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 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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 IHCnuclei 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 IHCnuclei 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 OHCsvs . CDDP - exposed IHCs ) . J Higher magnification images showing representative OHCnuclei 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 IHCnuclei 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 IHCnuclei 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 OHCsvs. CDDP-exposed IHCs).J Higher magnification images showing representative OHCnuclei 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.


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 IHCnuclei from control and CDDP - exposed cells . Scale bar = 5 µm . C Histograms displaying green fluorescent signal intensity of x - projections of OHC and IHCnuclei 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 OHCsvs . 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 OHCnucleus from the basal region of the organ of Corti culture treated with 10 µM CDDP for 1 day . Scale bar = 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

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 IHCnuclei from control and CDDP-exposed cells. Scale bar=5 µm.C Histograms displaying green fluorescent signal intensity of x-projections of OHC and IHCnuclei 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 OHCsvs. 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 OHCnucleus 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 IHCnuclei 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 IHCsvs . control IHCs ; CDDP - exposed OHCsvs . control OHCs ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O

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 IHCnuclei 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 IHCsvs. control IHCs; CDDP-exposed OHCsvs. 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

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).


A 3D images of OHCnuclei 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 , CDDPvs . control , or CDDPvs . CDDP + KU55933 ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

A 3D images of OHCnuclei 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, CDDPvs. control, or CDDPvs. 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 , CDDPvs . control , or CDDPvs . CDDP + KU55933 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

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, CDDPvs. control, or CDDPvs. CDDP+KU55933).


E 3D images of OHCnuclei 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 , CDDPvs . control ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

E 3D images of OHCnuclei 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, CDDPvs. control).


A Representative auditory brainstem response ( ABR ) waveforms evoked by 16 kHz tone - bursts in p53wt mice treated with saline ( black plot ) or CDDP ( green plot ) , and p53 - / - mice treated with saline ( light blue plot ) or CDDP ( dark blue plot ) for 5 days . B ABR thresholds recorded in p53wt mice before ( gray plot ) and after 5 days of saline ( black plot ) or CDDP treatments ( green plot ) , and ABR thresholds recorded in p53 - / - mice before ( light gray plot ) , and after 5 days of saline ( light blue plot ) or CDDP treatment ( dark blue plot ) . C Mean ABR threshold from 4 kHz to 32 kHz derived from B . Data are expressed as mean ± SEM ( saline treated - group : n = 7 , CDDP treated - group : n = 12 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * P ≤ 0 . 008 , p53wt + CDDP , d5 vs . p53wt , before or p53wt + CDDP , d5 vs . p53 - / - + CDDP , d5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O

A Representative auditory brainstem response (ABR) waveforms evoked by 16 kHz tone-bursts in p53wt mice treated with saline (black plot) or CDDP (green plot), and p53-/- mice treated with saline (light blue plot) or CDDP (dark blue plot) for 5 days.B ABR thresholds recorded in p53wt mice before (gray plot) and after 5 days of saline (black plot) or CDDP treatments (green plot), and ABR thresholds recorded in p53-/- mice before (light gray plot), and after 5 days of saline (light blue plot) or CDDP treatment (dark blue plot).C Mean ABR threshold from 4 kHz to 32 kHz derived from B. Data are expressed as mean ± SEM (saline treated-group: n=7, CDDP treated-group: n=12). One-way ANOVA test followed by post hoc Tukey's test (**P ≤ 0.008, p53wt+CDDP, d5 vs. p53wt, before or p53wt+CDDP, d5 vs. p53-/- + CDDP, d5).


D Representative scanning electron microscopy micrographs showing the basal regions of cochleae from CDDP - treated p53wt and p53 - / - mice after 5 days . Scale bar = 15µm .

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D Representative scanning electron microscopy micrographs showing the basal regions of cochleae from CDDP-treated p53wt and p53-/- mice after 5 days. Scale bar=15µm.


E Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from the cochlear apex provided from saline - treated p53wt mice ( black bars ) , CDDP - treated p53wt mice ( green bars ) or p53 - / - mice ( blue bars ) , after 5 days ( n = 5 per group ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 008 , p53wt + CDDP , d5 vs . p53wt + saline , d5 or p53wt + CDDP , d5 vs . p53 - / - + CDDP , d5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O

E Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apex provided from saline-treated p53wt mice (black bars), CDDP-treated p53wt mice (green bars) or p53-/- mice (blue bars), after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, p53wt+CDDP, d5 vs. p53wt+saline, d5 or p53wt+CDDP, d5 vs. p53-/- + CDDP, d5).


F ABR thresholds from p53wt mice recorded prior to ( gray plot ) or after 5 days systemic treatment with : DMSO ( yellow plot ) , CDDP + DMSO ( green plot ) , CDDP + PFT - α ( pink plot ) . G Mean ABR threshold from 4 kHz to 32 kHz derived from F . Data are expressed as mean ± SEM ( DMSO treated - group : n = 7 ; CDDP + DMSO treated - group : n = 12 ; CDDP + PFT - α - treated group : n = 12 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P ≤ 0 . 0005 , CDDP + DMSO , d5 vs . before or CDDP + DMSO , d5 vs . CDDP + PFT - α , d5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

F ABR thresholds from p53wt mice recorded prior to (gray plot) or after 5 days systemic treatment with: DMSO (yellow plot), CDDP+DMSO (green plot), CDDP+PFT-α (pink plot).G Mean ABR threshold from 4 kHz to 32 kHz derived from F. Data are expressed as mean ± SEM (DMSO treated-group: n=7; CDDP+DMSO treated-group: n=12; CDDP+ PFT-α-treated group: n=12). One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0005, CDDP+DMSO, d5 vs. before or CDDP+DMSO, d5 vs. CDDP+PFT-α, d5).


H Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from the cochlear apical end taken from p53wt mice treated with : DMSO ( yellow bars ) , CDDP + DMSO ( green bars ) , CDDP + PFT - α ( pink bars ) , after 5 days ( n = 5 per group ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 008 , CDDP + DMSO , d5 vs . DMSO , d5 or CDDP + DMSO , d5 vs . CDDP + PFT - α , d5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

H Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apical end taken from p53wt mice treated with: DMSO (yellow bars), CDDP+DMSO (green bars), CDDP+PFT-α (pink bars), after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP+DMSO, d5 vs. DMSO, d5 or CDDP+DMSO, d5 vs. CDDP+PFT-α, d5).


I ABR thresholds from p53wt mice recorded prior to ( gray plot and black plot for left and right ear , respectively ) or after 5 days of systemic treatment with CDDP plus intratympanic injection of DMSO into the left ear ( green plot ) , and of PFT - α into the right ear ( pink plot ) . J Mean ABR threshold from 4 kHz to 32 kHz derived from I . Data are expressed as mean ± SEM ( n = 14 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P = 0 . 0003 , CDDP + left ear , DMSOvs . before , left ear or CDDP + left ear , DMSOvs . CDDP + right ear , PFT - α .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

I ABR thresholds from p53wt mice recorded prior to (gray plot and black plot for left and right ear, respectively) or after 5 days of systemic treatment with CDDP plus intratympanic injection of DMSO into the left ear (green plot), and of PFT-α into the right ear (pink plot).J Mean ABR threshold from 4 kHz to 32 kHz derived from I. Data are expressed as mean ± SEM (n=14). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0003 , CDDP+left ear, DMSOvs. before, left ear or CDDP + left ear, DMSOvs. CDDP + right ear, PFT-α.


K Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from the cochlear apical end taken from the DMSO - treated left ear ( green bars ) and PFT - α treated right ear ( pink bars ) of the same CDDP - treated p53wt mice after 5 days ( n = 5 per group ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 008 , CDDP + right ear , PFT - α vs . CDDP + left ear , DMSO ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O

K Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apical end taken from the DMSO-treated left ear (green bars) and PFT-α treated right ear (pink bars) of the same CDDP-treated p53wt mice after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP + right ear, PFT-α vs. CDDP+left ear, DMSO).


A ABR thresholds recorded prior to ( gray plot ) or at day 28 in HBCx - 90 ( TP53 wt ) bearing - mice received systemic treatment with : DMSO ( black plot ) , PFT - α ( yellow plot ) , CDDP ( green plot ) , CDDP + PFT - α ( pink plot ) . B Mean ABR threshold from 4 kHz to 32 kHz derived from A . Data are expressed as mean ± SEM ( before : n = 20 ; DMSO : n = 5 ; PFT - α : n = 5 ; CDDP : n = 5 ; CDDP + PFT - α : n = 5 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P = 0 . 0002 , CDDP vs . before ; CDDP vs . CDDP + PFT - α . E ABR thresholds recorded prior to ( gray plot ) or at day 35 in HBCx - 14 ( TP53 mutant ) bearing - mice received systemic treatment with : DMSO ( black plot ) , PFT - α ( yellow plot ) , CDDP ( green plot ) , CDDP + PFT - α ( pink plot ) . F Mean ABR threshold from 4 kHz to 32 kHz derived from E . Data are expressed as mean ± SEM ( before : n = 30 ; DMSO : n = 5 ; PFT - α : n = 5 ; CDDP : n = 10 ; CDDP + PFT - α : n = 10 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P = 0 . 0003 , CDDPvs . before or CDDPvs . CDDP + PFT - α .

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A ABR thresholds recorded prior to (gray plot) or at day 28 in HBCx-90 (TP53 wt) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).B Mean ABR threshold from 4 kHz to 32 kHz derived from A. Data are expressed as mean ± SEM (before: n=20; DMSO: n=5; PFT-α: n=5; CDDP: n=5; CDDP+PFT-α: n=5). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0002, CDDP vs. before; CDDP vs. CDDP+PFT-α.E ABR thresholds recorded prior to (gray plot) or at day 35 in HBCx-14 (TP53 mutant) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).F Mean ABR threshold from 4 kHz to 32 kHz derived from E. Data are expressed as mean ± SEM (before: n=30; DMSO: n=5; PFT-α: n=5; CDDP: n=10; CDDP+PFT-α: n=10). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0003, CDDPvs. before or CDDPvs. CDDP+PFT-α.


C Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1 . 1 , 2 . 6 , and 3 . 5 mm from the cochlear apical end . These cochleae ( n = 5 per group ) were collected at 28 days from the beginning of treatment with DMSO ( black bars ) , PFT - α ( yellow bars ) , CDDP ( green bars ) , or CDDP + PFT - α ( pink bars ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 007 , CDDP , d28 vs . DMSO , d28 or CDDP , d28 vs . CDDP + PFT - α , d28 ) . G Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1 . 1 , 2 . 6 , and 3 . 5 mm from the cochlear apical end . These cochleae ( n = 5 per group ) were collected at 35 days from the beginning of treatment with DMSO ( black bars ) , PFT - α ( yellow bars ) , CDDP ( green bars ) , or CDDP + PFT - α ( pink bars ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 008 , CDDP , d35 vs . DMSO , d35 or CDDP , d35 vs . CDDP + PFT - α , d35 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

C Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 28 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.007, CDDP, d28 vs. DMSO, d28 or CDDP, d28 vs. CDDP+PFT-α, d28).G Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 35 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP, d35 vs. DMSO, d35 or CDDP, d35 vs. CDDP+PFT-α, d35).


D Tumor growth curves alongside images of dissected tumors collected on day 28 . Note the partial inhibition of growth in CDDP and CDDP + PFT - α - treated mice . H Tumor growth curves alongside images of dissected tumors collected on day 35 . Note the complete disappearance of tumors at day 35 and the significantly reduced tumor regrowth up to day 70 in CDDP + PFT - α - treated mice .

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D Tumor growth curves alongside images of dissected tumors collected on day 28. Note the partial inhibition of growth in CDDP and CDDP+PFT- α-treated mice.H Tumor growth curves alongside images of dissected tumors collected on day 35. Note the complete disappearance of tumors at day 35 and the significantly reduced tumor regrowth up to day 70 in CDDP+PFT- α-treated mice.


A Representative confocal images of microvessels in transversal tumor sections from HBCx - 14 ( TP53mutant ) tumors treated with either DMSO , CDDP or a combination of CDDP and PFT - α . The sections were immunolabeled for CD31 ( red ) and viewed with a 20x objective . The basal - like breast cancer cells were immunolabeled in green with an antibody against cytokeratin 5 and 8 . Upper right panel is a 2D projection from the white boxed area in upper left panel . The red area corresponds to CD31 labeled endothelial area and the white area represents the lumen area . The white line shows the vessel perimeter . Scale bars : left panel = 50 µm , right panels = 35 µm . The tumor samples were collected at day 21 . B Histograms representing the percentage of vascular area calculated using 2D reconstruction image analysis and the formula : vessel / vascular area = area of CD31 - positive objects + lumen area per field area × 100 % . The both HBCx - 14 ( TP53mutant ) and HBCx - 90 ( TP53wt ) tumors from the different treated groups were collected at day 21 ( n = 4 sections / tumor and 3 - 4 tumors / group ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * * P ≤ 0 . 0004 , CDDPvs . DMSO or CDDPvs . CDDP + PFT - α ) .

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A Representative confocal images of microvessels in transversal tumor sections from HBCx-14 (TP53mutant) tumors treated with either DMSO, CDDP or a combination of CDDP and PFT-α. The sections were immunolabeled for CD31 (red) and viewed with a 20x objective. The basal-like breast cancer cells were immunolabeled in green with an antibody against cytokeratin 5 and 8. Upper right panel is a 2D projection from the white boxed area in upper left panel. The red area corresponds to CD31 labeled endothelial area and the white area represents the lumen area. The white line shows the vessel perimeter. Scale bars: left panel=50 µm, right panels=35 µm. The tumor samples were collected at day 21.B Histograms representing the percentage of vascular area calculated using 2D reconstruction image analysis and the formula: vessel/vascular area = area of CD31-positive objects + lumen area per field area × 100 %. The both HBCx-14 (TP53mutant) and HBCx-90 (TP53wt) tumors from the different treated groups were collected at day 21 (n=4 sections/tumor and 3-4 tumors/group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (***P ≤0.0004, CDDPvs. DMSO or CDDPvs. CDDP+PFT-α).


C Representative confocal images of cleaved caspase 3 positive cells ( red ) in the transversal tumor sections and viewed with a 20x objective . The stromal compartments were immunolabeled in green with an antibody against vimentin . HBCx - 14 ( TP53mutant ) tumors from the different treated groups were collected at day 18 . Scale bar = 50 µm . c - cas - 3 : cleaved caspase 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Representative confocal images of cleaved caspase 3 positive cells (red) in the transversal tumor sections and viewed with a 20x objective. The stromal compartments were immunolabeled in green with an antibody against vimentin. HBCx-14 (TP53mutant) tumors from the different treated groups were collected at day 18. Scale bar=50 µm. c-cas-3: cleaved caspase 3.


D Representative western blot analysis using antibodies against β - actin , p53 and p21 in tumor extracts from HBCx - 14 ( TP53mutant ) and HBCx - 90 ( TP53 wt ) tumors treated with either DMSO or CDDP and collected at day 18 . Note the higher CDDP - induced increase of p53 and p21 expression in TP53wt HBCx - 90tumors .

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D Representative western blot analysis using antibodies against β-actin, p53 and p21 in tumor extracts from HBCx-14 (TP53mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO or CDDP and collected at day 18. Note the higher CDDP-induced increase of p53 and p21 expression in TP53wt HBCx-90tumors.


E , F Representative western blot analysis using antibodies against β - actin , p - Chk1 , Beclin 1 , LC3 - I / II and Rab7 in tumor extracts from HBCx - 14 ( TP53mutant ) and HBCx - 90 ( TP53 wt ) tumors treated with either DMSO , PFT - α , CDDP or a combination of CDDP and PFT - α . The tumor samples were collected at day 18 . G , H , I Histograms representing the levels of Beclin 1 , LC3 - II and Rab7 in HBCx - 14 and HBCx - 90tumors treated with the different regimens ( n = 3 - 4 tumors per group , all experiments were performed in triplicate ) . Actin served as a loading control . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( G : * P = 0 . 03 , * * P = 0 . 006 , * * * P ≤ 0 . 0004 ; H : * P = 0 . 02 , * * * P = 0 . 0005 ; I : * P = 0 . 03 , * * * P ≤ 0 . 0006 , CDDPvs . DMSO or CDDPvs . CDDP + PFT - α ) .

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E, F Representative western blot analysis using antibodies against β-actin, p-Chk1, Beclin 1, LC3-I/II and Rab7 in tumor extracts from HBCx-14 (TP53mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO, PFT-α, CDDP or a combination of CDDP and PFT-α. The tumor samples were collected at day 18.G, H, I Histograms representing the levels of Beclin 1, LC3-II and Rab7 in HBCx-14 and HBCx-90tumors treated with the different regimens (n=3-4 tumors per group, all experiments were performed in triplicate). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (G: *P = 0.03, **P =0.006 , ***P ≤ 0.0004 ; H: *P =0.02, ***P = 0.0005 ; I: *P =0.03, ***P ≤ 0.0006 , CDDPvs. DMSO or CDDPvs. CDDP+PFT-α).


To verify the specificity of Cx43 - CreERT mice for astrocytes , mice were crossed to a tdTomato reporter line to induce Cre - dependent tdTomato expression after tamoxifen administration . TdTomato fluorescence was enhanced with an antibody against red fluorescent protein , and astrocytes were stained with antibodies against GFAP or S100β ( not shown ) . Merged images and quantification show efficient and widespread tdTomato expression specific for astrocytes ( arrowheads ) in 8 - month and 11 - month old animals . Expression was small in neurons ( stained with NeuN , not shown ) and negligible in NG2 cells and oligodendrocytes . Scale bars , 250 µm ( upper panel ) and 50 µm ( lower panel ) .

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To verify the specificity of Cx43-CreERT mice for astrocytes, mice were crossed to a tdTomato reporter line to induce Cre-dependent tdTomato expression after tamoxifen administration. TdTomato fluorescence was enhanced with an antibody against red fluorescent protein, and astrocytes were stained with antibodies against GFAP or S100β (not shown). Merged images and quantification show efficient and widespread tdTomato expression specific for astrocytes (arrowheads) in 8-month and 11-month old animals. Expression was small in neurons (stained with NeuN, not shown) and negligible in NG2 cells and oligodendrocytes. Scale bars, 250 µm (upper panel) and 50 µm (lower panel).


The majority of peri - plaque astrocytes in APP / PS1 - Stat3WT were positive for activated / phosphorylated Stat3 ( pStat3 ) in the cortex ( Cx ) and hippocampus ( HC ) , whereas the fraction of pStat3 - positive astrocytes was significantly lower in APP / PS1 - Stat3KO mice in both regions ( n = 8 mice ( 3 females and 5 males ) per group ; age , 8 months ; Mann - Whitney test ) . The occurrence of pStat3 in WT - Stat3KO and WT - Stat3WT mice was negligible ( n = 8 mice ( 4 females and 4 males ) per group ; age , 8 months ; Mann - Whitney test ) .

The majority of peri-plaque astrocytes in APP/PS1-Stat3WT were positive for activated/phosphorylated Stat3 (pStat3) in the cortex (Cx) and hippocampus (HC), whereas the fraction of pStat3-positive astrocytes was significantly lower in APP/PS1-Stat3KO mice in both regions (n = 8 mice (3 females and 5 males) per group; age, 8 months; Mann-Whitney test). The occurrence of pStat3 in WT-Stat3KO and WT-Stat3WT mice was negligible (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test).


Examples of Stat3 activation in APP / PS1 - Stat3KO and APP / PS1 - Stat3WT . D , pStat3 ( arrowheads ) was abundantly present in reactive astrocytes ( identified by GFAP ) around plaques ( identified by IC16 ) . E , only few astrocytes were positive for pStat3 in APP / PS1 - Stat3WT mice . Scale bars , 50 µm .

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Examples of Stat3 activation in APP/PS1-Stat3KO and APP/PS1-Stat3WT. D, pStat3 (arrowheads) was abundantly present in reactive astrocytes (identified by GFAP) around plaques (identified by IC16). E, only few astrocytes were positive for pStat3 in APP/PS1-Stat3WT mice. Scale bars, 50 µm.


in postmortem brain sections from AD patients , we detected nuclear pStat3 ( arrows ) in the majority of peri - plaque reactive astrocytes ( identified by GFAP ) in the cortex and hippocampus ( plaques were stained with methoxy - XO4 , arrowheads ) . Scale bar , 50 µm .

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in postmortem brain sections from AD patients, we detected nuclear pStat3 (arrows) in the majority of peri-plaque reactive astrocytes (identified by GFAP) in the cortex and hippocampus (plaques were stained with methoxy-XO4, arrowheads). Scale bar, 50 µm.


Overall astrocytic and microglial coverage , as assessed by GFAP and Iba1 stainings , remained unchanged in APP / PS1 - Stat3WT vs . APP / PS1 - Stat3KO mice ( n = 8 mice ( 4 females and 4 males ) per group ; age , 8 months ; Mann - Whitney test for each comparison ) . Scale bars , 500 µm .

Overall astrocytic and microglial coverage, as assessed by GFAP and Iba1 stainings, remained unchanged in APP/PS1-Stat3WT vs. APP/PS1-Stat3KO mice (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test for each comparison). Scale bars, 500 µm.


The volume of peri - plaque reactive astrocytes was increased by the Stat3 deletion ( Mann - Whitney test ) , and there was a nonsignificant trend towards more astrocytes branches and junctions and longer total process length ( Mann - Whitney test ; APP / PS1 - Stat3WT , n = 5 ( 2 female and 3 male ) mice ; APP / PS1 - Stat3KO , n = 7 ( 3 female and 4 male ) mice ; age , 8 - 10 months ) . Scale bar , 20 µm .

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The volume of peri-plaque reactive astrocytes was increased by the Stat3 deletion (Mann-Whitney test), and there was a nonsignificant trend towards more astrocytes branches and junctions and longer total process length (Mann-Whitney test; APP/PS1-Stat3WT, n = 5 (2 female and 3 male) mice; APP/PS1-Stat3KO, n = 7 (3 female and 4 male) mice; age, 8-10 months). Scale bar, 20 µm.


Microglial volume was not affected by the Stat3 deletion ( Mann - Whitney test ) , but peri - plaque microglia had significantly more microglial branches and junctions per plaque , and the total process length of peri - plaque microglia was increased ( Mann - Whitney test ; same mice as in C - D ) . Scale bar , 20 µm .

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Microglial volume was not affected by the Stat3 deletion (Mann-Whitney test), but peri-plaque microglia had significantly more microglial branches and junctions per plaque, and the total process length of peri-plaque microglia was increased (Mann-Whitney test; same mice as in C-D). Scale bar, 20 µm.


Aβ plaque burden , as assessed by plaque load and size using an anti - Aβ antibody , was strongly reduced in APP / PS1 - Stat3KO vs . APP / PS1 - Stat3WT mice ( Mann - Whitney test ; scale bar , 300 µm ) .

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Aβ plaque burden, as assessed by plaque load and size using an anti-Aβ antibody, was strongly reduced in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 300 µm).


Plaque density , assessed by staining brain sections with thioflavin ( yellow ) , remained unchanged in APP / PS1 - Stat3KO vs . APP / PS1 - Stat3WT mice ( Mann - Whitney test ; scale bar , 250 µm ) .

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Plaque density, assessed by staining brain sections with thioflavin (yellow), remained unchanged in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 250 µm).


Electrochemiluminescence ELISA after sequential extraction from whole - brain homogenates using RIPA and SDS buffer revealed that soluble Aβ1 - 42 and Aβ1 - 40 were significantly reduced ( Mann - Whitney test ) , whereas there was a nonsignificant trend towards reduced levels of insoluble Aβ1 - 42 and Aβ1 - 40 ( Mann - Whitney test ) .

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Electrochemiluminescence ELISA after sequential extraction from whole-brain homogenates using RIPA and SDS buffer revealed that soluble Aβ1-42 and Aβ1-40 were significantly reduced (Mann-Whitney test), whereas there was a nonsignificant trend towards reduced levels of insoluble Aβ1-42 and Aβ1-40 (Mann-Whitney test).


Western Blot quantification of full - length amyloid precursor protein ( APP ) and its C - terminal fragments ( CTF ) showed no differences between both groups ( Mann - Whitney test ; for all datasets , n = 9 male mice for both groups ( age , 11 months ) ) .

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Western Blot quantification of full-length amyloid precursor protein (APP) and its C-terminal fragments (CTF) showed no differences between both groups (Mann-Whitney test; for all datasets, n = 9 male mice for both groups (age, 11 months)).


Internalization of Aβ ( stained with IC16 antibody or methoxy - XO4 ) was assessed using an engulfment assay , in which glial and Aβ structures were surface - rendered and Aβ volumes co - localized with glial volumes were quantified . Scale bars , 10 µm .

Internalization of Aβ (stained with IC16 antibody or methoxy-XO4) was assessed using an engulfment assay, in which glial and Aβ structures were surface-rendered and Aβ volumes co-localized with glial volumes were quantified. Scale bars, 10 µm.


Microglia ( left Y axes ) from APP / PS1 mice internalized significantly more Aβ positive for IC16 when Stat3 was deleted in astrocytes ( Mann - Whitney test ) , whereas no changes were seen in astrocytes ( right axes ; APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 11 ( 5 female and 6 male ) mice ; age , 11 months ; Mann - Whitney test ) .

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Microglia (left Y axes) from APP/PS1 mice internalized significantly more Aβ positive for IC16 when Stat3 was deleted in astrocytes (Mann-Whitney test), whereas no changes were seen in astrocytes (right axes; APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 11 (5 female and 6 male) mice; age, 11 months; Mann-Whitney test).


Microglia ( left Y axes ) from APP / PS1 mice internalized significantly more Aβ positive for methoxy - XO4 when Stat3 was deleted in astrocytes ( Mann - Whitney test ) , whereas no changes were seen in astrocytes ( right axes ; APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 11 ( 5 female and 6 male ) mice ; age , 11 months ; Mann - Whitney test ) .

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Microglia (left Y axes) from APP/PS1 mice internalized significantly more Aβ positive for methoxy-XO4 when Stat3 was deleted in astrocytes (Mann-Whitney test), whereas no changes were seen in astrocytes (right axes; APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 11 (5 female and 6 male) mice; age, 11 months; Mann-Whitney test).


Western Blot quantification of protein levels of the Aβ - degrading enzymes neprilysin / CD10 and CD36 , as well as the Aβ - binding apoliprotein E ( apoE ) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE ( APP / PS1 - Stat3WT , n = 9 ( 5 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 9 ( 5 female and 4 male ) mice ; age , 11 months ; Mann - Whitney test for all comparisons ) . In contrast , TREM2 expression remained unchanged ( APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 7 ( 4 female and 3 male ) mice ; age , 11 months ; Mann - Whitney test ) .

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Western Blot quantification of protein levels of the Aβ-degrading enzymes neprilysin/CD10 and CD36, as well as the Aβ-binding apoliprotein E (apoE) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE (APP/PS1-Stat3WT, n = 9 (5 female and 4 male) mice; APP/PS1-Stat3KO, n = 9 (5 female and 4 male) mice; age, 11 months; Mann-Whitney test for all comparisons). In contrast, TREM2 expression remained unchanged (APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 7 (4 female and 3 male) mice; age, 11 months; Mann-Whitney test).


Western Blot of protein levels of the Aβ - degrading enzymes neprilysin / CD10 and CD36 , as well as the Aβ - binding apoliprotein E ( apoE ) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE ( APP / PS1 - Stat3WT , n = 9 ( 5 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 9 ( 5 female and 4 male ) mice ; age , 11 months ; Mann - Whitney test for all comparisons ) . In contrast , TREM2 expression remained unchanged ( APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 7 ( 4 female and 3 male ) mice ; age , 11 months ; Mann - Whitney test ) .

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Western Blot of protein levels of the Aβ-degrading enzymes neprilysin/CD10 and CD36, as well as the Aβ-binding apoliprotein E (apoE) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE (APP/PS1-Stat3WT, n = 9 (5 female and 4 male) mice; APP/PS1-Stat3KO, n = 9 (5 female and 4 male) mice; age, 11 months; Mann-Whitney test for all comparisons). In contrast, TREM2 expression remained unchanged (APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 7 (4 female and 3 male) mice; age, 11 months; Mann-Whitney test).


Quantitative PCR from cortex of APP / PS1 - Stat3KO compared to APP / PS1 - Stat3WT mice revealed lower expression of ' A1 ' markers Amigo2 and C3 , whereas Ggta1 remained unchanged . In turn , the ' A2 ' marker Tm4sf1 was upregulated and there was a nonsignificant trend for a higher expression of B3gnt5 ( n = 6 mice ( 3 females and 3 males ) per group ; age , 8 months ; Mann - Whitney test ) .

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Quantitative PCR from cortex of APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice revealed lower expression of 'A1' markers Amigo2 and C3, whereas Ggta1 remained unchanged. In turn, the 'A2' marker Tm4sf1 was upregulated and there was a nonsignificant trend for a higher expression of B3gnt5 (n = 6 mice (3 females and 3 males) per group; age, 8 months; Mann-Whitney test).


Confirming lower expression of the ' A1 ' marker C3d , western blot analysis indicated lower protein levels of C3d in APP / PS1 - Stat3KO . APP / PS1 - Stat3WT , n = 6 ( 2 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 3 female and 3 male ) mice ; age , 8 months ; Mann - Whitney test ) .

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Confirming lower expression of the 'A1' marker C3d, western blot analysis indicated lower protein levels of C3d in APP/PS1-Stat3KO. APP/PS1-Stat3WT, n = 6 (2 female and 4 male) mice; APP/PS1-Stat3KO, n = 6 (3 female and 3 male) mice; age, 8 months; Mann-Whitney test).


Immunohistochemistry using an antibody against C3d revealed that lower expression of C3d particularly occurred in peri - plaque reactive astrocytes ( arrows ; plaques were visualized with methoxy - XO4 , arrowheads ; scale bars , 50 µm ; APP / PS1 - Stat3WT , n = 6 ( 2 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 3 female and 3 male ) mice ; age , 8 months ; Mann - Whitney test ) .

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Immunohistochemistry using an antibody against C3d revealed that lower expression of C3d particularly occurred in peri-plaque reactive astrocytes (arrows; plaques were visualized with methoxy-XO4, arrowheads; scale bars, 50 µm; APP/PS1-Stat3WT, n = 6 (2 female and 4 male) mice; APP/PS1-Stat3KO, n = 6 (3 female and 3 male) mice; age, 8 months; Mann-Whitney test).


Whole - brain levels of the pro - inflammatory cytokines IL - 1β and TNF - α were significantly reduced in APP / PS1 - Stat3KO mice ( Mann - Whitney test ) , whereas no changes were seen for IL - 10 ( APP / PS1 - Stat3WT , n = 13 ( 6 female and 7 male ) mice ; APP / PS1 - Stat3KO , n = 13 ( 8 female and 5 male ) mice ; age , 11 months ; Mann - Whitney test ) .

Whole-brain levels of the pro-inflammatory cytokines IL-1β and TNF-α were significantly reduced in APP/PS1-Stat3KO mice (Mann-Whitney test), whereas no changes were seen for IL-10 (APP/PS1-Stat3WT, n = 13 (6 female and 7 male) mice; APP/PS1-Stat3KO, n = 13 (8 female and 5 male) mice; age, 11 months; Mann-Whitney test).


These changes were paralleled by a decrease in the area covered by dystrophic neurites in APP / PS1 - Stat3KO compared to APP / PS1 - Stat3WT mice , as assessed by LAMP1 staining ( Mann - Whitney test ; n = 10 male mice for both groups ; age , 8 months ; scale bars , 300 µm ) .

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These changes were paralleled by a decrease in the area covered by dystrophic neurites in APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice, as assessed by LAMP1 staining (Mann-Whitney test; n = 10 male mice for both groups; age, 8 months; scale bars, 300 µm).


For in vivo two - photon imaging , astrocytes ( arrows ) and neurons ( arrowheads ) were labeled with the calcium indicator OGB - 1 , and astrocytes were co - labeled with sulforhodamine 101 ( SR101 ; arrows ) . Aβ plaques were labeled with the intravital dye methoxy - XO4 ( open arrowheads ) . Scale bar , 50 µm .

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For in vivo two-photon imaging, astrocytes (arrows) and neurons (arrowheads) were labeled with the calcium indicator OGB-1, and astrocytes were co-labeled with sulforhodamine 101 (SR101; arrows). Aβ plaques were labeled with the intravital dye methoxy-XO4 (open arrowheads). Scale bar, 50 µm.


Calcium imaging of anesthetized animals showed that the hyperactivity of astrocytes in APP / PS1 - Stat3KO mice was reduced to levels comparable to WT - Stat3WT mice , but significantly increased in APP / PS1 - Stat3WT mice ( One - Way ANOVA followed by Bonferroni ' s multiple comparison test ; n = 6 mice ( 3 female and 3 male ) for each group ; age , 8 months ) .

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Calcium imaging of anesthetized animals showed that the hyperactivity of astrocytes in APP/PS1-Stat3KO mice was reduced to levels comparable to WT-Stat3WT mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; n = 6 mice (3 female and 3 male) for each group; age, 8 months).


Similarly , neuronal activity was also reduced to levels comparable to WT - Stat3WT mice in APP / PS1 - Stat3KO mice , but significantly increased in APP / PS1 - Stat3WT mice ( One - Way ANOVA followed by Bonferroni ' s multiple comparison test ; same mice as in B ) . The cumulative distributions of neuronal calcium transients in APP / PS1 - Stat3KO mice were not different from those of WT - Stat3WT mice ( p = 0 . 31 , Kolmogorov - Smirnov test ) , but significantly different from those of APP / PS1 - Stat3WT mice ( p = 0 . 001 , Kolmogorov - Smirnov test ) .

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Similarly, neuronal activity was also reduced to levels comparable to WT-Stat3WT mice in APP/PS1-Stat3KO mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; same mice as in B). The cumulative distributions of neuronal calcium transients in APP/PS1-Stat3KO mice were not different from those of WT-Stat3WT mice (p = 0.31, Kolmogorov-Smirnov test), but significantly different from those of APP/PS1-Stat3WT mice (p = 0.001, Kolmogorov-Smirnov test).


Two - photon imaging of these mice confirmed that astrocytic hyperactivity were reduced by MRS2179 ( APP / PS1 - Stat3WT , n = 6 ( 4 female and 2 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 2 female and 4 male ) mice ; age , 8 months ; Mann - Whitney test ) .

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Two-photon imaging of these mice confirmed that astrocytic hyperactivity were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test).


Two - photon imaging of these mice confirmed that propagating calcium waves were reduced by MRS2179 ( APP / PS1 - Stat3WT , n = 6 ( 4 female and 2 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 2 female and 4 male ) mice ; age , 8 months ; Mann - Whitney test ) .

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Two-photon imaging of these mice confirmed that propagating calcium waves were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test).


MRS2179 ( APP / PS1 - Stat3WT , n = 6 ( 4 female and 2 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 2 female and 4 male ) mice ; age , 8 months ; Mann - Whitney test ) . This network normalization induced a reduction of activated Stat3 ( pStat3 ) in astrocytes assessed in fixed brain sections of the same mice ( Mann - Whitney test ) . Scale bars , 50 µm .

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MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test). This network normalization induced a reduction of activated Stat3 (pStat3) in astrocytes assessed in fixed brain sections of the same mice (Mann-Whitney test). Scale bars, 50 µm.


Spatial learning and memory were assessed in the Morris Water Maze paradigm . APP / PS1 - Stat3KO mice showed faster latencies to reach the hidden platform compared with APP / PS1 - Stat3WT on days 4 and 5 , but were similar to WT - Stat3WT and WT - Stat3KO mice ( Two - way repeated - measures ANOVA followed by Bonferroni post - hoc test ; P values are for APP / PS1 - Stat3KO vs . APP / PS1 - Stat3WT mice ) .

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Spatial learning and memory were assessed in the Morris Water Maze paradigm. APP/PS1-Stat3KO mice showed faster latencies to reach the hidden platform compared with APP/PS1-Stat3WT on days 4 and 5, but were similar to WT-Stat3WT and WT-Stat3KO mice (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test; P values are for APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice).


The area under the curve ( AUC ) for the latency to reach the hidden platform was similar in APP / PS1 - Stat3WT compared to WT - Stat3WT and WT - Stat3KO mice , but significantly higher in APP / PS1 - Stat3KO mice ( Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ) .

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The area under the curve (AUC) for the latency to reach the hidden platform was similar in APP/PS1-Stat3WT compared to WT-Stat3WT and WT-Stat3KO mice, but significantly higher in APP/PS1-Stat3KO mice (Kruskal-Wallis test followed by Dunn's multiple comparisons test).


The swimming velocity was similar in all groups ( Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ) .

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The swimming velocity was similar in all groups (Kruskal-Wallis test followed by Dunn's multiple comparisons test).


In the probe trial 24 h after the last training day , the time mice spent in the target quadrant ( TQ ) was different from chance in all groups except for APP / PS1 - Stat3WT mice ( p < 0 . 05 , one - tailed one - sample t test ) . APP / PS1 - Stat3KO , WT - Stat3WT and WT - Stat3KO mice spent significantly more time in the target quadrant compared to the mean of all other quadrants ( AO ) , whereas APP / PS1 - Stat3WT spent equal times in the target and all other quadrants ( Wilcoxon matched - pairs signed rank test for each comparison ; APP / PS1 - Stat3WT , n = 15 ( 8 female and 7 male ) mice ; APP / PS1 - Stat3KO , n = 15 ( 9 female and 6 male ) mice ; age , 8 - 9 months ) .

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In the probe trial 24 h after the last training day, the time mice spent in the target quadrant (TQ) was different from chance in all groups except for APP/PS1-Stat3WT mice (p < 0.05, one-tailed one-sample t test). APP/PS1-Stat3KO, WT-Stat3WT and WT-Stat3KO mice spent significantly more time in the target quadrant compared to the mean of all other quadrants (AO), whereas APP/PS1-Stat3WT spent equal times in the target and all other quadrants (Wilcoxon matched-pairs signed rank test for each comparison; APP/PS1-Stat3WT, n = 15 (8 female and 7 male) mice; APP/PS1-Stat3KO, n = 15 (9 female and 6 male) mice; age, 8-9 months).


APP / PS1 mice were systemically treated with SH - 4 - 54 for 6 weeks and compared to age - matched APP / PS1 mice treated with vehicle . In the Morris Water Maze test , treatment with SH - 4 - 54 led to significantly faster latencies to reach the hidden platform on the last training day ( Two - way repeated - measures ANOVA followed by Bonferroni post - hoc test ) .

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APP/PS1 mice were systemically treated with SH-4-54 for 6 weeks and compared to age-matched APP/PS1 mice treated with vehicle. In the Morris Water Maze test, treatment with SH-4-54 led to significantly faster latencies to reach the hidden platform on the last training day (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test).


The area under the curve ( AUC ) for the latency to reach the hidden platform was also smaller in APP / PS1 mice treated with the Stat3 inhibitor ( Mann - Whitney test ) .

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The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (Mann-Whitney test).


The swimming velocity was not affected by the treatment ( Mann - Whitney test ) .

The swimming velocity was not affected by the treatment (Mann-Whitney test).


In the probe trial test , APP / PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant ( TQ ) compared to the mean of all other quadrants ( AO ) , whereas APP / PS1 mice treated with vehicle spent equal times in the target and all other quadrants ( Wilcoxon matched - pairs signed rank test for all comparisons ; APP / PS1 - Stat3WT , n = 12 ( 5 female and 7 male ) mice ; APP / PS1 - Stat3KO , n = 12 ( 4 female and 8 male ) mice ; age , 8 months ) .

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In the probe trial test, APP/PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant (TQ) compared to the mean of all other quadrants (AO), whereas APP/PS1 mice treated with vehicle spent equal times in the target and all other quadrants (Wilcoxon matched-pairs signed rank test for all comparisons; APP/PS1-Stat3WT, n = 12 (5 female and 7 male) mice; APP/PS1-Stat3KO, n = 12 (4 female and 8 male) mice; age, 8 months).


( E ) To verify target engagement in the brain , the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two - photon microscopy of calcium activity . Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons ( Mann - Whitney test ) .

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(E) To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium activity. Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons (Mann-Whitney test).


SH - 4 - 54 significantly decreased plaque growth , as assessed by IC16 immunohistochemistry , while plaque load remained unchanged .

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SH-4-54 significantly decreased plaque growth, as assessed by IC16 immunohistochemistry, while plaque load remained unchanged.


SH - 4 - 54 significantly There was also no significant change in dystrophic neurite area during the treatment time ( Mann - Whitney test for all comparisons ; scale bars , 500 µm ) .

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SH-4-54 significantly There was also no significant change in dystrophic neurite area during the treatment time (Mann-Whitney test for all comparisons; scale bars, 500 µm).


The fraction of pStat3 - positive astrocytes in the peri - plaque region was strongly reduced by the treatment with the Stat3 inhibitor ( arrowheads indicate pStat3 signals ; scale bars , 100 µm ; Mann - Whitney test ) .

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The fraction of pStat3-positive astrocytes in the peri-plaque region was strongly reduced by the treatment with the Stat3 inhibitor (arrowheads indicate pStat3 signals; scale bars, 100 µm; Mann-Whitney test).


While no changes were seen in morphological parameters of peri - plaque astrocytes , there was a significant increase in the process length of near - plaque microglia , indicating higher microglial complexity ( Mann - Whitney test for all comparisons ; APP / PS1 - Stat3WT , n = 12 ( 6 female and 6 male ) mice ; APP / PS1 - Stat3KO , n = 12 ( 7 female and 5 male ) mice ; age , 8 months ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

While no changes were seen in morphological parameters of peri-plaque astrocytes, there was a significant increase in the process length of near-plaque microglia, indicating higher microglial complexity (Mann-Whitney test for all comparisons; APP/PS1-Stat3WT, n = 12 (6 female and 6 male) mice; APP/PS1-Stat3KO, n = 12 (7 female and 5 male) mice; age, 8 months).


( B ) Electropherograms of DNA sequencing , family pedigrees , and audiograms of two unrelated patients . Electropherograms show a heterozygous c . 3610C > T substitution resulting in a stop codon ( R1204X ) . Arrows in family pedigrees indicate the patients identified in the present study . In audiograms : red and blue indicate right and left ; respectively , solid and dashed lines and square brackets show air conduction hearing and bone conduction hearing ; respectively . The bold line ( at the age of 48 years ) and thin line ( at the age of 50 years ) in the audiogram of patient 1 indicate progressive hearing loss ( green arrow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Electropherograms of DNA sequencing, family pedigrees, and audiograms of two unrelated patients. Electropherograms show a heterozygous c.3610C>T substitution resulting in a stop codon (R1204X). Arrows in family pedigrees indicate the patients identified in the present study. In audiograms: red and blue indicate right and left; respectively, solid and dashed lines and square brackets show air conduction hearing and bone conduction hearing; respectively. The bold line (at the age of 48 years) and thin line (at the age of 50 years) in the audiogram of patient 1 indicate progressive hearing loss (green arrow).


GFP - tagged DIA1 , DIA1 ( M1190D ) , and DIA1 ( R1204X ) plasmids were transfected into MDCKmCherry - ESPIN1 cells cultured on membrane inserts . Twenty - four hours after transfection , a reconstructed lateral view of fixed cells was generated using a confocal laser fluorescence microscope . Arrows indicate the elongated microvilli visualized by mCherry - ESPIN1 with GFP - hDIA1 ( R1204X ) expression . Scale bars : 5 μm . Representative of 3 experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GFP-tagged DIA1, DIA1(M1190D), and DIA1(R1204X) plasmids were transfected into MDCKmCherry-ESPIN1 cells cultured on membrane inserts. Twenty-four hours after transfection, a reconstructed lateral view of fixed cells was generated using a confocal laser fluorescence microscope. Arrows indicate the elongated microvilli visualized by mCherry-ESPIN1 with GFP-hDIA1(R1204X) expression. Scale bars: 5 μm. Representative of 3 experiments.


Various GFP - tagged DIA1 plasmids were transfected into HeLa cells , and fixed 24 h after transfection . Fixed cells were stained with Alexa568 - conjugated phalloidin and DAPI ( A , B : representative of 5 experiments ) . ( A ) Stress fiber formation was observed with a confocal laser microscope . Note the induction of stress fibers by GFP - DIA1 ( M1190D ) ( asterisks ) . Arrow and arrowheads indicate a plasma membrane ( PM ) localization of GFP - DIA1 ( M1190D ) and a dot - like localization of GFP - DIA1 ( R1204X ) adjacent to the PM , respectively . Inset in the top panel of DIA1 ( R1204X ) shows the magnified image of the region indicated by rectangle . Scale bars : 10 μm . .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(A) Stress fiber formation was observed with a confocal laser microscope. Note the induction of stress fibers by GFP-DIA1(M1190D) (asterisks). Arrow and arrowheads indicate a plasma membrane (PM) localization of GFP-DIA1(M1190D) and a dot-like localization of GFP-DIA1(R1204X) adjacent to the PM, respectively. Inset in the top panel of DIA1 (R1204X) shows the magnified image of the region indicated by rectangle. Scale bars: 10 μm..


Various GFP - tagged DIA1 plasmids were transfected into HeLa cells , and fixed 24 h after transfection . Fixed cells were stained with Alexa568 - conjugated phalloidin and DAPI ( A , B : representative of 5 experiments ) . ( B ) Microvilli with or without GFP - DIA1 ( R1204X ) expression were observed under a confocal laser microscope . Note elongated and thick microvilli ( arrowheads ) in GFP - DIA1 ( R1204X ) - transfected cells , but not in non - transfected cells ( circles ) . 3D movies are available in Movie EV1 ( R1204X ) , Movie EV2 ( WT ) , and Movie EV3 ( ttaa ) . Scale bar : 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(B) Microvilli with or without GFP-DIA1(R1204X) expression were observed under a confocal laser microscope. Note elongated and thick microvilli (arrowheads) in GFP-DIA1(R1204X)- transfected cells, but not in non-transfected cells (circles). 3D movies are available in Movie EV1 (R1204X), Movie EV2 (WT), and Movie EV3 (ttaa). Scale bar: 10 μm.


( B ) Purified GST and GST - tagged DID proteins ( 50 nM ) were mixed with biotin - labeled DADs ( WT ; R1204X ; and M1190D , 50 nM ) in binding buffer . After rotation , streptavidin - coupled magnetic beads were added to the solution , and the mixture was agitated . The material absorbed to the beads was eluted in Laemmli ' s sample buffer , and were subjected to SDS - PAGE , followed by immunoblotting using an HRP - conjugated GST Ab . Comparable levels of input ( GSTproteins ) are confirmed in the right panel . Representative of 5 experiments .

(B) Purified GST and GST-tagged DID proteins (50 nM) were mixed with biotin-labeled DADs (WT; R1204X; and M1190D, 50 nM) in binding buffer. After rotation, streptavidin-coupled magnetic beads were added to the solution, and the mixture was agitated. The material absorbed to the beads was eluted in Laemmli's sample buffer, and were subjected to SDS-PAGE, followed by immunoblotting using an HRP-conjugated GST Ab. Comparable levels of input (GSTproteins) are confirmed in the right panel. Representative of 5 experiments.


GFP - tagged DIA1 ( WT ) , DIA1 ( R1204X ) , and DIA1 ( M1190D ) were transfected into XTC cells . XTC cells were spread on the PLL - coated coverslips and time - lapse images were acquired every 300 ms , illuminating the restricted areas near the cell edges , by using a single - molecule speckle microscope . Representative images are shown for DIA1 ( WT ) ( A , Movie EV5 ) , DIA1 ( R1204X ) ( B , Movie EV6 ) , and DIA1 ( M1190D ) ( C , Movie EV7 ) . Circles indicate speckles showing directional movement over a distance of several micrometers . Trajectories of these speckles are also illustrated , and crosses were plotted at the end of the directional movement . Speckles showing directional movement were frequently observed in cells expressing GFP - DIA1 ( R1204X ) ( B ) , whereas such speckles were scarce in cells expressing GFP - DIA1 ( WT ) ( A ) . However , the density of these speckles in GFP - DIA1 ( R1204X ) - expressing cells appeared lower than that in GFP - DIA1 ( M1190D ) - expressing cells , which is considered a fully active mutant ( C ) . To evaluate the frequency of speckles indicating directional movement , we performed a quantitative analysis ( D ) . The number of speckles showing directional movement during 3 continuous planes was counted and were normalized by the fluorescent intensity of each cell , which corresponds to the expression level of the GFP - tagged protein . * P = 0 . 0288 and * * P = 0 . 0002 Bonferroni ' s post hoc test following one - way ANOVA ( WT ; n = 5 , R1204X ; n = 6 , M1190D ; n = 6 ) . Time is in seconds . Scale bars : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GFP-tagged DIA1(WT), DIA1(R1204X), and DIA1(M1190D) were transfected into XTC cells. XTC cells were spread on the PLL-coated coverslips and time-lapse images were acquired every 300 ms, illuminating the restricted areas near the cell edges, by using a single-molecule speckle microscope. Representative images are shown for DIA1(WT) (A, Movie EV5), DIA1(R1204X) (B, Movie EV6), and DIA1(M1190D) (C, Movie EV7). Circles indicate speckles showing directional movement over a distance of several micrometers. Trajectories of these speckles are also illustrated, and crosses were plotted at the end of the directional movement. Speckles showing directional movement were frequently observed in cells expressing GFP-DIA1(R1204X) (B), whereas such speckles were scarce in cells expressing GFP-DIA1(WT) (A). However, the density of these speckles in GFP-DIA1(R1204X)-expressing cells appeared lower than that in GFP-DIA1(M1190D)-expressing cells, which is considered a fully active mutant (C). To evaluate the frequency of speckles indicating directional movement, we performed a quantitative analysis (D). The number of speckles showing directional movement during 3 continuous planes was counted and were normalized by the fluorescent intensity of each cell, which corresponds to the expression level of the GFP-tagged protein. *P= 0.0288 and **P= 0.0002 Bonferroni's post hoc test following one-way ANOVA (WT; n = 5, R1204X; n = 6, M1190D; n = 6). Time is in seconds. Scale bars: 5 µm.


Cochleae from control , heterozygous Dia1 - KO ( Dia1 - / + ) and homozygous Dia1 - KO ( Dia1 - / - ) mice were fixed at 5 weeks of age ( A , B ; representative of n = 6 ) . ( A ) Paraffin - embedded 4 μm sections of cochlea were stained with hematoxylin and eosin . No hair cell ( HC ) loss ( either IHC or OHC ) is observed at the basal turn of the Dia1 - / + or Dia1 - / - cochleae . No obvious differences are observed in the spiral ganglion ( SV ) , spiral ligament ( SL ) , stria vascularis ( SV ) , and Reissner ' s membrane ( RM ) in Dia1 - / + , or Dia1 - / - mice compared with control mice . Lower panels ( Scale bars : 20 µm ) are magnified images of the region indicated rectangle in upper panels ( Scale bars : 100 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cochleae from control, heterozygous Dia1-KO (Dia1-/+) and homozygous Dia1-KO (Dia1-/-) mice were fixed at 5 weeks of age (A, B; representative of n=6).(A) Paraffin-embedded 4 μm sections of cochlea were stained with hematoxylin and eosin. No hair cell (HC) loss (either IHC or OHC) is observed at the basal turn of the Dia1-/+ or Dia1-/-cochleae. No obvious differences are observed in the spiral ganglion (SV), spiral ligament (SL), stria vascularis (SV), and Reissner's membrane (RM) in Dia1-/+, or Dia1-/-mice compared with control mice. Lower panels (Scale bars: 20 µm) are magnified images of the region indicated rectangle in upper panels (Scale bars: 100 µm).


Cochleae from control , heterozygous Dia1 - KO ( Dia1 - / + ) and homozygous Dia1 - KO ( Dia1 - / - ) mice were fixed at 5 weeks of age ( A , B ; representative of n = 6 ) . ( B ) Basal turns of the dissected cochleae were immunostained using a Myo7a Ab followed by an Alexa488 secondary Ab with DAPI as a nuclear counterstain . No HC loss ( either IHC or OHC ) is observed in Dia1 - / - mice . Scale bars : 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cochleae from control, heterozygous Dia1-KO (Dia1-/+) and homozygous Dia1-KO (Dia1-/-) mice were fixed at 5 weeks of age (A, B; representative of n=6).(B) Basal turns of the dissected cochleae were immunostained using a Myo7a Ab followed by an Alexa488 secondary Ab with DAPI as a nuclear counterstain. No HC loss (either IHC or OHC) is observed in Dia1-/-mice. Scale bars: 20 µm.


( C ) ABR thresholds ( 10 , 20 , 40 kHz ) in control and Dia1 - / - mice at 5 weeks of age ( dB SPL , mean SE ) . No hearing loss was observed in Dia1 - / - mice ( n = 8 ) compared with control mice ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) ABR thresholds (10, 20, 40 kHz) in control and Dia1-/-mice at 5 weeks of age (dB SPL, mean SE). No hearing loss was observed in Dia1-/-mice (n = 8) compared with control mice (n = 4).


( B ) Expression of FLAG - tagged DIA1 ( R1204X ) in the inner ear and heart of TG , but not WT , mice is confirmed by immunoblotting using a FLAG Ab . Representative of 3 experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Expression of FLAG-tagged DIA1(R1204X) in the inner ear and heart of TG, but not WT, mice is confirmed by immunoblotting using a FLAG Ab. Representative of 3 experiments.


( C ) Expression levels of FLAG - tagged DIA1 ( R1204X ) relative to endogenous Dia1 in the inner ear , thymus , and heart were determined by immunoblotting using a Dia1 / DIA1 Ab ( inner ear ; n = 4 , thymus and heart ; n = 3 ) , and quantified in the inner ear and thymus ( * P = 0 . 0226 and * * P = 0 . 0016 by two - tailed Student ' s t - test ) . Lysates of HEK293 cells overexpressing equal amounts of GFP - Dia1 and GFP - DIA1 ( determined using a GFP Ab : right end ) are detected with equal affinity by the Dia1 / DIA1 antibody . Comparable loading of proteins is confirmed using an α − tubulin or GAPDH Ab .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Expression levels of FLAG-tagged DIA1(R1204X) relative to endogenous Dia1 in the inner ear, thymus, and heart were determined by immunoblotting using a Dia1/DIA1 Ab (inner ear; n = 4, thymus and heart; n = 3), and quantified in the inner ear and thymus (*P= 0.0226 and **P= 0.0016 by two-tailed Student's t-test). Lysates of HEK293 cells overexpressing equal amounts of GFP-Dia1 and GFP-DIA1 (determined using a GFP Ab: right end) are detected with equal affinity by the Dia1/DIA1 antibody. Comparable loading of proteins is confirmed using an α−tubulin or GAPDH Ab.


( A ) , Age - related click and pure tone - burst ( 8 , 16 , 24 , 32 kHz ) ABR thresholds ( dB SPL , mean SE ) in DIA1 ( R1204X ) - TG ( 5 weeks , n = 6 ; 10 weeks , n = 16 ; 25 weeks , n = 42 ) and control mice ( 5 weeks , n = 6 ; 10 weeks , n = 16 ; 25 weeks , n = 33 ) . Note the progressive hearing loss of DIA1 ( R1204X ) - TG mice starting at high frequency sounds from 10 weeks . * * P = 0 . 0039 ( 24 kHz ) and * * P < 0 . 0001 ( 32 kHz ) at 10 weeks and * * P < 0 . 0001 ( click ) , * * P < 0 . 0001 ( 8 kHz ) , * * P = 0 . 0001 ( 16 kHz ) , * * P < 0 . 0001 ( 24 kHz ) , and * * P < 0 . 0001 ( 32 kHz ) at 25 weeks by Bonferroni ' s post hoc test following two - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A), Age-related click and pure tone-burst (8, 16, 24, 32 kHz) ABR thresholds (dB SPL, mean SE) in DIA1(R1204X)-TG (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 42) and control mice (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 33). Note the progressive hearing loss of DIA1(R1204X)-TG mice starting at high frequency sounds from 10 weeks. **P= 0.0039 (24 kHz) and **P< 0.0001 (32 kHz) at 10 weeks and **P< 0.0001 (click), **P< 0.0001 (8 kHz), **P= 0.0001 (16 kHz), **P< 0.0001 (24 kHz), and **P< 0.0001 (32 kHz) at 25 weeks by Bonferroni's post hoc test following two-way ANOVA.


( B ) Representative SEM images of the organ of Corti at the basal turn obtained from DIA1 ( R1204X ) - TG and control mice at 5 , 10 , and 25 weeks of age . White and red circles show IHC and OHC losses , respectively . Note OHC - dominant HC loss . Scale bars : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative SEM images of the organ of Corti at the basal turn obtained from DIA1(R1204X)-TG and control mice at 5, 10, and 25 weeks of age. White and red circles show IHC and OHC losses, respectively. Note OHC-dominant HC loss. Scale bars: 5 µm.


( C ) The percentages of remaining OHC and IHC in each turn in DIA1 ( R1204X ) - TG and control mice at 25 weeks of age ( n = 5 ) . Schematic illustration shows the regions of the cochlea divided into three parts : Ap ; apical turn , Md ; middle turn , Bs ; basal turn . * * P = 0 . 0017 ( OHC at Md ) , * * P < 0 . 0001 ( OHC at Bs ) , * P = 0 . 0117 ( IHC at Ap ) , * P = 0 . 0330 ( IHC at Md ) , and * * P = 0 . 0003 ( IHC at Bs ) by Bonferroni ' s post hoc test following two - way ANOVA .

(C) The percentages of remaining OHC and IHC in each turn in DIA1(R1204X)-TG and control mice at 25 weeks of age (n = 5). Schematic illustration shows the regions of the cochlea divided into three parts: Ap; apical turn, Md; middle turn, Bs; basal turn. **P= 0.0017 (OHC at Md), **P< 0.0001 (OHC at Bs), *P= 0.0117 (IHC at Ap), *P= 0.0330 (IHC at Md), and **P= 0.0003 (IHC at Bs) by Bonferroni's post hoc test following two-way ANOVA.


( D ) DPOAE ( f2 frequency at 8 , 12 , 16 , 20 kHz ) amplitude ( dB SPL , mean SE ) in DIA1 ( R1204X ) - TG ( n = 37 ) and control mice ( n = 30 ) at 25 weeks of age . * P = 0 . 0127 , * * P = 0 . 0029 ( at 12 kHz ) , and * * P = 0 . 0013 ( at 16 kHz ) by Bonferroni ' s post hoc test following two - way ANOVA .

(D) DPOAE (f2 frequency at 8, 12, 16, 20 kHz) amplitude (dB SPL, mean SE) in DIA1(R1204X)-TG (n = 37) and control mice (n = 30) at 25 weeks of age. *P= 0.0127, **P= 0.0029 (at 12 kHz), and **P= 0.0013 (at 16 kHz) by Bonferroni's post hoc test following two-way ANOVA.


( E ) High magnification SEM images of OHCs and IHCs at the basal turn of cochlea obtained from DIA1 ( R1204X ) - TG and control mice at 25 weeks of age . Note the sparse and short ( arrow ) stereocilia of OHC , and sparse and fused ( arrowheads ) stereocilia of IHC in DIA1 ( R1204X ) - TG mice . Scale bars : 1 µm in OHCs and 5 µm in IHCs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) High magnification SEM images of OHCs and IHCs at the basal turn of cochlea obtained from DIA1(R1204X)-TG and control mice at 25 weeks of age. Note the sparse and short (arrow) stereocilia of OHC, and sparse and fused (arrowheads) stereocilia of IHC in DIA1(R1204X)-TG mice. Scale bars: 1 µm in OHCs and 5 µm in IHCs.


TRIP6 promotes YAP activity by inhibiting LATS1 / 2 ( A ) Full length ( WT ) , the amino - terminal half ( 1 - 277 ) , or the carboxy - terminal half ( 278 - 476 ) of TRIP6 were tested for binding to LATS2 by immunoprecipitation . FLAG - TRIP6 variants were co - expressed with LATS2 - GFP in HEK293 cells , anti - FLAG or control ( IgG ) antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS2 - GFP and FLAG - TRIP6 . Schematic diagram depicts TRIP6 domains ( NES : Nuclear Export Signal ; LIM : LIM domain ; PDZ : PDZ domain binding motif ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TRIP6 promotes YAP activity by inhibiting LATS1/2 (A) Full length (WT), the amino-terminal half (1-277), or the carboxy-terminal half (278-476) of TRIP6 were tested for binding to LATS2 by immunoprecipitation. FLAG-TRIP6 variants were co-expressed with LATS2-GFP in HEK293 cells, anti-FLAG or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS2-GFP and FLAG-TRIP6. Schematic diagram depicts TRIP6 domains (NES: Nuclear Export Signal; LIM: LIM domain; PDZ: PDZ domain binding motif).


( B ) Lysates from MCF10A cells were subjected to immunoprecipitation using anti - TRIP6 or control ( IgG ) antibodies , and immune complexes and lysates were probed for TRIP6 and LATS1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Lysates from MCF10A cells were subjected to immunoprecipitation using anti-TRIP6 or control (IgG) antibodies, and immune complexes and lysates were probed for TRIP6 and LATS1.


( C ) FLAG - TRIP6 was tested for binding to various LATS2 - GFP deletion mutants as described in part ( A ) . Schematic diagram of LATS2 shows MOB1 binding domain , and the autophosphorylation ( S872 ) and MST1 / 2 phosphorylation sites ( T1041 ) in the kinase domain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) FLAG-TRIP6 was tested for binding to various LATS2-GFP deletion mutants as described in part (A). Schematic diagram of LATS2 shows MOB1 binding domain, and the autophosphorylation (S872) and MST1/2 phosphorylation sites (T1041) in the kinase domain.


The regions marked in green depict TRIP6 binding sites on LATS2 ( D ) Lysates from HEK293A cells transfected with control or FLAG - TRIP6 plasmid were analyzed by western blotting using the indicated antibodies ( quantification is shown in ( F ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The regions marked in green depict TRIP6 binding sites on LATS2 (D) Lysates from HEK293A cells transfected with control or FLAG-TRIP6 plasmid were analyzed by western blotting using the indicated antibodies (quantification is shown in (F)).


( E ) Lysates from control ( WT ) or CRISPR generated TRIP6 null ( TRIP6 - KO ) HEK293A cells were analyzed by western blotting using the indicated antibodies ( quantification shown in ( G ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Lysates from control (WT) or CRISPR generated TRIP6 null (TRIP6-KO) HEK293A cells were analyzed by western blotting using the indicated antibodies (quantification shown in (G)).


( F ) The relative levels of LATS1 activating phosphorylation ( pLATS1 - 1079 , 909 ) and YAP S127 inhibitory phosphorylation from ( D ) were measured relative to LATS1 and YAP levels respectively . ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The relative levels of LATS1 activating phosphorylation (pLATS1-1079, 909) and YAP S127 inhibitory phosphorylation from (D) were measured relative to LATS1 and YAP levels respectively. (Mean ± SD; n=3; **P≤0.01, ***P≤0.001, T-test)


( G ) The levels of LATS1 activating phosphorylation and YAP inactivating phosphorylation from part ( E ) were quantified ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) The levels of LATS1 activating phosphorylation and YAP inactivating phosphorylation from part (E) were quantified (Mean ± SD; n=3; *P≤0.05, T-test).