netrin-1, DCC induces apoptosis, unless mutated on its D1290 caspase cleavage site.

B. DCC and netrin-1 expression (FACS analysis) in B-cells lymphocytes isolated from DCC+/+ and DCCmut/mut mice spleen and lymph nodes. Results are presented as expression ratios between means expression levels in mutant animals (n=3) and control ones (n=3). No significant difference was observed between wild-type and mutant animals.

C-E. Incidence and characterization of lymphoid tumoral proliferations in DCC mutant mice. C. DCCmut/mut mouse with splenomegaly and systemic lymphadenopathy. Left panel: black and white arrows indicate respectively the inguinal and mesenteric lymph nodes. Right panel: enlarged spleen in a lymphoma-bearing mutant mouse (left) as compared to control organ (right). D. Incidence of lymphoid proliferations and lymphoma in DCC+/+ (n=19) and DCCmut/mut (n=29) mice. Tumors classification was performed blinded to genotype according to anatomopathologists consensus (29). *: p=0.036; one-tailed Fisher’s exact test. E. Histological analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice. Sections from control spleen, low-grade FL and high-grade DLBCL were stained with hematoxylin-eosin-safran or with antibodies to B220 or Ki67. w: white pulp, r: red pulp, cr: compressed red pulp.
Targeting netrin-1/DCC interaction in Diffuse Large-B and Mantle Cell Lymphoma (2015)
Figure 2

Figure 2. Alterations of netrin-1 and DCC expression levels in human DLBCL and MCL tip the scales towards loss of DCC-induced apoptosis.

A-B. Analysis of DCC (A) and netrin-1 (B) expression in a cohort of normal tonsils (n=9), Germinal-Center-B-Cell-like-DLBCL (n=13), Activated-B-Cell-like-DLBCL (n=18) and MCL (n=36). Quantification of genes expression was performed by Q-RT-PCR, relatively to HPRT housekeeping gene. **: p<0.005; ****: p<0.0001; two-sided Mann-Whitney U-test.

C-D. Analysis of netrin-1 expression in DLBCL (C) and MCL (D) by immunohistochemistry. C. 21 DLBCL patients biopsies were used to build a TMA. Normal tissues (normal tonsils, reactive lymph nodes, muscle, liver) are included (the left-most spot, upper panel, corresponds to liver). Enlarged representative image of netrin-1 expression in DLBCL is shown (lower panel). D. Representative images of netrin-1 expression in paraffin-embedded biopsies of MCL (n=9). Five tumors were positive for netrin-1 (left panel) and four were negative (right panel).
Targeting netrin-1/DCC interaction in Diffuse Large-B and Mantle Cell Lymphoma (2015)
Figure 3

Figure 3. Disruption of netrin-1 autocrine loop with an interfering-antibody triggers lymphoma cells death in vitro and slows down tumor growth in vivo.

A. Expression of netrin-1 and DCC in lymphoma cell lines. Quantification was performed by Q-RT-PCR in 21 lymphoma cell lines. HPRT housekeeping gene was used as a standardization control. Netrin-1 and DCC levels are indicated as follow: −, not detectable; + to ++++, moderate to very high expression. ABC and GC-DLBCL cell lines are highlighted respectively in blue and green, MCL ones are in grey. Expression of netrin-1 (in green) by immunofluorescence using netrin-1 antibody on OCI-Ly3 and Granta-519 cell lines is shown on the right panel. Nuclei are counterstained in blue by Hoechst.

B. Caspase-3 activity in Granta-519 cells after transfection of scrambled siRNA (si-Ctl) or netrin-1 siRNA (si-netrin-1), with or without DCC siRNA (si-DCC). Results are means+/-std of four independent experiments. $: p=0.03; *: p=0.02; two-sided Mann-Whitney U-test.

C. Schematic model of impact of restoration of DCC-induced cell death by using a netrin-1 targeting antibody.

D. Effect of net-1 mAb on Granta-519 (left panel) and OCI-Ly3 (right panel) cell density. Results are means+/-std indexed to control of four independent experiments. $: p=0.05; *: p=0.03; two-sided Mann-Whitney U-test.

E. Effect of net-1 mAb on induction of Granta-519 cells apoptosis, detected by TUNEL staining. Left panel: representative images are shown. TUNEL positive cells are labelled in red. Nuclei are counterstained in blue by Hoechst staining. Right panel: quantification of one representative experiment out of three performed is exposed. Results are presented as percentage of TUNEL-positive cells per field. *: p=0.04; two-sided Mann-Whitney U-test.

F. Caspase-3 activity in Granta-519 cells treated with net-1 mAb antibody or with an unrelated Ig-G1 antibody (Ctl), with or without silencing of DCC via siRNA (siDCC). Results are means+/-std of four independent experiments. $: p=0.05; *: p=0.02; two-sided Mann-Whitney U-test.

G-H. Effect of netrin-1 interfering antibody net-1 mAb on OCI-Ly3 (G) and Granta-519 (H) xenograft tumors growth. Results are presented as means+/-95% CIs of tumor growth rates from the beginning of treatment. *:p=0.02; **:p=0.01; $: p=0.007; $$:p=0.002; ***: p=0.0002; ****:p<0.0001; two-sided Student t-test (Granta-519) or Welch t-test (OCI-Ly3). I. Caspase-3 activity in tumors from control (n=4) or netrin-1-interfering antibody (n=4) treated mice. Tumors resection was performed at the end of the treatment. Results are means +/-std of caspase-3 activity. *:p=0.02; two-sided Mann-Whitney test.
Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy (2016)
Figure 1

Figure 1. Dose- and time-dependent CDDP-induced apoptosis of sensory hair cells in organ of Corti cultures.

A Confocal images showing the basal regions of cochlear explants treated with either culture medium alone or medium containing 5 µM, 10 µM, or 20 µM CDDP for 3 days. The explants were labeled with Myosin 7A (red) to identify hair cells and NF200 (green) to highlight auditory nerve fibers. Scale bar=20 µm. nf: nerve fibers, OHCs: outer hair cells, IHCs: inner hair cells.

B Dose-response curves of CDDP-induced loss of OHCs (blue line) and IHCs (red line) in basal cochlear regions. Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point).

C Effect over time on OHCs and IHCs treated with either culture medium alone (light blue and red lines for OHCs and IHCs, respectively), or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively). Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point).

D Representative Western blot analysis using antibodies against β-actin and cleaved caspase 3 in whole cochlear extracts.

E Histogram representing the change in cleaved caspase 3 expression levels over time in control and CDDP group (n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test (*P ≤ 0.028, **P=0.0007, cochleae at the different times after CDDP exposure vs. control 12h).

F, G Confocal images showing the basal region of cochlear explants treated with either culture medium alone or containing 10 µM CDDP for 1 (F) or 2 (G) days. Hair cells were identified using Myosin 7A (red), phosphatidylserine sites on the cell membrane surface were detected using fluorochrome-labeled Annexin V (green in F), and apoptotic DNA fragmentation was identified using a TUNEL apoptosis kit (green in G). The white arrow heads indicate cell surface Annexin V-positive labeling (lower left) and TUNEL-positive nuclei (lower right). Scale bar=15 µm.

Data information: all experiments were performed in triplicate. In B and C, continuous lines are sigmoidal fits to the data.
Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy (2016)
Figure 2

Figure 2. Distribution of γH2AX and 53BP1 foci induced by CDDP in IHCs versus OHCs.

A, F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and γH2AX (green in A), or Myosin 7A (red) and 53BP1 (green in F). The white arrow heads indicate CDDP-induced increase of γH2AX foci in both OHCs and IHCs. Scale bars: A and F=24 µm.

B, G Higher magnification images of representative OHC and IHC nuclei from all conditions tested. Scale bar=5 µm.

C, H Histograms displaying green fluorescent signal intensity of x-projections and 3D reconstruction images from OHC and IHC nuclei presented in B and G, respectively. F0 corresponds to background noise, gray dashed lines represent the threshold used to detect specific foci labeling. 3D images were reconstructed according to the threshold defined in the histograms of all conditions tested. Scale bar=5 µm.

D, E, I Quantification analysis of γH2AX foci number (D); total volume of foci per nucleus (E); 53BP1 foci number (I) from OHCs and IHCs treated with either culture medium alone (light blue and red lines for OHCs and IHCs, respectively), or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively, n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test (D: **P=0.007; E and I: **P ≤ 0.009. CDDP-exposed OHCs vs. CDDP-exposed IHCs).

J Higher magnification images showing representative OHC nuclei from the basal region of the organ of Corti treated with 10 µM CDDP for 1 day and immunolabeled for 53BP1 (red), γH2AX (green) and counterstained with Hoechst (blue). The 3D image shows co-localization of smaller sized 53BP1 foci within the γH2AX foci. Scale bar=5 µm.

Data information: all experiments were performed in triplicate.
Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy (2016)
Figure 3

Figure 3. Activation of the ATM-Chk2-p53 pathway upon CDDP treatment.

A Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-ATM (green). Scale bar=15 µm.

B Higher magnification images showing representative OHC and IHC nuclei from control and CDDP-exposed cells. Scale bar=5 µm.

C Histograms displaying green fluorescent signal intensity of x-projections of OHC and IHC nuclei from control and CDDP-exposed cells.

D Quantification analysis of p-ATM foci numbers in both OHCs and IHCs exposed to either culture medium alone (light blue and red lines for OHCs and IHCs, respectively) or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively, n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test (**P<0.008, ***P=0.00005, CDDP-exposed OHCs vs. CDDP-exposed IHCs).

E Higher magnification confocal and 3D images showing the co-localization of p-ATM foci (green) with 53BP1 foci (red) in an OHC nucleus from the basal region of the organ of Corti culture treated with 10 µM CDDP for 1 day. Scale bar=5 µm.

F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-Chk2 (green). Scale bar=15 µm.

G Histograms of green fluorescent signal intensity from OHC and IHC nuclei of a CDDP-treated organ of Corti explant.

H Quantification of Chk2-positive nuclei in both IHCs (red bars) and OHCs (blue bars) from control and CDDP-treated organ of Corti cultures (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. Kruskal–Wallis test followed by post hoc Dunn's test (*P=0.045, ***P=0.0002, CDDP-exposed IHCs vs. control IHCs; CDDP-exposed OHCs vs. control OHCs).

I Representative western blots using antibodies against p-p53 (serine 15), p53 and β-actin in control and 10 µM CDDP-exposed whole cochlear extracts.

J-K Histograms representing the levels of total p53 protein (J) and p53 phosphorylation (K) in control (gray bars) and CDDP-exposed cochleae (green bars, n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test (J: *P ≤ 0.023; K: *P=0.032, **P=0.007, ***P ≤ 0.0008, cochleae at the different times after CDDP exposure vs. control 12h).

Data information: all experiments were performed in triplicate.
Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy (2016)
Figure 4

Figure 4. Effect of ATM or p53 inhibition on DNA damage repair and hair cell survival.

A 3D images of OHC nuclei taken from the basal regions of organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.

B Quantification analysis of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test (***P ≤ 0.0004, CDDP vs. control, or CDDP vs. CDDP+KU55933).

C Confocal images showing the basal region of the organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 5 days and immunolabeled for Myosin 7A (red). Scale bar=24 µm.

D Histograms representing the numbers of surviving IHCs (red bars) and OHCs (blue bars) for all treatment conditions after 5 days (n=5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test (*P=0.03, **P =0.004, ***P ≤ 0.0008, CDDP vs. control, or CDDP vs. CDDP+KU55933).

E 3D images of OHC nuclei from the basal regions of organ of Corti cultures treated with culture medium alone, 100 µM PFT-α, 10 µM CDDP or 10 µM CDDP in combination with 100 µM PFT-α for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.

F Quantification of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test (***P ≤ 0.0007, CDDP vs. control).

Data information: all experiments were performed in triplicate.