( E ) Endogenous PINK1 gradually accumulated after CCCP treatment . first through fifth lanes show endogenous PINK1 , sixth lane shows overexpressed untagged PINK1 . Note that asterisk indicates a cross - reacting band because it was not affected by overproduction of untagged PINK1 .

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(E) Endogenous PINK1 gradually accumulated after CCCP treatment. The first through the fifth lanes show endogenous PINK1, and the sixth lane shows overexpressed untagged PINK1. Note that the asterisk indicates a cross-reacting band because it was not affected by overproduction of untagged PINK1.


( F ) Subcellular fractionation of endogenous PINK1 . Intact SH - SY5Y were treated with CCCP or DMSO subjected to fractionation experiments ( same sample as Fig . 1 B ) . I , C , M indicate input , cytosol - rich supernatant , mitochondria - rich pellet , respectively .

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(F) Subcellular fractionation of endogenous PINK1. Intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments (same sample as Fig. 1 B). I, C, and M indicate input, cytosol-rich supernatant, and the mitochondria-rich membrane pellet, respectively.


( G ) HeLa were treated with CCCP cycloheximide as depicted , followed by immunoblotting with indicated antibodies . LDH , lactate dehydrogenase . ( F G ) Asterisks indicate a cross - reacting band .

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(G) HeLa cells were treated with CCCP and cycloheximide as depicted, followed by immunoblotting with the indicated antibodies. LDH, lactate dehydrogenase. (F and G) Asterisks indicate a cross-reacting band.


( H ) N - terminal 34 aa of PINK1 recruited GFP to mitochondria both in absence presence of CCCP . top panel shows control HeLa expressing only GFP . ( B , C , H ) Higher magnification views of boxed areas are shown in insets . Bars , 10 µm .

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(H) N-terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP. The top panel shows control HeLa cells expressing only GFP. (B, C, and H) Higher magnification views of the boxed areas are shown in the insets. Bars, 10 µm.


( A ) PINK1 knockout ( PINK1 − / − ) or control ( PINK1 + / + ) MEFs were transfected with HA - Parkin , treated with CCCP , subjected to immunocytochemistry with indicated antibodies . Higher magnification views of boxed areas are shown in insets . ( B ) number of MEFs with Parkin localized to mitochondria was counted as in Fig . 3 A .

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(A) PINK1 knockout (PINK1−/−) or control (PINK1+/+) MEFs were transfected with HA-Parkin, treated with CCCP, and subjected to immunocytochemistry with the indicated antibodies. Higher magnification views of the boxed areas are shown in the insets. (B) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig. 3 A.


( C D ) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1 − / − MEFs . MEFs stably expressing GFP - Parkin were treated with CCCP for 4 h subjected to immunoblotting ( C )

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(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 4 h and subjected to immunoblotting (C)


( C D ) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1 − / − MEFs . MEFs stably expressing GFP - Parkin were treated with CCCP for 24 h , followed by immunocytochemistry ( D ) . IB , immunoblot ; Ub , ubiquitylation .

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(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 24 h, followed by immunocytochemistry (D). IB, immunoblot; Ub, ubiquitylation.


( E ) number of MEFs without anti - Tom20 antibody - detectable mitochondria was counted as in Fig . 3 A . In example figure ( left ) , blue arrowheads indicate without anti - Tom20 antibody - detectable mitochondria , red arrowheads indicate harboring anti - Tom20 antibody - detectable mitochondria .

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(E) The number of MEFs without anti-Tom20 antibody-detectable mitochondria was counted as in Fig. 3 A. In the example figure (left), blue arrowheads indicate cells without anti-Tom20 antibody-detectable mitochondria, and red arrowheads indicate cells harboring anti-Tom20 antibody-detectable mitochondria.


( A ) Schematic depiction of pathogenic deletion mutants of PINK1 used in this study . MTS , mitochondria - targeting ; TMD , transmembrane domain . ( B ) Subcellular localization of Parkin in PINK1 − / − complemented by various pathogenic deletion mutants of PINK1 - Flag . were treated with CCCP . Higher magnification views of boxed areas are shown in insets . ( C ) number of with Parkin - positive mitochondria was counted as in Fig . 3 A . Error bars represent mean ± SD values of least three experiments .

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(A) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study. MTS, mitochondria-targeting sequence; TMD, transmembrane domain. (B) Subcellular localization of Parkin in PINK1−/− cells complemented by various pathogenic and deletion mutants of PINK1-Flag. Cells were treated with CCCP. Higher magnification views of the boxed areas are shown in the insets. (C) The number of cells with Parkin-positive mitochondria was counted as in Fig. 3 A. Error bars represent the mean ± SD values of least three experiments.


( D ) PINK1 − / − MEFs complemented by various PINK1 mutants were treated with CCCP subjected to immunoblotting using anti - Parkin or anti - Flag ( tag of PINK1 ) antibodies . IB , immunoblot ; Ub , ubiquitylation .

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(D) PINK1−/− MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti-Parkin or anti-Flag (tag of PINK1) antibodies. IB, immunoblot; Ub, ubiquitylation.


( A ) Immunoblot with anti - LC3 antibody shows that NO donors reduced LC3 - II in rat cortical neurons decreased autophagosome synthesis in bafilomycin A1 - treated rat cortical neurons HeLa .

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(A) Immunoblot analysis with anti-LC3 antibody shows that NO donors reduced LC3-II levels in rat primary cortical neurons and decreased autophagosome synthesis in bafilomycin A1-treated rat primary cortical neurons and HeLa cells.


( B - E ) Confocal microscopy of autophagic flux by automated Cellomics microscopy show that NO donors reduced autolysosome numbers in mRFP - GFP - LC3 HeLa grown in full medium ( FM ) ( B D ) or starved with HBSS ( C E ) . in HBSS had more autolysosomes compared to FM ( C ) . Autolysosome number in untreated in FM is set at 100 % .

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(B-E) Confocal microscopy images and analysis of autophagic flux by automated Cellomics microscopy show that NO donors reduced autolysosome numbers in mRFP-GFP-LC3 HeLa cells grown in full medium (FM) (B and D) or starved with HBSS (C and E). Cells in HBSS had more autolysosomes compared to FM (C). Autolysosome number in untreated cells in FM is set at 100%.


( F ) NO donors increased EGFP - HDQ74 in a dose - dependent manner in EGFP - HDQ74 - transfected Atg5 + / + , but not in Atg5 − / − , MEFs . Atg5 − / − MEFs had more EGFP - HDQ74 than Atg5 + / + MEFs .

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(F) NO donors increased EGFP-HDQ74 aggregates in a dose-dependent manner in EGFP-HDQ74-transfected Atg5+/+, but not in Atg5−/−, MEFs. Atg5−/− MEFs had more EGFP-HDQ74 aggregates than Atg5+/+ MEFs.


( G H ) Confocal microscopy ( H ) immunofluorescence with anti - Atg16 antibody ( G ) show that NO donors reduced of HeLa with Atg16 - positive structures ( arrows ) under starvation . Graphical data denote mean ± .

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(G and H) Confocal microscopy images (H) and immunofluorescence analysis with anti-Atg16 antibody (G) show that NO donors reduced the percentage of HeLa cells with Atg16-positive structures (arrows) under starvation. Graphical data denote mean ± SEM.


( A ) NO donors reduced JNK1 phosphorylation in HeLa , as analyzed by immunoprecipitation with anti - JNK1 agarose - conjugated beads immunoblotting with anti - phospho - JNK antibody .

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(A) NO donors reduced JNK1 phosphorylation in HeLa cells, as analyzed by immunoprecipitation with anti-JNK1 agarose-conjugated beads and immunoblotting with anti-phospho-JNK antibody.


( B ) NO donors reduced Bcl - 2 phosphorylation in HeLa , as analyzed by immunoblotting with anti - phospho - Bcl - 2 antibody .

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(B) NO donors reduced Bcl-2 phosphorylation in HeLa cells, as analyzed by immunoblotting with anti-phospho-Bcl-2 antibody.


C ) Immunoblot with anti - phospho - Bcl - 2 antibody shows that DETA NONOate could not reduce phospho - Bcl - 2 in Flag - CA JNK1 - transfected HeLa , where phospho - Bcl - 2 was higher compared to mock - transfected .

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C) Immunoblot analysis with anti-phospho-Bcl-2 antibody shows that DETA NONOate could not reduce phospho-Bcl-2 in Flag-CA JNK1-transfected HeLa cells, where phospho-Bcl-2 was higher compared to mock-transfected cells.


( D E ) Immunoprecipitation with anti - Flag M2 affinity agarose gel immunoblotting with anti - Myc antibody shows that NO donors increased interaction of Flag - Beclin 1 with WT Myc - Bcl - 2 ( D ) , but not with AAA Myc - Bcl - 2 ( E ) , in HeLa transfected with WT Myc - Bcl - 2 ( D ) or AAA Myc - Bcl - 2 ( E ) along with either empty Flag or Flag - Beclin 1 . Asterisk denotes IgG band ( E ) .

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(D and E) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Myc antibody shows that NO donors increased the interaction of Flag-Beclin 1 with WT Myc-Bcl-2 (D), but not with AAA Myc-Bcl-2 (E), in HeLa cells transfected with WT Myc-Bcl-2 (D) or AAA Myc-Bcl-2 (E) along with either empty Flag or Flag-Beclin 1. Asterisk denotes IgG band (E).


( F ) Immunoprecipitation with anti - Flag M2 affinity agarose gel immunoblotting with anti - Vps34 antibody shows that NO donors decreased Flag - Beclin 1 - hVps34 interaction in HeLa transfected with hVps34 along with either empty Flag or Flag - Beclin 1 .

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(F) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that NO donors decreased Flag-Beclin 1-hVps34 interaction in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1.


( G ) Immunoprecipitation with anti - Flag M2 affinity agarose gel immunoblotting with anti - Vps34 antibody shows that AAA Myc - Bcl - 2 decreased Flag - Beclin 1 - hVps34 interaction in presence ( right panel ) or absence ( left panel ) of DETA NONOate in HeLa transfected with hVps34 along with either empty Flag or Flag - Beclin 1 , with pcDNA3 . 1 or AAA Myc - Bcl - 2 . Graphical data denote mean ± .

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(G) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that AAA Myc-Bcl-2 decreased Flag-Beclin 1-hVps34 interaction in the presence (right panel) or absence (left panel) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1, and with pcDNA3.1 or AAA Myc-Bcl-2. Graphical data denote mean ± SEM.


( A ) Immunoblot with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated WT Bcl - 2 AAA Bcl - 2 MEFs .

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(A) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Bcl-2 and AAA Bcl-2 MEFs.


( B C ) NO donors decreased EGFP - LC3 vesicles in EGFP - LC3 - transfected WT Bcl - 2 AAA Bcl - 2 MEFs ( C ) . were acquired by a confocal microscope ( B ) . WT Bcl - 2 AAA Bcl - 2 MEFs were analyzed separately .

(B and C) NO donors decreased EGFP-LC3 vesicles in EGFP-LC3-transfected WT Bcl-2 and AAA Bcl-2 MEFs (C). Images were acquired by a confocal microscope (B). WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.


( D ) NO donors increased EGFP - HDQ74 in EGFP - HDQ74 - transfected WT Bcl - 2 AAA Bcl - 2 MEFs . WT Bcl - 2 AAA Bcl - 2 MEFs were analyzed separately .

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(D) NO donors increased EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected WT Bcl-2 and AAA Bcl-2 MEFs. WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.


( E ) Immunoblot with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated WT Jnk , Jnk1 − / − , Jnk2 − / − MEFs .

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(E) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Jnk, Jnk1−/−, and Jnk2−/− MEFs.


( F ) Immunoblot analyses with anti - phospho - S6K anti - phospho - S6 antibodies show that NO donors activated mTORC1 in HeLa .

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(F) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in HeLa cells.


( G ) Confocal microscope of immunofluorescence with anti - phospho - S6 antibody show that DETA NONOate increased S6 phosphorylation ; arrowhead shows a where this effect was not observed .

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(G) Confocal microscope images of immunofluorescence with anti-phospho-S6 antibody show that DETA NONOate increased S6 phosphorylation; arrowhead shows a cell where this effect was not observed.


( H ) Immunoblot analyses with anti - phospho - S6K anti - phospho - S6 antibodies show that NO donors activated mTORC1 in WT Bcl - 2 AAA Bcl - 2 MEFs .

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(H) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in WT Bcl-2 and AAA Bcl-2 MEFs.


( I ) Confocal microscope of immunofluorescence with anti - phospho - mTOR anti - LAMP1 antibodies in HeLa show that DETA NONOate increased phospho - mTOR but did not alter its distribution with lysosomes . Graphical data denote mean ± .

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(I) Confocal microscope images of immunofluorescence with anti-phospho-mTOR and anti-LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho-mTOR but did not alter its distribution with lysosomes. Graphical data denote mean ± SEM.


( A ) Immunoblot analyses with anti - phospho - S6K anti - phospho - S6 antibodies show that NO donors activated mTORC1 in Tsc2 + / + MEFs , but not in Tsc2 − / − , MEFs .

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(A) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Tsc2+/+ MEFs, but not in Tsc2−/−, MEFs.


( B ) NO donors reduced phosphorylation of IKKα / β AMPKα in MEFs , as analyzed by immunoblotting with anti - phospho - IKKα / β anti - phospho - AMPKα antibodies , respectively .

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(B) NO donors reduced phosphorylation of IKKα/β and AMPKα in MEFs, as analyzed by immunoblotting with anti-phospho-IKKα/β and anti-phospho-AMPKα antibodies, respectively.


( C ) Immunoblot analyses with anti - phospho - S6K anti - phospho - S6 antibodies show that NO donors activated mTORC1 in Ikkβ + / + MEFs , but not in Ikkβ − / − MEFs .

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(C) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Ikkβ+/+ MEFs, but not in Ikkβ−/− MEFs.


( D ) Immunoblot with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated Tsc2 + / + Tsc2 − / − MEFs .

(D) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated Tsc2+/+ and Tsc2−/− MEFs.


( E ) DETA NONOate decreased EGFP - LC3 vesicles in Tsc2 + / + Tsc2 − / − MEFs transfected with EGFP - LC3 , along with either pcDNA3 . 1 or AAA Myc - Bcl - 2 . Although it further reduced EGFP - LC3 vesicles in Tsc2 + / + MEFs expressing AAA Myc - Bcl - 2 compared to mock - transfected Tsc2 + / + MEFs , this effect was not seen in Tsc2 − / − MEFs . Tsc2 + / + Tsc2 − / − MEFs were analyzed separately . Graphical data denote mean ± .

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(E) DETA NONOate decreased EGFP-LC3 vesicles in Tsc2+/+ and Tsc2−/−MEFs transfected with EGFP-LC3, along with either pcDNA3.1 or AAA Myc-Bcl-2. Although it further reduced EGFP-LC3 vesicles in Tsc2+/+MEFs expressing AAA Myc-Bcl-2 compared to mock-transfected Tsc2+/+MEFs, this effect was not seen in Tsc2−/−MEFs. Tsc2+/+ and Tsc2−/−MEFs were analyzed separately. Graphical data denote mean ± SEM.


( A - D ) Immunoblot analyses with anti - LC3 , along with anti - nNOS , anti - iNOS , anti - eNOS antibodies , show decreased LC3 - II in stable HEK293 lines overexpressing nNOS ( A D ) , iNOS - GFP ( B D ) , or eNOS ( C D ) compared to HEK293 control in presence or absence of bafilomycin A1 .

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(A-D) Immunoblot analyses with anti-LC3, along with anti-nNOS, anti-iNOS, and anti-eNOS antibodies, show decreased LC3-II levels in stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and D), or eNOS (C and D) compared to HEK293 control cells in the presence or absence of bafilomycin A1.


( E F ) mCherry - LC3 vesicles were reduced in mCherry - LC3 - transfected stable HEK293 NOS lines compared to HEK293 control cultured in HBSS , an effect that was restored in NOS by L - NAME ( F ) . were acquired by a confocal microscope ( E ) .

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(E and F) mCherry-LC3 vesicles were reduced in mCherry-LC3-transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS, an effect that was restored in the NOS cells by L-NAME (F). Images were acquired by a confocal microscope (E).


( G H ) Confocal microscopy ( G ) immunofluorescence with anti - Atg16 antibody show that stable HEK293 NOS lines had fewer Atg16 - positive structures under starvation , compared to HEK293 control ( H ) . Graphical data denote mean ± .

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(G and H) Confocal microscopy images (G) and immunofluorescence analysis with anti-Atg16 antibody show that stable HEK293 NOS cell lines had fewer Atg16-positive structures under starvation, compared to HEK293 control cells (H). Graphical data denote mean ± SEM.


( A - F ) Immunoblot analyses with anti - nNOS , anti - iNOS , anti - eNOS , anti - phospho - JNK1 , anti - phospho - Bcl - 2 , anti - phospho - S6K , anti - phospho - S6 antibodies show that stable HEK293 lines overexpressing nNOS ( A D ) , iNOS - GFP ( B E ) , or eNOS ( C F ) inhibited both JNK1 - Bcl - 2 mTORC1 pathways compared to HEK293 control under basal ( FM ) ( A - C ) or starvation ( HBSS ) ( D - F ) conditions . Graphical data denote mean ± .

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(A-F) Immunoblot analyses with anti-nNOS, anti-iNOS, anti-eNOS, anti-phospho-JNK1, anti-phospho-Bcl-2, anti-phospho-S6K, and anti-phospho-S6 antibodies show that stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and E), or eNOS (C and F) inhibited both the JNK1-Bcl-2 and mTORC1 pathways compared to the HEK293 control cells under basal (FM) (A-C) or starvation (HBSS) (D-F) conditions. Graphical data denote mean ± SEM.


( A ) Immunoblot analyses with anti - LC3 antibody show that rapamycin ( 8 hr ) L - NAME ( 24 hr ) increased autophagosome synthesis in bafilomycin A1 - treated mouse cortical neurons HeLa .

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(A) Immunoblot analyses with anti-LC3 antibody show that rapamycin (8 hr) and L-NAME (24 hr) increased autophagosome synthesis in bafilomycin A1-treated mouse primary cortical neurons and HeLa cells.


( B C ) Confocal microscopy ( B ) of autophagic flux by automated Cellomics microscope ( C ) show that L - NAME increased autolysosomes in mRFP - GFP - LC3 HeLa .

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(B and C) Confocal microscopy images (B) and analysis of autophagic flux by automated Cellomics microscope (C) show that L-NAME increased autolysosomes in mRFP-GFP-LC3 HeLa cells.


( D ) L - NAME reduced EGFP - HDQ74 in EGFP - HDQ74 - transfected Atg5 + / + , but not in Atg5 − / − , MEFs .

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(D) L-NAME reduced EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected Atg5+/+, but not in Atg5−/−, MEFs.


( E ) Drosophila expressing mutant huntingtin exon 1 ( Q120 ) shows a significant decrease in neurodegeneration ( p < 0 . 001 , paired t ) upon L - NAME treatment compared to DMSO .

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(E) Drosophila expressing mutant huntingtin exon 1 (Q120) shows a significant decrease in neurodegeneration (p < 0.001, paired t test) upon L-NAME treatment compared to DMSO.


( F ) Expression of an endogenous negative regulator of NOS activity ( NOS4 ) significantly attenuates neurodegeneration ( p < 0 . 05 , paired t ) in Drosophila expressing mutant huntingtin exon 1 ( Q120 ) .

(F) Expression of an endogenous negative regulator of NOS activity (NOS4) significantly attenuates neurodegeneration (p < 0.05, paired t test) in Drosophila expressing mutant huntingtin exon 1 (Q120).


( G - I ) from retina of transgenic HD zebrafish show mutant huntingtin ( arrows ) ( G ) . Treatment with rapamycin or L - NAME reduced number of ( H ) . L - NAME did not reduce in presence of NH4Cl , which increased aggregate ( I ) .

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(G-I) Images from the retina of transgenic HD zebrafish show mutant huntingtin aggregates (arrows) (G). Treatment with rapamycin or L-NAME reduced the number of aggregates (H). L-NAME did not reduce aggregates in the presence of NH4Cl, which increased aggregate count (I).


IFM of KIF13A - YFP transfected control Rab - knockdown HeLa . TN : average tubule number ( mean ± , n = 3 ) . B , C Graphs represent measurement of KIF13A - positive TN ( B ) TL ( C ) in HeLa of Fig 1A ( mean ± ) . n = 3 . nc : total number of . nt : total number of tubules . arrowheads arrows point to KIF13A - / Rab22A - positive tubular REs E / SEs respectively . Scale bars : 10 μm .

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IFM images of KIF13A-YFP transfected control and Rab-knockdown HeLa cells. TN: average tubule number (mean±SEM, n=3). B, C Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 1A (mean± SEM). n=3. nc: total number of cells. nt: total number of tubules.arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm.


IFM of KIF13A - YFP mCherry - Rab7A / 11A / 22A transfected HeLa arrowheads arrows point to KIF13A - / Rab22A - positive tubular REs E / SEs respectively . Scale bars : 10

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

IFM images of KIF13A-YFP and mCherry-Rab7A/11A/22A transfected HeLa cells arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Live imaging of GFP / mCherry - Rab22A with respect to mCherry - Rab11A or GFP - Rab7A in HeLa . Magnified view of insets ( at 0 , 20 , 40 seconds ) are shown separately ) , arrowheads arrows point to KIF13A - / Rab22A - positive tubular REs E / SEs respectively . Scale bars : 10

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

Live cell imaging of GFP/mCherry-Rab22A with respect to mCherry-Rab11A or GFP-Rab7A in HeLa cells. Magnified view of insets (at 0, 20, 40 seconds) are shown separately ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Pull - down of different His - KIF13A domains using HeLa lysate then probed with indicated Rab . bead bound His - KIF13A in each pull - down was shown as coomassie gel . * , non - specific bands .

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

Pull-down of different His-KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead bound His-KIF13A in each pull-down was shown as coomassie gel. *, non-specific bands.


IFM of KIF13A - YFP mCherry - Rab22AWT / Q64L / S19N cotransfected HeLa . Arrowheads arrows point to KIF13A - positive tubular REs E / SEs respectively . colocalization coefficient ( r , in mean ± ) between two markers was indicated separately . Scale bars : 10 μm Graphs represent measurement of KIF13A - positive TN ( B ) TL ( C ) in HeLa of Fig 2A . n = 3 . nc : total number of . nt : total number of

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

IFM images of KIF13A-YFP and mCherry-Rab22AWT/Q64L/S19N cotransfected HeLa cells. Arrowheads and arrows point to the KIF13A-positive tubular REs and E/SEs respectively. The colocalization coefficient (r, in mean±SEM) between two markers was indicated separately. Scale bars: 10 μm Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 2A. n=3. nc: total number of cells. nt: total number of tubules


Subcellular fractionation of HeLa to probe localization of Rab22A ( red box ) with respect to other organelle specific . * , non - specific bands .

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

Subcellular fractionation of HeLa cells to probe the localization of Rab22A (red box) with respect to other organelle specific proteins. *, non-specific bands.


IFM of KIF13A - YFP transfected control Rab22A - , BLOC - 1Mu - , BLOC - 2HPS6 - knockdown HeLa . were stained for AP - 1 ( γ ) or internalized with Tf - Alexa fluor 594 arrowheads arrows point to KIF13A - / Rab22A - positive tubular REs E / SEs respectively . Scale bars : 10

IFM images of KIF13A-YFP transfected control and Rab22A-, BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells. Cells were stained for AP-1 (γ) or internalized with Tf-Alexa fluor 594 arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Graphs represent measurement of KIF13A - positive TN ( B ) TL ( C ) in HeLa of Fig 3A . n = 3 . nc : total number of . nt : total number of

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

Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 3A. n=3. nc: total number of cells. nt: total number of tubules


Immunoblotting of in control Rab22A - , BLOC - 1 - , BLOC - 2 - depleted HeLa . * , non - specific bands . band intensities were quantified indicated on gels .

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

Immunoblotting analysis of proteins in control and Rab22A-, BLOC-1-, BLOC-2-depleted HeLa cells. *, non-specific bands. Protein band intensities were quantified and indicated on the gels.


IFM of mCherry - Rab22A KIF13A - YFP cotransfected control BLOC - 1Mu - , BLOC - 2HPS6 - knockdown HeLa ) , arrowheads arrows point to KIF13A - / Rab22A - positive tubular REs E / SEs respectively . Scale bars : 10

IFM images of mCherry-Rab22A and KIF13A-YFP cotransfected control and BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


BF IFM of control Rab22A - depleted melanocytes . colocalization coefficient ( r , in mean ± ) between two markers indicated separately . Nuclei are stained with Hoechst33258 ) , arrows indicate melanocyte pigmentation arrowheads point to localization of TYRP1 / GFP - Rab22A . Scale bars : 10

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

BF and IFM images of control and Rab22A-depleted melanocytes. The colocalization coefficient (r, in mean±SEM) between the two markers indicated separately. Nuclei are stained with Hoechst33258 ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm


Graph represents quantified melanin content in control sh , Rab22A sh BLOC - 1 - melanocytes . n = 3 . fold - change in melanin content ( mean ± ) indicated

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 B-MEASURED_VAR O O O O O O

Graph represents the quantified melanin content in control sh, Rab22A sh and BLOC-1- melanocytes. n=3. The fold-change in melanin content (mean±SEM) indicated separately


Immunoblotting of in Control sh Rab22A sh melanocytes ) , band intensities were quantified indicated on

Immunoblotting analysis of proteins in Control sh and Rab22A sh melanocytes ), protein band intensities were quantified and indicated on the gels


EM of control Rab22A - knockdown MNT - 1 melanocytes . Arrowheads indicate PMEL fibrils . Scale bars : 1 μm . I , II , III , IV : stages of melanosomes . M : mitochondria . Magnified view of insets are shown separately they emphasize stage I melanosomes in both conditions . Note : presence of hybrid stage I / II melanosomes in Rab22A - knockdown . Insets ( b ) - ( d ) ( g ) - ( h ) are from different of respective condition . Scale bars : 200 nm .

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

EM of control and Rab22A-knockdown MNT-1 melanocytes. Arrowheads indicate PMEL fibrils. Scale bars: 1 μm. I, II, III, IV: stages of melanosomes. M: mitochondria. Magnified view of insets are shown separately and they emphasize the stage I melanosomes in both conditions. Note: the presence of hybrid stage I/II melanosomes in Rab22A-knockdown cells. Insets (b)-(d) and (g)-(h) are from different cells of respective condition. Scale bars: 200 nm.


BF IFM of GFP - Rab22A transfected BLOC - 1 - ( Mu ) melanocytes ) , arrows indicate melanocyte pigmentation arrowheads point to localization of TYRP1 / GFP - Rab22A . Scale bars : 10

BF and IFM images of GFP-Rab22A transfected BLOC-1-(Mu) melanocytes ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm


Immunoblotting of in BLOC - 1 - BLOC - 1R melanocytes band intensities were quantified indicated on

Immunoblotting analysis of proteins in BLOC-1- and BLOC-1R melanocytes protein band intensities were quantified and indicated on the gels


Subcellular fractionation of control Rab22A - , BLOC - 1 - , BLOC - 2 - knockdown HeLa probed fractions for pallidin , dysbindin muted . Red colored box emphasizes BLOC - 1 association in respective types .

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

Subcellular membrane fractionation of control and Rab22A-, BLOC-1-, BLOC-2-knockdown HeLa cells and probed the fractions for pallidin, dysbindin and muted. Red colored box emphasizes BLOC-1 membrane association in the respective cell types.


Cytosol - fractionation of homogenates from HeLa ( B as indicated . band intensities were quantified indicated association on

Cytosol-membrane fractionation of homogenates from HeLa cells (B as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels


Cytosol - fractionation of homogenates fro melanocyte as indicated . band intensities were quantified indicated association on

Cytosol-membrane fractionation of homogenates fro melanocyte as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels


Pull - down of His - Rab22AWT using HeL lysate bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

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

Pull-down of His-Rab22AWT using HeL lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


Pull - down of His - Rab22AWT usin melanocyte ( E ) lysate . In E , beads were preloaded with GTPγS or GDP bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

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

Pull-down of His-Rab22AWT usin melanocyte (E) lysate. In E, the beads were preloaded with GTPγS or GDP the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


Pull - down of different His - KIF13A domains using HeLa lysate bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

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

Pull-down of different His-KIF13A domains using HeLa cell lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


C . Biotinylation of centrosome microtubules by V5 - BirA * - AURKA in different cycle stages . After 18 h biotin incubation , HEK293T : : V5 - BirA * - AURKA were fixed stained for fusion with V5 antibody , biotinylated with streptavidin centrosome with gamma - tubulin antibody . was stained with DAPI . 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 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

C. Biotinylation of the centrosome and microtubules by V5-BirA*-AURKA in different cell cycle stages. After 18 h biotin incubation, HEK293T::V5-BirA*-AURKA were fixed and stained for the fusion protein with V5 antibody, biotinylated proteins with the fluorescent streptavidin and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 5 μm.


D . Control biotin - treated HEK293T stably expressing V5 - BirA * V5 - BirA * - AURKA were lysed biotinylated were precipitated by streptavidin beads . initial sample ( initial ) captured biotinylated ( pulldown ) were run on an SDS gel immunoblotted with streptavidin antibodies against V5 vinculin ( loading control ) .

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

D. Control and biotin-treated HEK293T cells stably expressing V5-BirA* and V5-BirA*-AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads. The initial sample (initial) and captured biotinylated proteins (pulldown) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin (loading control).


C . AURKA interacts with centriolar satellite PCM1 , CEP131 , CEP72 , CSPP1 , KIAA0586 / Talpid3 CP110 . HEK293T were transiently transfected with FLAG - BirA * or FLAG - BirA * - AURKA . Following 18 h biotin incubation , were lysed , biotinylated were precipitated by streptavidin beads . initial sample immunoprecipitated biotinylated were run on a gel immunoblotted with streptavidin antibodies against FLAG , CEP72 , CEP131 , PCM1 , CEP63 , CSPP1 , KIAA0586 / Talpid3 , CP110 , BBS4 SSX2IP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. AURKA interacts with the centriolar satellite proteins PCM1, CEP131, CEP72, CSPP1, KIAA0586/Talpid3 and CP110. HEK293T cells were transiently transfected with FLAG-BirA* or FLAG-BirA*-AURKA. Following 18 h biotin incubation, cells were lysed, and biotinylated proteins were precipitated by streptavidin beads. The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG, CEP72, CEP131, PCM1, CEP63, CSPP1, KIAA0586/Talpid3, CP110, BBS4 and SSX2IP.


E . Endogenous interaction of PCM1 AURKA . Lysates from synchronous serum - starved HEK293T were immunoprecipitated with AURKA antibody rabbit IgG control . initial sample immunoprecipitated were run on a gel immunoblotted with antibodies against PCM1 AURKA .

O O O O O O O O O O O O O O O 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. Endogenous interaction of PCM1 and AURKA. Lysates from synchronous and serum-starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA.


F . AURKA co - localize with PCM1 at peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain . U2OS were co - transfected with GFP - PCM1 - FKBP HA - Kif5b ( 1 - 269 a . a . ) - FRB treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours . that were not treated with rapamycin were processed in parallel as a control . Fixed were stained with antibodies against GFP , AURKA gamma - tubulin . was stained with DAPI . edges are outlined . Scale bar , 10 μm . G . of pericentrosomal of AURKA gamma tubulin for F . n > 25 per experiment . Data represent mean value from two experiments per condition ± ( * * * * p < 0 . 0001 ; * * p < 0 . 01 ; Unpaired Student ' s t - . ) .

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

F. AURKA co-localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain. U2OS cells were co-transfected with GFP-PCM1-FKBP and HA-Kif5b(1-269 a.a.)-FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours. Cells that were not treated with rapamycin were processed in parallel as a control. Fixed cells were stained with antibodies against GFP, AURKA and gamma-tubulin. DNA was stained with DAPI. Cell edges are outlined. Scale bar, 10 μm. G. Quantification of pericentrosomal levels of AURKA and gamma tubulin for F. n>25 cells per experiment. Data represent mean value from two experiments per condition ± SEM (****p < 0.0001; **p < 0.01; Unpaired Student's t-test.).


A , B . Representative of basal of ( A ) AURKA ( B ) phospho - AURKA ( p - AURKA ) in control PCM1 - depleted . RPE1 were transfected with control or PCM1 siRNA # 1 or siRNA # 2 for 48 h . Following 24 h serum starvation , were fixed stained with indicated antibodies . Centrosomal AURKA p - AURKA intensities were measured from maximum projections , average means of in control were normalized to 1 . n > 500 per experiment . Data for ( A ) AURKA ( B ) p - AURKA represent mean value from three experiments per condition ± ( * * * * p < 0 . 0001 , Unpaired Student ' s t - . ) Scale bar , 10

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B. Representative images and quantification of basal body levels of (A) AURKA and (B) phospho-AURKA (p-AURKA) in control and PCM1-depleted cells. RPE1 cells were transfected with control or PCM1 siRNA #1 or siRNA #2 for 48 h. Following 24 h serum starvation, cells were fixed and stained with the indicated antibodies. Centrosomal AURKA and p-AURKA fluorescence intensities were measured from maximum projections, and average means of the levels in control cells were normalized to 1. n>500 cells per experiment. Data for (A) AURKA and (B) p-AURKA represent mean value from three experiments per condition ± SEM (****p < 0.0001, Unpaired Student's t-test.) Scale bar, 10 μm


D . Cycloheximide chase experiment for of AURKA half - life . were transfected with control or PCM1 siRNA # 1 for 48 h , then treated with 200 nM cycloheximide along with serum starvation for indicated time points . AURKA intensities were quantified by immunoblotting for AURKA vinculin ( loading control ) AURKA were normalized to vinculin . Data represent mean value from three experiments per condition ± ( * * p < 0 . 01 , Unpaired Student ' s t - )

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 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

D. Cycloheximide chase experiment for quantification of AURKA half-life. Cells were transfected with control or PCM1 siRNA#1 for 48 h, then treated with 200 nM cycloheximide along with serum starvation for indicated time points. AURKA intensities were quantified by immunoblotting for AURKA and vinculin (loading control) and AURKA levels were normalized to vinculin levels. Data represent mean value from three experiments per condition ± SEM (**p < 0.01, Unpaired Student's t-test)


A . Representative immunofluorescence of cilium assembly experiments in control PCM1 - depleted RPE1 treated with DMSO or MLN8237 . were transfected with control or PCM1 siRNA # 1 for 48 h treated with DMSO ( vehicle control ) or 0 . 5 μM MLN8237 in serum starvation medium for 24 h . were fixed immunostained for cilium with acetylated tubulin antibody ( Acet - tub ) centrosome with gamma - tubulin antibody . was stained with DAPI . Scale bar , 10 μm . B . of ciliogenesis efficiency for A . n > 100 per experiment . Data represent mean value from three experiments per condition ± ( * * * p < 0 . 001 ; * * p < 0 . 01 ; ns , non - significant , Unpaired Student ' s t - . ) . C . of cilium for A . n > 100 per experiment . Data represent mean value from three experiments per condition ± ( * * * * p < 0 . 0001 ; ns , non - significant , Unpaired Student ' s t - . ) .

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 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

A. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or MLN8237. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO (vehicle control) or 0.5 μM MLN8237 in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm. B. Quantification of ciliogenesis efficiency for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; **p < 0.01; ns, non-significant, Unpaired Student's t-test.). C. Quantification of cilium length for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (****p < 0.0001; ns, non-significant, Unpaired Student's t-test.).


D . Representative immunofluorescence of cilium assembly experiments in control PCM1 - depleted RPE1 treated with DMSO or tubacin . were transfected with control or PCM1 siRNA # 1 for 48 h treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h . were fixed immunostained for cilium with Arl13b acetylated tubulin antibody ( Acet - tub ) centrosome with gamma - tubulin antibody . was stained with DAPI . Scale bar , 10 μm E . of ciliogenesis efficiency for D . n > 100 per experiment . Data represent mean value from three experiments per condition ± ( * p < 0 . 05 ; ns , non - significant , Unpaired Student ' s t - ) .

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 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

D. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or tubacin. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with Arl13b and acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm E. Quantification of ciliogenesis efficiency for D. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (*p < 0.05; ns, non-significant, Unpaired Student's t-test).


F . Representative immunofluorescence of IFT88 at centrosome cilium . RPE1 were transfected with control or PCM1 siRNA # 1 for 48 h , serum starved for 24 h treated with DMSO or 0 . 5 μM MLN8237 . were fixed immunostained with antibodies against IFT88 , acetylated tubulin gamma tubulin . was stained with DAPI . Scale bar , 10 μm . G . of IFT88 centrosomal ciliary for F . IFT88 intensities at centrosome axoneme were measured from maximum projections , average means of in control were normalized to 1 . Ciliary was calculated by dividing ciliary to cilium . n > 100 per experiment . Data represent mean value from three experiments per condition ± ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , Unpaired Student ' s t - )

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

F. Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237. Cells were fixed and immunostained with antibodies against IFT88, acetylated tubulin and gamma tubulin. DNA was stained with DAPI. Scale bar, 10 μm. G. Quantification of IFT88 centrosomal intensity and ciliary concentration for F. IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections, and average means of the levels in control cells were normalized to 1. Ciliary concentration was calculated by dividing ciliary fluorescence intensity to the cilium length. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test)


H , I . of cilium disassembly centrosomal AURKA after serum stimulation . RPE1 were transfected with control or PCM1 siRNA # 1 for 48 h , serum starved for 24 h treated with DMSO or 0 . 5 μM MLN8237 in serum stimulation medium for 2 h 24 h . were fixed immunostained with antibodies against acetylated tubulin gamma tubulin , of ciliated were quantified . x - axis indicates hours after serum stimulation . ( H ) n > 100 per experiment . Data represent mean value from three experiments per condition ± . ( I ) n > 95 per experiment . Data represent mean value from three experiments per condition ± ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , Unpaired Student ' s t - ) .

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

H, I. Quantification of cilium disassembly and centrosomal AURKA levels after serum stimulation. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237 in serum stimulation medium for 2 h and 24 h. Cells were fixed and immunostained with antibodies against acetylated tubulin and gamma tubulin, and percentage of ciliated cells were quantified. x-axis indicates the hours after serum stimulation. (H) n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM. (I) n>95 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test).


( A , B ) C57BL / 6 mice were left untreated ( NT ) or were intraperitoneally treated with indicated amounts of AG for 3 d . Lung sections stained with Haematoxylin eosin ( H & E ) ( A ) of lung lesion burden from H & E - stained sections ( B ) . Data information : Data are means + SD of indicated numbers of mice from 1 of n = 3 independent experiments with similar results each symbol represents 1 mouse . Data are representative of n = 3 independent experiments . One - way ANOVA followed by Dunnett ' s post hoc were used for statistical , respectively . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

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(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d. Lung sections stained with Haematoxylin and eosin(H&E) (A) and quantification of lung lesion burden from H&E-stained sections (B). Data information: Data are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.


( D - G ) H & E of lung sections from mice 4 after intranasal infection with M . bovis BCG ( D E ) or Mtb H37Rv ( F G ) in absence or presence of intranasally administrated AG aptamers ( 1 μg ) once at a 1 week - interval . of lung inflamed regions shown in ( E G ) . Data information : Data in are means + SD of indicated numbers of mice from 1 of n = 3 independent experiments with similar results each symbol represents 1 mouse . Data are representative of n = 3 independent experiments . One - way ANOVA followed by Dunnett ' s post hoc Student ' s t were used for statistical , respectively . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

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(D-G) H&E staining of lung sections from mice 4 weeks after intranasal infection with M. bovis BCG (D and E) or Mtb H37Rv (F and G) in the absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Quantification of lung inflamed regions shown in (E and G). Data information: Data in are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test and Student's t test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.


( B ) Quantitative polymerase chain reaction ( qPCR ) of Mmps including Mmp2 , Mmp9 , Mmp10 , Mmp12 Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG ( 1 μg / ml ) for indicated times . ( C ) Immunoblots of supernatants to analyze secreted MMP2 , MMP9 , MMP10 , MMP12 MMP13 by mouse peritoneal macrophages stimulated with AG ( 1 μg / mL ) for indicated times ; GADPH of lysates served as a loading control . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates each symbol represents mean of technical triplicates . Two - way ANOVA followed by Tukey ' s post hoc was used for statistical . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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(B) Quantitative polymerase chain reaction (qPCR) analysis of Mmps including Mmp2, Mmp9, Mmp10, Mmp12 and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG (1 μg/ml) for indicated times. (C) Immunoblots of cell supernatants to analyze secreted MMP2, MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/mL) for indicated times; GADPH of cell lysates served as a loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


( H ) qPCR of Mmps including Mmp9 , Mmp10 , Mmp12 , Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h ( MOI = 5 ) in absence or presence of AG aptamers ( 1 μg / ml ) . ( I ) Immunoblots of supernatants to analyze secreted MMP9 , MMP10 , MMP12 MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times ( MOI = 5 ) in absence or presence of AG aptamers ( 0 . 5 μg / ml ) ; GADPH of lysates served as loading control . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates each symbol represents mean of technical triplicates . Two - way ANOVA followed by Tukey ' s post hoc was used for statistical . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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(H) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h (MOI=5) in absence or presence of AG aptamers (1 μg/ml). (I) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in the absence or presence of AG aptamers (0.5 μg/ml); GADPH of cell lysates served as the loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


( G ) Immunoblots of supernatants to analyze secreted MMP9 , MMP10 , MMP12 , MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times ( MOI = 5 ) in absence or presence of inhibitor targeting ERK ( PD98059 ) at of 10 μM ; GADPH of lysates served as loading control .

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(G) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control.


( A , B ) C57BL / 6 mice were left untreated ( NT ) or were intraperitoneally treated with indicated amounts of AG for 3 d in absence or presence of MMP inhibitor marimastat ( 10 mg / kg ) given intraperitoneally prior to AG stimulation . Lung sections stained with H & E ( A ) of lung lesion burden from H & E stained sections ( B ) . Data information : Data in ( A ) are representative of n = 3 independent experiments . Data in ( B ) are means + SD of indicated numbers of mice from one of n = 3 independent experiments each symbol represents data from 1 mouse . One - way ANOVA followed by Dunnett ' s post hoc ( B ) was used for statistical . ns , not significant ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

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(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E (A) and quantification of lung lesion burden from H&E stained sections (B). Data information: Data in (A) are representative of n=3 independent experiments. Data in (B) are means + SD of indicated numbers of mice from one of n=3 independent experiments and each symbol represents data from 1 mouse. One-way ANOVA followed by Dunnett's post hoc test (B) was used for statistical analysis. ns, not significant; ****, p<0.0001. Scale bar, 200 μm.


( A ) Surface plasmon resonance ( SPR ) of direct interaction of AG with galectin - 9 . Curve fittings to a 1 : 1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines .

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(A) Surface plasmon resonance (SPR) assay of the direct interaction of AG with galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.


( C ) SPR of interaction of AG with CRD2 of galectin - 9 . Curve fittings to a 1 : 1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines .

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(C) SPR assay of the interaction of AG with CRD2 of galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.


( I ) Immunoblot of lysates of shCtrl shGalectin - 9 THP - 1 stimulated with AG ( 1 μg / ml ) for indicated times . Data are representative of n = 3 independent experiments .

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(I) Immunoblot of lysates of shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for indicated times. Data are representative of n=3 independent experiments.


( K ) qRT - PCR detection of MMP transcripts including Mmp9 , Mmp10 , Mmp12 in shCtrl shGalectin - 9 THP - 1 stimulated with AG ( 1 μg / ml ) for 24 h in absence or presence of ERK inhibitor PD98059 ( 10 μM ) . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates each symbol represents mean of technical triplicates . Two - way ANOVA followed by Dunnett ' s post hoc were used for statistical . ns , not significant ; * , p < 0 . 05 ; * * * , p < 0 . 001 * * * * , p < 0 . 0001 .

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(K) qRT-PCR detection of MMP transcripts including Mmp9, Mmp10, and Mmp12 in shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for 24 h in absence or presence of ERK inhibitor PD98059 (10 μM). Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001.


( D ) Confocal microscopy of mouse peritoneal macrophages left untreated ( NC ) ( upper row ) or stimulated with AG ( 1 μg / mL ) for 2 h ( middle row ) or infected with H37Rv for 3 h ( MOI = 5 ) ( bottom row ) , with anti - Galectin - 9 anti - TAK1 antibody . DAPI , nuclei , blue . Scale bar , 5 μm . Data in right graph show mean + SD of n = 12 fields from three independent experiments . symbols indicate colocalization of at least 10 in each field .

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(D) Confocal microscopy of mouse peritoneal macrophages left untreated (NC) (upper row) or stimulated with AG (1 μg/mL) for 2 h (middle row) or infected with H37Rv for 3 h (MOI=5) (bottom row), staining with anti-Galectin-9 and anti-TAK1 antibody. DAPI, nuclei, blue. Scale bar, 5 μm. Data in the right graph show mean + SD of n=12 fields from three independent experiments. The symbols indicate the colocalization ratio of at least 10 cells in each field.


( F ) Immunoblots of lysates of peritoneal macrophages isolated from WT or Galectin - 9 KO mice stimulated with AG ( 1 μg / ml ) in absence or presence of TAK1 inhibitor 5Z - 7 - OZ ( 1 μM ) for indicated times . Data are representative of n = 3 independent experiments .

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(F) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) in absence or presence of TAK1 inhibitor 5Z-7-OZ (1 μM) for indicated times. Data are representative of n=3 independent experiments.


( B , WT or Galectin - 9 KO mice were intraperitoneally treated with AG for 3 d in absence or presence of MMP inhibitor marimastat ( 10 mg / kg ) given intraperitoneally prior to AG stimulation . Lung sections stained with H & E Data information : Data are representative of n = 3 independent experiments .

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(B, WT or Galectin-9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments.


( D , WT or Galectin - 9 KO mice were intranasally infected with H37Rv for 4 in absence or presence of intranasally administrated AG aptamers ( 1 μg ) once at a 1 week - interval . Lung sections stained with H & E Data information : Data are representative of n = 3 independent experiments .

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(D, WT or Galectin-9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments.


Recovery of infectious recombinant MHV - GFP . culture supernatants containing produced after rescue from two MHV - GFP YAC clones ( Clone1 , Clone 2 ) were used to infect 17Cl - 1 . At 48 hours post - infection , infected were visualised for GFP expression ( left panels ) by bright field microscopy ( right panels ) . Mock represents 17Cl - 1 inoculated with supernatant from BHK - MHV - N electroporated without viral RNAs .

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Recovery of infectious recombinant MHV-GFP. Cell culture supernatants containing viruses produced after virus rescue from two MHV-GFP YAC clones (Clone1, Clone 2) were used to infect 17Cl-1 cells. At 48 hours post-infection, infected cells were visualised for GFP expression (left panels) and by bright field microscopy (right panels). Mock represents 17Cl-1 cells inoculated with supernatant from BHK-MHV-N cells electroporated without viral RNAs.


Replication kinetics of parental MHV - GFP recombinant MHV - GFP Clone 1 Clone 2 . L929 were infected at an MOI of 0 . 1 . culture supernatants were harvested at indicated time points post - infection titrated by plaque . Data are representative of three independent experiments with two replicates per in each experiment . Error bars show standard deviation ( SD ) from mean . pfu / ml , plaque forming units per millilitre .

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Replication kinetics of parental MHV-GFP and recombinant MHV-GFP Clone 1 and Clone 2. L929 cells were infected at an MOI of 0.1. Cell culture supernatants were harvested at indicated time points post-infection and titrated by plaque assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. pfu/ml, plaque forming units per millilitre.


Rescue of recombinant MERS - CoV MERS - CoV - GFP . Following delivery of viral RNAs into BHK - 21 via electroporation were co - cultivated with VeroB4 supernatants containing recombinant produced were used to infect new VeroB4 . Infected were visualised by bright field microscopy for recombinant MERS - CoV ( upper panel ; 5 days post infection ) , for GFP expression for recombinant MERS - CoV - GFP ( lower panel , 3 days post infection ) . Mock represents VeroB4 inoculated with supernatant from BHK - 21 electroporated without viral RNAs .

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Rescue of recombinant MERS-CoV and MERS-CoV-GFP. Following delivery of viral RNAs into BHK-21 cells via electroporation the cells were co-cultivated with VeroB4 cells and supernatants containing recombinant viruses produced were used to infect new VeroB4 cells. Infected cells were visualised by bright field microscopy for recombinant MERS-CoV (upper panel; 5 days post infection), and for GFP expression for recombinant MERS-CoV-GFP (lower panel, 3 days post infection). Mock represents VeroB4 cells inoculated with supernatant from BHK-21 cells electroporated without viral RNAs.


Replication kinetics of MERS - CoV - EMC ( laboratory adapted isolate ) , rMERS - CoV rMERS - CoV - GFP . VeroB4 were infected at an MOI of 0 . 01 . culture supernatants were harvested at indicated time points post infection titrated by TCID50 . Data are representative of three independent experiments with two replicates per in each experiment . Error bars show standard deviation ( SD ) from mean . TCID50 / ml , 50 % tissue culture infectious dose per millilitre .

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Replication kinetics of the MERS-CoV-EMC (laboratory adapted isolate), rMERS-CoV and rMERS-CoV-GFP. VeroB4 cells were infected at an MOI of 0.01. Cell culture supernatants were harvested at indicated time points post infection and titrated by TCID50 assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. TCID50/ml, 50% tissue culture infectious dose per millilitre.


Rescue of recombinant rSARS - CoV - 2 rSARS - CoV - 2 - GFP . experimental setting is illustrated in upper panel . Two clones of each construct 1 - 6 were used to prepare YAC DNAs in - vitro transcribed viral genome RNAs were electroporated into BHK - 21 / or BHK - SARS - N together with an mRNA encoding SARS - CoV - 2 N . Electroporated were co - cultivated with VeroE6 in a T25 flask culture supernatants were transferred to 12 - well plates to perform plaque for rSARS - CoV - 2 constructs 1 - 3 microscopy for rSARS - CoV - 2 - GFP constructs 4 - 6 . Plaque assays are shown in lower panel for SARS - CoV - 2 clones 1 . 1 , 2 . 2 , 3 . 1 . VeroE6 were infected with 10 − 1 , 10 − 2 , 10 − 3 dilutions of 1 ml supernatant from rescue experiments are compared to non - infected ( Mock ) . expression of GFP in SARS - CoV - 2 - GFP infected VeroE6 is shown for clones 4 . 1 , 5 . 2 , 6 . 1 . Mock represents non - infected VeroE6 .

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Rescue of recombinant rSARS-CoV-2 and rSARS-CoV-2-GFP. The experimental setting is illustrated in the upper panel. Two clones of each construct 1-6 were used to prepare YAC DNAs and in-vitro transcribed viral genome RNAs were electroporated into BHK-21 cells and/or BHK-SARS-N together with an mRNA encoding the SARS-CoV-2 N protein. Electroporated cells were co-cultivated with VeroE6 cells in a T25 flask and cell culture supernatants were transferred to 12-well plates to perform plaque assay for rSARS-CoV-2 constructs 1-3 and fluorescence microscopy for rSARS-CoV-2-GFP constructs 4-6. Plaque assays are shown in the lower panel for SARS-CoV-2 clones 1.1, 2.2, and 3.1. VeroE6 cells were infected with 10−1, 10−2, and 10−3 dilutions of 1 ml supernatant from the individual rescue experiments and are compared to non-infected cells (Mock). The expression of GFP in SARS-CoV-2-GFP infected VeroE6 cells is shown for clones 4.1, 5.2, and 6.1. Mock represents non-infected VeroE6 cells.


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

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


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

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


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

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


D Representative Western blot using antibodies against β - actin caspase 3 in whole cochlear extracts . E Histogram representing change in caspase 3 expression over time in control CDDP group ( n = 6 cochleae per condition per time point ) . Actin served as a loading control . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( * P ≤ 0 . 028 , * * P = 0 . 0007 , cochleae at different times after CDDP exposure vs . control 12h ) .

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


F , G Confocal showing basal region of cochlear explants treated with either culture medium alone or containing 10 µM CDDP for 1 ( F ) or 2 ( G ) days . Hair were identified using Myosin 7A ( red ) , phosphatidylserine sites on surface were detected using fluorochrome - labeled Annexin V ( green in F ) , apoptotic fragmentation was identified using a TUNEL apoptosis kit ( green in G ) . arrow heads indicate surface Annexin V - positive labeling ( lower left ) 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 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 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 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 showing basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day immunolabeled for Myosin 7A ( red ) γH2AX ( green in A ) , or Myosin 7A ( red ) 53BP1 ( green in F ) . arrow heads indicate CDDP - induced increase of γH2AX foci in both OHCs IHCs . Scale bars : A F = 24 µm . B , G Higher magnification of representative OHC IHCnuclei from conditions tested . Scale bar = 5 µm . C , H Histograms displaying green signal of x - projections 3D reconstruction from OHC IHCnuclei presented in B G , respectively . F0 corresponds to background noise , gray dashed lines represent threshold used to detect specific foci labeling . 3D were reconstructed according to threshold defined in histograms of conditions tested . Scale bar = 5 µm . D , E , I of γH2AX foci number ( D ) ; total volume of foci per nucleus ( E ) ; 53BP1 foci number ( I ) from OHCs IHCs treated with either culture medium alone ( blue red lines for OHCs IHCs , respectively ) , or 10 µM CDDP ( blue red lines for OHCs IHCs , respectively , n = 50 nuclei per condition per time point ) . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( D : * * P = 0 . 007 ; E I : * * P ≤ 0 . 009 . CDDP - exposed OHCsvs . CDDP - exposed IHCs ) . J Higher magnification showing representative OHCnuclei from basal region of organ of Corti treated with 10 µM CDDP for 1 day immunolabeled for 53BP1 ( red ) , γH2AX ( green ) counterstained with Hoechst ( blue ) . 3D image shows co - localization of smaller sized 53BP1 foci within 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

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 showing basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day immunolabeled for Myosin 7A ( red ) p - ATM ( green ) . Scale bar = 15 µm . B Higher magnification showing representative OHC IHCnuclei from control CDDP - exposed . Scale bar = 5 µm . C Histograms displaying green signal of x - projections of OHC IHCnuclei from control CDDP - exposed . D of p - ATM foci numbers in both OHCs IHCs exposed to either culture medium alone ( blue red lines for OHCs IHCs , respectively ) or 10 µM CDDP ( blue red lines for OHCs IHCs , respectively , n = 50 nuclei per condition per time point ) . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( * * P < 0 . 008 , * * * P = 0 . 00005 , CDDP - exposed OHCsvs . CDDP - exposed IHCs ) . E Higher magnification confocal 3D showing co - localization of p - ATM foci ( green ) with 53BP1 foci ( red ) in an OHCnucleus from basal region of 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 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 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 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 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 showing basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day immunolabeled for Myosin 7A ( red ) p - Chk2 ( green ) . Scale bar = 15 µm . G Histograms of green signal from OHC IHCnuclei of a CDDP - treated organ of Corti explant . H of Chk2 - positive nuclei in both IHCs ( red bars ) OHCs ( blue bars ) from control CDDP - treated organ of Corti cultures ( n = 50 nuclei per condition per time point ) . Data are expressed as mean ± . Kruskal - Wallis followed by post hoc Dunn ' s ( * 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 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 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 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 β - actin in control 10 µM CDDP - exposed whole cochlear extracts . J - K Histograms representing of total p53 ( J ) p53 phosphorylation ( K ) in control ( gray bars ) CDDP - exposed cochleae ( green bars , n = 6 cochleae per condition per time point ) . Actin served as a loading control . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( J : * P ≤ 0 . 023 ; K : * P = 0 . 032 , * * P = 0 . 007 , * * * P ≤ 0 . 0008 , cochleae at 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 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 O O O O O O O O O O O O O O O O O O O O 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 of OHCnuclei taken from 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 immunolabeled for 53BP1 ( green ) Hoechst 33342 ( blue ) . Scale bar = 5 µm . B of 53BP1 foci number per nucleus in both IHCs ( red bars ) OHCs ( blue bars ) for conditions ( n = 50 nuclei per condition per time point ) . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( * * * 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 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 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 showing basal region 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 5 days immunolabeled for Myosin 7A ( red ) . Scale bar = 24 µm . D Histograms representing numbers of surviving IHCs ( red bars ) OHCs ( blue bars ) for treatment conditions after 5 days ( n = 5 cochleae per condition per time point ) . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( * 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 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 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 of OHCnuclei from 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 immunolabeled for 53BP1 ( green ) Hoechst 33342 ( blue ) . Scale bar = 5 µm . F of 53BP1 foci number per nucleus in both IHCs ( red bars ) OHCs ( blue bars ) for conditions ( n = 50 nuclei per condition per time point ) . Data are expressed as mean ± . One - way ANOVA followed by post hoc Tukey ' s ( * * * 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 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 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 ) , p53 - / - mice treated with saline ( blue plot ) or CDDP ( blue plot ) for 5 days . B ABR thresholds recorded in p53wt mice before ( gray plot ) after 5 days of saline ( black plot ) or CDDP treatments ( green plot ) , ABR thresholds recorded in p53 - / - mice before ( gray plot ) , after 5 days of saline ( blue plot ) or CDDP treatment ( blue plot ) . C Mean ABR threshold from 4 kHz to 32 kHz derived from B . Data are expressed as mean ± ( saline treated - group : n = 7 , CDDP treated - group : n = 12 ) . One - way ANOVA followed by post hoc Tukey ' s ( * * 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 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 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 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 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 showing basal regions of cochleae from CDDP - treated p53wt 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 of surviving hair in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from 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 ± . Kruskal - Wallis followed by post hoc Dunn ' s ( * * 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 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 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 ± ( DMSO treated - group : n = 7 ; CDDP + DMSO treated - group : n = 12 ; CDDP + PFT - α - treated group : n = 12 ) . One - way ANOVA followed by post hoc Tukey ' s ( * * * 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 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 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).