Figure 4A Confocal immunofluorescence microscopy images of P1 . HTR cells transfected with IDO1 . WT or IDO1 . Y149F and co - stained with antibodies recognizing IDO1 ( green ) and EEA1 ( red ) . Nuclei were stained with DAPI ( blue ) . The merge imagine is shown for all stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm . B Pearson ' s coefficient of IDO1 colocalization with EEA1 in P1 . HTR cells transfected as in A , with each point representing colocalization within an image stack . Data represent mean ± SD ( n > 24 cells from three independent experiments ) . Statistical analysis was performed using two - tailed unpaired Student ' s t - test . * * * P < 0 . 001 . C Distribution of IDO1 . WT and the IDO1 . Y149F mutants in homogenates of P1 . HTR cells along the isopycnic sucrose gradient as in Fig 3D . Fractions were immunoblotted using antibodies specific for IDO1 . D The distribution of IDO1 . WT and IDO1 . Y149F proteins along the isopycnic sucrose gradient is shown in term of densitometric analysis in each fraction ( mean ± SD of three experiments ) .

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Figure 4A Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT or IDO1.Y149F and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.B Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected as in A, with each point representing colocalization within an image stack. Data represent mean ± SD (n > 24 cells from three independent experiments). Statistical analysis was performed using two-tailed unpaired Student's t-test. ***P < 0.001.C Distribution of IDO1.WT and the IDO1.Y149F mutants in homogenates of P1.HTR cells along the isopycnic sucrose gradient as in Fig 3D. Fractions were immunoblotted using antibodies specific for IDO1.D The distribution of IDO1.WT and IDO1.Y149F proteins along the isopycnic sucrose gradient is shown in term of densitometric analysis in each fraction (mean ± SD of three experiments).


Figure 5 A Pull - down assay of lysates from untransfected P1 . HTR cells with unphosphorylated ( YENM ) and tyrosine - phosphorylated ( pYENM ) IDO1 peptides , analyzed by immunoblotting with anti - p85 , - p110α , - p110β , - p110γ , and - p110δ antibodies . Whole cell lysates ( WCL ) were used as control of lysate amount . B Confocal immunofluorescence microscopy images of P1 . HTR cells transfected with IDO1 . WT incubated with class IA PI3K p110 isoform inhibitors ( indicated ) or medium alone ( Ctrl ) for 24 h and co - stained with antibodies recognizing IDO1 ( green ) and EEA1 ( red ) . Nuclei were stained with DAPI ( blue ) . The merge imagine is shown for all stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm . C Pearson ' s coefficient of IDO1 colocalization with EEA1 in P1 . HTR cells transfected and treated as in ( B ) , with each point representing colocalization within an image stack . Data represent mean ± SD ( n > 20 cells from two independent experiments ) . D Tyrosine phosphatase activity in anti - IDO1 immunoprecipitates from P1 . HTR cells transfected with IDO1 . WT cells and treated with PI3K catalytic inhibitors ( indicated ) or medium alone ( ctrl ) . Data represent the mean ± SD ( n = 3 technical replicates ) .

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Figure 5 A Pull-down assay of lysates from untransfected P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated (pYENM) IDO1 peptides, analyzed by immunoblotting with anti-p85, -p110α, -p110β, -p110γ, and -p110δ antibodies. Whole cell lysates (WCL) were used as control of lysate amount. B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT incubated with class IA PI3K p110 isoform inhibitors (indicated) or medium alone (Ctrl) for 24 h and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.C Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected and treated as in (B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from two independent experiments).D Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from P1.HTR cells transfected with IDO1.WT cells and treated with PI3K catalytic inhibitors (indicated) or medium alone (ctrl). Data represent the mean ± SD (n = 3 technical replicates).


Figure 6A Confocal immunofluorescence microscopy images of freshly purified pDCs co - stained with antibodies recognizing IDO1 ( green ) and markers of intracellular organelles and structures ( indicated ; red ) . Nuclei were stained with DAPI ( blue ) . The merge imagine is shown for all stainings or without DAPI ( no DAPI ) . Scale bar , 5 µm . B Confocal immunofluorescence microscopy images of pDCs incubated for 24 h with TGF - β or IFN - γ and co - stained with antibodies recognizing IDO1 ( green ) and EEA1 ( red ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 5 µm . C Pearson ' s coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF - β or IFN - γ ( as in B ) , with each point representing colocalization within an image stack . Data represent mean ± SD ( n > 20 cells from three independent experiments ) .

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Figure 6A Confocal immunofluorescence microscopy images of freshly purified pDCs co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 5 µm.B Confocal immunofluorescence microscopy images of pDCs incubated for 24 h with TGF-β or IFN-γ and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). Scale bar, 5 µm. C Pearson's coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF-β or IFN-γ (as in B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from three independent experiments).


Figure 1 ( C ) The Hippo WW domain - containing proteins show binding specificity to the known Hippo PY motif - containing proteins . TAP - MS analysis of a series of WW domain - containing proteins were performed and their binding with the indicated Hippo PY motif - containing proteins was summarized in a heatmap . ( E ) Validation of the binding specificity for the Hippo WW domain - containing proteins . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay . ( F ) Validation of the binding specificity for the derived WW domains from the Hippo WW domain - containing proteins . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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Figure 1(C) The Hippo WW domain-containing proteins show binding specificity to the known Hippo PY motif-containing proteins. TAP-MS analysis of a series of WW domain-containing proteins were performed and their binding with the indicated Hippo PY motif-containing proteins was summarized in a heatmap.(E) Validation of the binding specificity for the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.(F) Validation of the binding specificity for the derived WW domains from the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


Figure 2 ( C - G ) Validation of the identified 9 - amino acid sequence in determining the Hippo WW domain binding specificity . The requirement of the identified 9 - amino acid sequence for AMOT association was respectively examined for TAZ ( C ) , TAZ - WW domain ( D ) , KIBRA ( E ) , YAP ( F ) and SAV1 ( G ) . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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Figure 2(C-G) Validation of the identified 9-amino acid sequence in determining the Hippo WW domain binding specificity. The requirement of the identified 9-amino acid sequence for AMOT association was respectively examined for TAZ (C), TAZ-WW domain (D), KIBRA (E), YAP (F) and SAV1 (G). HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


Figure 3 ( B ) STXBP4 forms a complex with several Hippo PY motif - containing proteins . Immunoprecipitation was performed with STXBP4 antibody . ( C ) The identified 9 - amino acid sequence is required for the association between STXBP4 and AMOT . HEK293T cells were transfected with the indicated STXBP4 mutants and subjected to the pulldown assay . ( E ) The YAP - WW1 / SMAD7 - PY and STXBP4 - WW / SMAD7 - PY complexes show similar cumulative average trend and average binding free energy ( ∆ G ) within standard deviation ( the shaded region ) of one another . ( F ) Loss of STXBP4 inhibits YAP phosphorylation and LATS activation . Western blotting was performed with the indicated antibodies . Loss of STXBP4 activates YAP . STXBP4 deficiency promotes YAP nuclear translocation ( G ) Loss of STXBP4 activates YAP . STXBP4 deficiency promotes YAP downstream gene transcription ( mean ± s . d . , n = 3 biological replicates ) ( H ) . Scale bar , 20 μm . * * * p < 0 . 001 ( Student ' s t - test ) . ( I ) WW domain is required for the STXBP4 - mediated YAP cytoplasmic translocation . STXBP4 KO cells were transfected with the indicated STXBP4 constructs and immunofluorescent staining was performed . HA - positive cells ( arrows ) from ~ 30 different views ( ~ 200 cells in total ) were randomly selected and quantified for YAP localization . Percentage of HA - positive cells with nuclear YAP enrichment is shown . Scale bar , 20 μm . ( J ) Loss of STXBP4 attenuates YAP phosphorylation as induced by actin cytoskeleton inhibition . The indicated cells were subjected to serum starvation ( treatment with no - serum medium for 12 hours ) , glucose starvation ( treatment with no - glucose medium for 6 hours ) or actin inhibition ( treatment with 0 . 5 μg / mL latrunculin B or 5 μM blebbistatin for 30 min ) . YAP phosphorylation was detected using phospho - tag gel , where the YAP phosphorylation level was indicated .

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Figure 3(B) STXBP4 forms a complex with several Hippo PY motif-containing proteins. Immunoprecipitation was performed with STXBP4 antibody.(C) The identified 9-amino acid sequence is required for the association between STXBP4 and AMOT. HEK293T cells were transfected with the indicated STXBP4 mutants and subjected to the pulldown assay.(E) The YAP-WW1/SMAD7-PY and STXBP4-WW/SMAD7-PY complexes show similar cumulative average trend and average binding free energy (∆G) within standard deviation (the shaded region) of one another.(F) Loss of STXBP4 inhibits YAP phosphorylation and LATS activation. Western blotting was performed with the indicated antibodies.Loss of STXBP4 activates YAP. STXBP4 deficiency promotes YAP nuclear translocation (G)Loss of STXBP4 activates YAP. STXBP4 deficiency promotes YAP downstream gene transcription (mean ± s.d., n=3 biological replicates) (H). Scale bar, 20 μm.*** p < 0.001 (Student's t-test).(I) WW domain is required for the STXBP4-mediated YAP cytoplasmic translocation. STXBP4 KO cells were transfected with the indicated STXBP4 constructs and immunofluorescent staining was performed. HA-positive cells (arrows) from ~30 different views (~200 cells in total) were randomly selected and quantified for YAP localization. Percentage of HA-positive cells with nuclear YAP enrichment is shown. Scale bar, 20 μm.(J) Loss of STXBP4 attenuates YAP phosphorylation as induced by actin cytoskeleton inhibition. The indicated cells were subjected to serum starvation (treatment with no-serum medium for 12 hours), glucose starvation (treatment with no-glucose medium for 6 hours) or actin inhibition (treatment with 0.5 μg/mL latrunculin B or 5 μM blebbistatin for 30 min). YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.


Figure 4 ( B ) STXBP4 forms a protein complex with α - catenin and a group of Hippo pathway proteins . ( C ) STXBP4 promotes the association of α - catenin with AMOT and LATS1 . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay . ( D ) Loss of STXBP4 diminishes the association of α - catenin with AMOT , LATS1 and YAP . HEK293A and STXBP4 KO cells were transfected with the SFB - tagged α - catenin construct and subjected to the pulldown assay . ( E ) STXBP4 induces the co - localization between α - catenin and AMOT as well as LATS1 and YAP . HEK293A cells were transfected with the indicated constructs and immunofluorescence was performed . Scale bar , 20 μm . Identification of several STXBP4 missense mutations that disrupt its interaction with α - catenin and AMOT . The identified missense mutations respectively disrupted the STXBP4 - α - catenin ( G ) and STXBP4 - AMOT ( H ) complex formation . ( I ) Inhibition of actin cytoskeleton promotes the STXBP4 - associated protein complex formation . HEK293A and the STXBP4 KO cells were subjected to immunoprecipitation using pre - immune serum and anti - STXBP4 serum under the indicated treatments . ( J ) The missense mutations of STXBP4 ( F ) diminished the ability of STXBP4 to rescue YAP phosphorylation in the STXBP4 KO cells with low actin cytoskeleton tension . YAP phosphorylation was detected using phospho - tag gel , where the YAP phosphorylation level was indicated .

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Figure 4(B) STXBP4 forms a protein complex with α-catenin and a group of Hippo pathway proteins.(C) STXBP4 promotes the association of α-catenin with AMOT and LATS1. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.(D) Loss of STXBP4 diminishes the association of α-catenin with AMOT, LATS1 and YAP. HEK293A and STXBP4 KO cells were transfected with the SFB-tagged α-catenin construct and subjected to the pulldown assay.(E) STXBP4 induces the co-localization between α-catenin and AMOT as well as LATS1 and YAP. HEK293A cells were transfected with the indicated constructs and immunofluorescence was performed. Scale bar, 20 μm.Identification of several STXBP4 missense mutations that disrupt its interaction with α-catenin and AMOT. The identified missense mutations respectively disrupted the STXBP4- α-catenin (G) and STXBP4-AMOT (H) complex formation.(I) Inhibition of actin cytoskeleton promotes the STXBP4-associated protein complex formation. HEK293A and the STXBP4 KO cells were subjected to immunoprecipitation using pre-immune serum and anti-STXBP4 serum under the indicated treatments.(J) The missense mutations of STXBP4 (F) diminished the ability of STXBP4 to rescue YAP phosphorylation in the STXBP4 KO cells with low actin cytoskeleton tension. YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.


Figure 5STXBP4 is downregulated in human kidney cancer . The mRNA level of STXBP4 is analyzed in the Firebrowse web database ( A ) , where 14 , 729 tumor sample data generated by TCGA were included . The first quartile , median and third quartile values were indicated as the boxplots . Outliers were plotted as individual points . Error bars indicated the standard deviation above and below the mean of the data . STXBP4 is downregulated in human kidney cancer . The expression of STXBP4 was also examined using kidney tissue microarray , where percentage of the indicated tissue samples with downregulated STXBP4 was shown ( B ) . The p value was calculated by using the paired Student ' s t - test . ( C ) Kaplan - Meier curves of overall survival of patients with ccRCC is stratified by STXBP4 expression level . Clinical data of STXBP4 were analyzed in TCGA - KIRC project containing total 611 patient samples . The p value was calculated by using the Log - rank ( Mantel - Cox ) test . ( D ) Immunohistochemical staining of STXBP4 and YAP were performed in a kidney cancer tissue microarray , where the indicated regions in the box were shown three times enlarged . Brown staining indicates positive immunoreactivity . Scale bar , 100 μm . ( E ) Correlation analyses between STXBP4 and YAP in human normal kidney and clear cell carcinoma samples are shown as tables . Statistical significance was determined by chi - square test . R , correlation coefficient . N , nuclear localization . C , cytoplasmic localization . ( F ) STXBP4 expression is examined in a panel of ccRCC cell lines by Western Blotting . ( G and H ) Both the association with α - catenin and the functional WW domain are required for the STXBP4 ' s tumor suppressive function in 786 - O cells . Overexpression of STXBP4 , but not the indicated STXBP4 missense mutants , significantly suppressed the 786 - O cell xenograft tumor formation . Xenograft tumors are shown in ( G ) , and the tumor weight is quantified in ( H ) ( n = 5 mice , mean ± s . d . ) . * * p < 0 . 01 ( Student ' s t - test ) . Scale bar , 1 cm .

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Figure 5STXBP4 is downregulated in human kidney cancer. The mRNA level of STXBP4 is analyzed in the Firebrowse web database (A), where 14,729 tumor sample data generated by TCGA were included. The first quartile, median and third quartile values were indicated as the boxplots. Outliers were plotted as individual points. Error bars indicated the standard deviation above and below the mean of the data.STXBP4 is downregulated in human kidney cancer. The expression of STXBP4 was also examined using kidney tissue microarray, where percentage of the indicated tissue samples with downregulated STXBP4 was shown (B). The p value was calculated by using the paired Student's t-test.(C) Kaplan-Meier curves of overall survival of patients with ccRCC is stratified by STXBP4 expression level. Clinical data of STXBP4 were analyzed in TCGA-KIRC project containing total 611 patient samples. The p value was calculated by using the Log-rank (Mantel-Cox) test.(D) Immunohistochemical staining of STXBP4 and YAP were performed in a kidney cancer tissue microarray, where the indicated regions in the box were shown three times enlarged. Brown staining indicates positive immunoreactivity. Scale bar, 100 μm.(E) Correlation analyses between STXBP4 and YAP in human normal kidney and clear cell carcinoma samples are shown as tables. Statistical significance was determined by chi-square test. R, correlation coefficient. N, nuclear localization. C, cytoplasmic localization.(F) STXBP4 expression is examined in a panel of ccRCC cell lines by Western Blotting.(G and H) Both the association with α-catenin and the functional WW domain are required for the STXBP4's tumor suppressive function in 786-O cells. Overexpression of STXBP4, but not the indicated STXBP4 missense mutants, significantly suppressed the 786-O cell xenograft tumor formation. Xenograft tumors are shown in (G), and the tumor weight is quantified in (H) (n = 5 mice, mean ± s.d.). ** p < 0.01 (Student's t-test). Scale bar, 1 cm.


Figure 2A , RT - qPCR ( n = 3 - 12 biological replicates ) analyses of CGN expression in different mouse organs at P21 . B . Western blot analyses of CGN expression in different mouse organs at P21 . G . Whole mount immunofluorescence of CGN expression in P3 , P14 and P21 mouse cochlea coimmunostained with cuticular plate marker LMO7 ( G ) . CGN expression levels at the cuticular plates were normalized to LMO7 immunofluorescent signals ( n = 130 - 193 hair cells from 3 - 4 cochleae ) . Data information : Data are presented as mean ± SEM ; one - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 .

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Figure 2A, RT-qPCR (n = 3-12 biological replicates) analyses of CGN expression in different mouse organs at P21.B. Western blot analyses of CGN expression in different mouse organs at P21.G. Whole mount immunofluorescence of CGN expression in P3, P14 and P21 mouse cochlea coimmunostained with cuticular plate marker LMO7 (G). CGN expression levels at the cuticular plates were normalized to LMO7 immunofluorescent signals (n = 130-193 hair cells from 3-4 cochleae). Data information: Data are presented as mean ± SEM; one-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.


Figure 3B . MDCK cells expressing WT or mutant CGN were immunolabeled with CGN or Flag antibodies . D . RT - qPCR of CGN expression in transfected MDCK cells ( n = 3 biological replicates ) . Data information : Data are presented as mean ± SEM ; unpaired Student ' s t - test was used in ( D ) . ns , not significant , P > 0 . 05 .

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Figure 3B. MDCK cells expressing WT or mutant CGN were immunolabeled with CGN or Flag antibodies.D. RT-qPCR of CGN expression in transfected MDCK cells (n = 3 biological replicates). Data information: Data are presented as mean ± SEM; unpaired Student's t-test was used in (D). ns, not significant, P > 0.05.


Figure 4A - C . DPOAE thresholds ( A ) , ABR thresholds ( B ) and ABR P1 amplitudes ( C ) of the 2 - month old control ( Cgnfl / fl ) and Cgn - cKO ( Cgnfl / fl ; Pou4f3creER / + ) mice . Control mice ( circle ) , n = 16 mice ( female = 8 , male = 8 ) ; Cgn - cKO mice ( square ) , n = 19 mice ( female = 11 , male = 8 ) . Data information : Data are presented as mean ± SEM ; two - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 . D - I . Growth curves of ABR P1 amplitudes at different cochlear frequencies ( 5 . 6 - 32 kHz ) of the 2 - month old control and Cgn - cKO mice . Control mice ( circle ) , n = 16 mice ( female = 8 , male = 8 ) ; Cgn - cKO mice ( square ) , n = 19 mice ( female = 11 , male = 8 ) . Data information : Data are presented as mean ± SEM ; two - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 . J . Whole mount immunofluorescence of the organ of Corti from 2 - month old control and Cgn - cKO mice . Phalloidin ( F - actin , magenta ) ; Myosin7a ( hair cells , green ) . White arrows label the lost OHCs .

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Figure 4A-C. DPOAE thresholds (A), ABR thresholds (B) and ABR P1 amplitudes (C) of the 2-month old control (Cgnfl/fl) and Cgn-cKO (Cgnfl/fl;Pou4f3creER/+) mice. Control mice (circle), n = 16 mice (female = 8, male = 8); Cgn-cKO mice (square), n = 19 mice (female = 11, male = 8). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.D-I. Growth curves of ABR P1 amplitudes at different cochlear frequencies (5.6 - 32 kHz) of the 2-month old control and Cgn-cKO mice. Control mice (circle), n = 16 mice (female = 8, male = 8); Cgn-cKO mice (square), n = 19 mice (female = 11, male = 8). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.J. Whole mount immunofluorescence of the organ of Corti from 2-month old control and Cgn-cKO mice. Phalloidin (F-actin, magenta); Myosin7a (hair cells, green). White arrows label the lost OHCs.


Figure 5C . Whole mount immunofluorescence of CGN expression in organ of Corti from P21 CgndelG mouse . D - I . DPOAE thresholds ( D , E ) , ABR thresholds ( F , G ) , and ABR P1 amplitudes at 32 kHz ( H , I ) of 2 - month old ( D , F , H ) or 6 - month old ( E , G , I ) CgndelG mice . Control Cgn + / + mice ( circle ) , n = 3 mice ( 2 - month , female = 1 , male = 2 ) or 15 mice ( 6 - month , female = 4 , male = 11 ) ; CgndelG / + mice ( triangle ) , n = 8 mice ( 2 - month , female = 4 , male = 4 ) or 20 mice ( 6 - month , female = 9 , male = 11 ) ; CgndelG / delG mice ( square ) , n = 7 mice ( 2 - month , female = 4 , male = 3 ) or 16 mice ( 6 - month , female = 6 , male = 10 ) . Cgn + / + vs CgndelG / delG , * ( red ) ; Cgn + / + vs CgndelG / + , * ( green ) . Data information : Data are presented as mean ± SEM ; two - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 . J . Whole mount immunofluorescence of the organ of Corti from 7 - month old CgndelG mice . White arrows indicate lost OHCs by F - actin labeling ( Phalloidin ) . K . Percentage of OHC loss in 7 - month old CgndelG mice ( n = 3 - 8 biological replicates from 3 cochleae ) . Data information : Data are presented as mean ± SEM ; two - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 .

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Figure 5C. Whole mount immunofluorescence of CGN expression in organ of Corti from P21 CgndelG mouse.D-I. DPOAE thresholds (D, E), ABR thresholds (F, G), and ABR P1 amplitudes at 32 kHz (H, I) of 2-month old (D, F, H) or 6-month old (E, G, I) CgndelG mice. Control Cgn+/+ mice (circle), n = 3 mice (2-month, female = 1, male = 2) or 15 mice (6-month, female = 4, male = 11); CgndelG/+ mice (triangle), n = 8 mice (2-month, female = 4, male = 4) or 20 mice (6-month, female = 9, male = 11); CgndelG/delG mice (square), n = 7 mice (2-month, female = 4, male = 3) or 16 mice (6-month, female = 6, male = 10). Cgn+/+ vs CgndelG/delG, * (red); Cgn+/+ vs CgndelG/+, * (green). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.J. Whole mount immunofluorescence of the organ of Corti from 7-month old CgndelG mice. White arrows indicate lost OHCs by F-actin labeling (Phalloidin).K. Percentage of OHC loss in 7-month old CgndelG mice (n = 3-8 biological replicates from 3 cochleae). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.


Figure 6F . Percentage of OHC loss in noise exposed CgndelG mice ( n = 5 - 9 cochleae ) . Data information : Data are presented as mean ± SEM ; one - way ANOVA was used in ( F ) . * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 .

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Figure 6F. Percentage of OHC loss in noise exposed CgndelG mice (n = 5-9 cochleae). Data information: Data are presented as mean ± SEM; one-way ANOVA was used in (F). ** P < 0.01, *** P < 0.001 and **** P < 0.0001.


Figure 7Whole mount immunofluorescence with LMO7 antibody on 2 - month old CgndelG mice at 32 kHz cochlear regionWhole mount immunofluorescence with Spectrin β II antibody on 2 - month old CgndelG mice at 32 kHz cochlear

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Figure 7Whole mount immunofluorescence with LMO7 antibody on 2-month old CgndelG mice at 32 kHz cochlear regionWhole mount immunofluorescence with Spectrin β II antibody on 2-month old CgndelG mice at 32 kHz cochlear region


Figure 8A . Low - magnification SEM images of hair bundles from 2 - month old Cgn + / + and CgndelG / delG mice . B . High - magnification SEM images of IHC hair bundles from 2 - month old Cgn + / + and CgndelG / delG mice . C . High - magnification SEM images of OHC hair bundles from 2 - month old Cgn + / + and CgndelG / delG mice . Shown are hair bundles of the second - row OHCs ( OHC2 ) .

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Figure 8A. Low-magnification SEM images of hair bundles from 2-month old Cgn+/+ and CgndelG/delG mice. B. High-magnification SEM images of IHC hair bundles from 2-month old Cgn+/+ and CgndelG/delG mice. C. High-magnification SEM images of OHC hair bundles from 2-month old Cgn+/+ and CgndelG/delG mice. Shown are hair bundles of the second-row OHCs (OHC2).


Figure 1A WT MDFs were treated ± TSI individually or in combination for 3 hrs . Whole cell lysates ( WCL ) and UBA - pull down ( UBA - PD ) fractions were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments . Samples of UBA - pull down in following figures were analysed in the same way unless otherwise indicated . B WT , Ripk3 - / - , Mlkl - / - , Tnfr1 - / - and Traf2 - / - MDFs were untreated ( UT ) or treated with TSI for 3 hrs . Nec - 1 and GSK872 were added to inhibit RIPK1 and RIPK3 kinase activities respectively . E RIPK3 - gyrase were inducibly expressed in Ripk3 - / - MDFs by doxycycline ( dox ) for 5 hrs , and cells were then treated ± combination of TSI , or coumermycin ( coum ) for 3 hrs .

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Figure 1A WT MDFs were treated ± TSI individually or in combination for 3 hrs. Whole cell lysates (WCL) and UBA-pull down (UBA-PD) fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments. Samples of UBA-pull down in following figures were analysed in the same way unless otherwise indicated.B WT, Ripk3-/-, Mlkl-/-, Tnfr1-/- and Traf2-/- MDFs were untreated (UT) or treated with TSI for 3 hrs. Nec-1 and GSK872 were added to inhibit RIPK1 and RIPK3 kinase activities respectively.E RIPK3-gyrase were inducibly expressed in Ripk3-/- MDFs by doxycycline (dox) for 5 hrs, and cells were then treated ± combination of TSI, or coumermycin (coum) for 3 hrs.


Figure 2B UBA - pull down from WT MDFs treated with TSI for 3 hrs were subjected to the DUBs shown in ( A ) . Beads eluates were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments . C 1 . 4 mL cleared cell lysate from 4 x 106 TSI - treated WT MDFs was split into three parts of the indicated volume , followed by UBA - pull down and DUB incubation . Bead eluates were analysed by Western blot and probed with the indicated antibodies . Representative of three independent experiments .

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Figure 2B UBA-pull down from WT MDFs treated with TSI for 3 hrs were subjected to the DUBs shown in (A). Beads eluates were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.C 1.4 mL cleared cell lysate from 4 x 106 TSI-treated WT MDFs was split into three parts of the indicated volume, followed by UBA-pull down and DUB incubation. Bead eluates were analysed by Western blot and probed with the indicated antibodies. Representative of three independent experiments.


Figure 3A WT MDFs were treated with TSI for indicated time . Cells were fractionated into cytosol and crude membrane , followed by UBA - pull down . All fractions were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments . B WT USP21 - CaaX and USP21C221R - CaaX ( CR ) were inducibly expressed in WT MDFs by doxycycline for 5 hrs . Ubiquitylated proteins were enriched followed by TSI stimulation and cellular fractionation . All fractions were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments .

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Figure 3A WT MDFs were treated with TSI for indicated time. Cells were fractionated into cytosol and crude membrane, followed by UBA-pull down. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.B WT USP21-CaaX and USP21C221R-CaaX (CR) were inducibly expressed in WT MDFs by doxycycline for 5 hrs. Ubiquitylated proteins were enriched followed by TSI stimulation and cellular fractionation. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


Figure 4A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline , at the same time cells were untreated ( UT ) or treated with TSQ for 6 hrs . Cells were fractionated into cytosol ( C ) and crude membrane ( M ) . Fractions were analysed by BN - or SDS - PAGE , Western blot and probed with the indicated antibodies . Representative of three independent experiments . B Cell lysates from ( A ) were subjected to UBA - pull down and analysed as described above . E HT29 cells were stimulated with TSI , ± NSA ( 500 nM ) , or left untreated ( UT ) for 16 hrs , followed by cellular fractionation . Fractions were analysed by BN - or SDS - PAGE , Western blot and probed with the indicated antibodies . Representative of three independent experiments . F Cell lysates from ( E ) were subjected to UBA - pulldown and analysed as described above .

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Figure 4A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, at the same time cells were untreated (UT) or treated with TSQ for 6 hrs. Cells were fractionated into cytosol (C) and crude membrane (M). Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. B Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.E HT29 cells were stimulated with TSI, ± NSA (500 nM), or left untreated (UT) for 16 hrs, followed by cellular fractionation. Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. F Cell lysates from (E) were subjected to UBA-pulldown and analysed as described above.


Figure 5A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline , cells were untreated or treated with TSQ for 6 hrs ( please note that the same WT control was used in Fig . EV3A ) . Cell death was measured by PI staining based on flow cytometry . Data are plotted as mean ± SEM of three independent experiments . B Cellular fractions from ( A ) were analysed by Western blot from BN - PAGE or SDS - PAGE using antibodies as indicated . The same WT control was used in Fig . 4A . Representative of three independent experiments . C Cell lysates from ( A ) were subjected to UBA - pull down and analysed as described above . D N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline overnight , and cells were treated with TSI for indicated time , followed by UBA - pull down . Representative of three independent experiments . E N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 16 hrs . Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline . Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 ( BAF ) , PS341 ( PS ) or left untreated ( UT ) . IDN - 6556 was added to all conditions to block apoptosis . Cells were collected 0 , 2 , 4 , 6 hrs after medium replacement , followed by UBA - pull down . Representative of three independent experiments .

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Figure 5A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, cells were untreated or treated with TSQ for 6 hrs (please note that the same WT control was used in Fig. EV3A). Cell death was measured by PI staining based on flow cytometry. Data are plotted as mean ± SEM of three independent experiments. B Cellular fractions from (A) were analysed by Western blot from BN-PAGE or SDS-PAGE using antibodies as indicated. The same WT control was used in Fig. 4A. Representative of three independent experiments. C Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.D N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline overnight, and cells were treated with TSI for indicated time, followed by UBA-pull down. Representative of three independent experiments. E N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 16 hrs. Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline. Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 (BAF), PS341 (PS) or left untreated (UT). IDN-6556 was added to all conditions to block apoptosis. Cells were collected 0, 2, 4, 6 hrs after medium replacement, followed by UBA-pull down. Representative of three independent experiments.


Figure 6B WT and 4KR mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 6 hrs and cells were untreated ( UT ) or treated with TSI , followed by UBA - pull down . Representative of three independent experiments .

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Figure 6B WT and 4KR mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 6 hrs and cells were untreated (UT) or treated with TSI, followed by UBA-pull down. Representative of three independent experiments.


Figure 7D MLKL - / - HT29 cells stably transfected with constructs encoding human MLKL - USP21 and MLKL - USP21C221R , were treated with doxycycline , NSA ( 1 μM ) , TSI or combinations thereof ( added simultaneously ) . Sytox Green positive cells were quantified in real time by live cell imaging . Data are plotted as mean ± SEM of six independent experiments . ( A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA ) . E Human MLKL - USP21 and MLKL - USP21C221R were inducibly expressed in MLKL - / - HT29 cells by doxycycline ( 10 ng / mL ) for 16 hrs , TSI was added for the indicated times but all samples were collected 25 hrs post - induction , followed by UBA - pulldown . Representative of three independent experiments .

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Figure 7D MLKL-/- HT29 cells stably transfected with constructs encoding human MLKL-USP21 and MLKL-USP21C221R, were treated with doxycycline, NSA (1 μM), TSI or combinations thereof (added simultaneously). Sytox Green positive cells were quantified in real time by live cell imaging. Data are plotted as mean ± SEM of six independent experiments. (A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA). E Human MLKL-USP21 and MLKL-USP21C221R were inducibly expressed in MLKL-/- HT29 cells by doxycycline (10 ng/mL) for 16 hrs, TSI was added for the indicated times but all samples were collected 25 hrs post-induction, followed by UBA-pulldown . Representative of three independent experiments.


Figure 3B Inhibition zone - based assay for synergy between AMSIN and HNP1 & 2 or lysozyme C ( abbreviated as hLZc ) to the two oral Streptococcus ( SSAN and SSAL ) . Inset ( upper panel , boxed ) , a representative example of the inhibition - zone test showing the synergy . Peptide doses used here were 0 . 05 nmol / well for HNP1 & 2 and hLZc at which no visible inhibition zones were observed for both of them . At the same dose , AMSIN produced a small inhibition zone . For synergetic observation , two peptides were applied to the same well according to the doses indicated . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test ( * * * P < 0 . 001 Note : the nanomolar concentration of HNP1 & 2 was calculated from the average molecular weight of HNP1 and HNP2 . C The FIC index assay for synergy between AMSIN and HNP1 & 2 or hLZc to SSAL and SSAN . The dashed line represents the cutoff of the FIC index ( 0 . 5 ) for synergy .

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Figure 3B Inhibition zone-based assay for synergy between AMSIN and HNP1&2 or lysozyme C (abbreviated as hLZc) to the two oral Streptococcus (SSAN and SSAL). Inset (upper panel, boxed), a representative example of the inhibition-zone test showing the synergy. Peptide doses used here were 0.05 nmol/well for HNP1&2 and hLZc at which no visible inhibition zones were observed for both of them. At the same dose, AMSIN produced a small inhibition zone. For synergetic observation, two peptides were applied to the same well according to the doses indicated. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test (***P<0.001 Note: the nanomolar concentration of HNP1&2 was calculated from the average molecular weight of HNP1 and HNP2.C The FIC index assay for synergy between AMSIN and HNP1&2 or hLZc to SSAL and SSAN. The dashed line represents the cutoff of the FIC index (0.5) for synergy.


Figure 4A Growth kinetic measurements of SM exposed to AMSIN or various antibiotics with known cellular targets . The peptide or the compounds used were 4 × CL . VAN , vancomycin ; RIF , rifampicin ; KAN , kanamycin . OD600 were calculated as measured values subtracting the background value from broth medium ( OD600 = 0 . 356 ) . D Scanning electron microscopic observation of AMSIN - induced BM deformation . The doses used were 10 × CL for AMSIN and vancomycin . E Cytoplasmic accumulation of the soluble cell - wall precursor UMP in vancomycin - treated ( blue line ) , AMSIN - treated ( red dashed line ) or not treated ( green line ) culture of MRSA P1374 . Inset : UMP identified by UPLC - HRMS with singlely and doubly protonated m / z observed . H ESI - MS detecting the non - covalent complex between AMSIN and UMP . The quadruply protonated complex ( m / z 1304 . 8 ) is observed . Inset : comparison of intensities of the complex between two ratios of UMP : AMSIN Here , AMSIN is abbreviated as AMS due to space limitation . J The dose - response unit curve of UMP binding to AMSIN , which is fitted by the BIAevaluation v2 . 0 . 1 . software using 1 : 1 Langmuir binding model .

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Figure 4A Growth kinetic measurements of SM exposed to AMSIN or various antibiotics with known cellular targets. The peptide or the compounds used were 4×CL. VAN, vancomycin; RIF, rifampicin; KAN, kanamycin. OD600 were calculated as measured values subtracting the background value from broth medium (OD600 = 0.356).D Scanning electron microscopic observation of AMSIN-induced BM deformation. The doses used were 10×CL for AMSIN and vancomycin.E Cytoplasmic accumulation of the soluble cell-wall precursor UMP in vancomycin-treated (blue line), AMSIN-treated (red dashed line) or not treated (green line) culture of MRSA P1374. Inset: UMP identified by UPLC-HRMS with singlely and doubly protonated m/z observed.H ESI-MS detecting the non-covalent complex between AMSIN and UMP. The quadruply protonated complex (m/z 1304.8) is observed. Inset: comparison of intensities of the complex between two ratios of UMP:AMSIN Here, AMSIN is abbreviated as AMS due to space limitation.J The dose-response unit curve of UMP binding to AMSIN, which is fitted by the BIAevaluation v2.0.1. software using 1:1 Langmuir binding model.


Figure 5B Hemolysis by AMSIN . Meucin - 18 was used as a positive control . Mean ± SD of three technical replicates is displayed . C Comparison of cytotoxic effects of AMSIN and LL - 37 on HL - 60 cells . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test or Mann - Whitney U test ( the latter indicated by a red asterisk ; * P < 0 . 05 , * * * P < 0 . 001 , ns : no significanceD An inhibition - zone assay showing the inability of AMSIN to bind DNA . The bacterium used was MRSA P1374 and the peptide dose was 1 . 0 nmol / well . Mean ± SD of three biological replicates is displayed . P values were obtained by Mann - Whitney U ( ns : no significanceE The effect of AMSIN on human ion channels expressed in the CNS and heart . The asterisks mark steady state current traces after administering 100 μM AMSIN . F Peptide - induced IL - 8 release . A549 cells were treated with AMSIN or LL - 37 for 24 h . IL - 8 levels in culture supernatants from the FBS - containing medium ( left ) or the serum - free medium ( right ) were measured with an ELISA , as described in Materials and Methods . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test or Mann - Whitney U test ( the latter indicated by a red asterisk or ns ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : no significance G Peptide - induced MCP - 1 release . HL - 60 cells were treated with AMSIN or LL - 37 for 24 h . MCP - 1 levels in culture supernatants from the FBS - containing medium ( left ) or the serum - free medium ( right ) were measured with an ELISA . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test or Mann - Whitney U test ( the latter indicated by a red asterisk or ns ; * P < 0 . 05 , * * P < 0 . 01 , ns : no significanceJ Survival after peritoneal infection with MRSA P1374 . The survival fraction of the vehicle - treated control group was zero out of 11 mice and the treatment group contained six mice and all survived .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B Hemolysis by AMSIN. Meucin-18 was used as a positive control. Mean ± SD of three technical replicates is displayed.C Comparison of cytotoxic effects of AMSIN and LL-37 on HL-60 cells. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk; *P<0.05, ***P<0.001, ns: no significanceD An inhibition-zone assay showing the inability of AMSIN to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann-Whitney U (ns: no significanceE The effect of AMSIN on human ion channels expressed in the CNS and heart. The asterisks mark steady state current traces after administering 100 μM AMSIN.F Peptide-induced IL-8 release. A549 cells were treated with AMSIN or LL-37 for 24 h. IL-8 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA, as described in Materials and Methods. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ***P<0.001, ns: no significance G Peptide-induced MCP-1 release. HL-60 cells were treated with AMSIN or LL-37 for 24 h. MCP-1 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ns: no significanceJ Survival after peritoneal infection with MRSA P1374. The survival fraction of the vehicle-treated control group was zero out of 11 mice and the treatment group contained six mice and all survived.


Figure 1A , The indicated cells co - transfected with p5HRE - Luc and either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( empty vector : EV ) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay . B , U2OS cells co - transfected with p5HRE - Luc and the indicated siRNA were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay . C , HCT116 p53 - / - cells co - transfected with p5HRE - Luc , either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) , and either pcDNA3 / p53 ( at two concentrations : + and + + ) or pcDNA3 ( EV ) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay and Western blotting using the indicated antibodies . D , HCT116 p53 + / + cells co - transfected with p5HRE - Luc , either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) , and either pcDNA3 / p53 R175H , R248W , R273H , or pcDNA3 ( EV ) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, The indicated cells co-transfected with p5HRE-Luc and either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (empty vector: EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay. B, U2OS cells co-transfected with p5HRE-Luc and the indicated siRNA were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.C, HCT116 p53-/- cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 (at two concentrations: + and ++) or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay and Western blotting using the indicated antibodies.D, HCT116 p53+/+ cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 R175H, R248W, R273H, or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.


Figure 2The indicated cells transiently transfected with either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) either pcDNA3 / p53 ( p53 ) or pcDNA3 ( EV ) ( C ) with the indicated siRNA were cultured under < 0 . 1 % O2 conditions for 24 hours and subjected to qRT - PCR for the indicated mRNAThe indicated cells transiently transfected with either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) to the transwell invasion assayThe indicated cells transiently transfected with either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) with the indicated siRNA or to the transwell invasion assayG , The number of metastatic colonies in lungs in the pulmonary metastasis model using the indicated cells stably transfected with ZBTB2 expression vector ( ZBTB2 ) or its empty vector ( EV ) ( left ) and the representative images of lungs ( right ) . The indicated cells stably transfected with the ZBTB2 expression vector ( ZBTB2 ) or its empty vector ( EV ) ( H ) were cultured under < 0 . 1 % O2 ( H ) for the indicated periods and subjected to the in vitro cell proliferation assay . The indicated cells stably transfected with the ZBTB2 expression vector ( ZBTB2 ) transiently transfected with siScr or siZBTB2 ( I ) the indicated oxygen conditions ( I ) for the indicated periods and subjected to the in vitro cell proliferation assay . J , K , Growth of the indicated tumor xenografts with or without ZBTB2 overexpression was analyzed .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O

Figure 2The indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) either pcDNA3/p53 (p53) or pcDNA3 (EV) (C) with the indicated siRNA were cultured under < 0.1% O2 conditions for 24 hours and subjected to qRT-PCR for the indicated mRNAThe indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) to the transwell invasion assayThe indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) with the indicated siRNA or to the transwell invasion assayG, The number of metastatic colonies in lungs in the pulmonary metastasis model using the indicated cells stably transfected with ZBTB2 expression vector (ZBTB2) or its empty vector (EV) (left) and the representative images of lungs (right).The indicated cells stably transfected with the ZBTB2 expression vector (ZBTB2) or its empty vector (EV) (H) were cultured under < 0.1% O2 (H) for the indicated periods and subjected to the in vitro cell proliferation assay.The indicated cells stably transfected with the ZBTB2 expression vector (ZBTB2) transiently transfected with siScr or siZBTB2 (I) the indicated oxygen conditions (I) for the indicated periods and subjected to the in vitro cell proliferation assay.J,K, Growth of the indicated tumor xenografts with or without ZBTB2 overexpression was analyzed.


Figure 3A , Tissue microarrays of human lung adenocarcinoma ( 243 samples ) stained with anti - ZBTB2 antibodies . High magnification images are shown in the left lower side of each image . Scale bar , 500 and 100 μm in the low and high magnification images , respectively . B , Fisher ' s exact test for percentage of CA9 - positive tumors in the four indicated groups . Percentages and absolute numbers of each group are indicated in the graph . C , Kaplan - Meier analysis of the disease - free survival of the lung cancer patients stratified by the expression levels of ZBTB2 and p53 status . D - F , TCGA - based Kaplan - Meier analysis of overall survival of lung adenocarcinoma patients stratified by the expression levels of ZBTB2 and those of BAX ( D ) , ZMAT3 ( E ) , and CEACAM1 ( F ) .

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Figure 3A, Tissue microarrays of human lung adenocarcinoma (243 samples) stained with anti-ZBTB2 antibodies. High magnification images are shown in the left lower side of each image. Scale bar, 500 and 100 μm in the low and high magnification images, respectively.B, Fisher's exact test for percentage of CA9-positive tumors in the four indicated groups. Percentages and absolute numbers of each group are indicated in the graph.C, Kaplan-Meier analysis of the disease-free survival of the lung cancer patients stratified by the expression levels of ZBTB2 and p53 status.D-F, TCGA-based Kaplan-Meier analysis of overall survival of lung adenocarcinoma patients stratified by the expression levels of ZBTB2 and those of BAX (D), ZMAT3 (E), and CEACAM1 (F).


Figure 4A , The indicated cells transiently transfected with pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) were cultured under the indicated oxygen conditions for 24 hours and subjected to Western blotting using the indicated antibodies . The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A and N803A ( E ) were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay . The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay . The indicated cells transiently transfected with pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A and N803A ( E ) were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated ( B , D , E , F ) The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay . The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay . H , After transient transfection with the expression vectors for ZBTB2 - V5 , ZBTB2 - myc , or their empty vector ( - ) , ZBTB2 - myc protein was immunoprecipitated using the anti - myc antibody and co - precipitated ZBTB2 - V5 was detected using the anti - V5 antibody ( upper ) . One - tenth of the whole cell lysate ( WCL ) was subjected to immunoblotting with the indicated antibodies ( lower ) . I , J , The indicated cells transiently transfected with or without either pcDNA4 / ZBTB2 - LgBiT or pcDNA4 / LgBiT and either pcDNA4 / ZBTB2 - SmBiT or pcDNA4 / SmBiT , as indicated , were cultured under < 0 . 1 % oxygen ( I ) or the indicated oxygen conditions ( J ) and subjected to the split luciferase complementation assay . K , HeLa cells overexpressing wildtype or the indicated mutant of ZBTB2 ( ZF2 or ZF3 - deletion ) were cultured under < 0 . 1 % oxygen conditions , and subjected to immunoprecipitation with anti - myc antibody . Coprecipitated DNA was subjected to the qRT - PCR experiment using primers against HRE regions of the STC1 promoter . L , The same experiment as I was conducted using the indicated combination of pcDNA4 / ZBTB2 - LgBiT ( WT ) , pcDNA4 / ZBTB2ΔZF2 - LgBiT ( ΔZF2 ) , or pcDNA4 / ZBTB2ΔZF3 - LgBiT ( ΔZF3 ) , and pcDNA4 / ZBTB2 - SmBiT ( WT ) , pcDNA4 / ZBTB2ΔZF2 - SmBiT ( ΔZF2 ) , or pcDNA4 / ZBTB2ΔZF3 - SmBiT ( ΔZF3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, The indicated cells transiently transfected with pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) were cultured under the indicated oxygen conditions for 24 hours and subjected to Western blotting using the indicated antibodies.The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.The indicated cells transiently transfected with pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated (B,D,E,F) The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.H, After transient transfection with the expression vectors for ZBTB2-V5, ZBTB2-myc, or their empty vector (-), ZBTB2-myc protein was immunoprecipitated using the anti-myc antibody and co-precipitated ZBTB2-V5 was detected using the anti-V5 antibody (upper). One-tenth of the whole cell lysate (WCL) was subjected to immunoblotting with the indicated antibodies (lower).I,J, The indicated cells transiently transfected with or without either pcDNA4/ZBTB2-LgBiT or pcDNA4/LgBiT and either pcDNA4/ZBTB2-SmBiT or pcDNA4/SmBiT, as indicated, were cultured under < 0.1% oxygen (I) or the indicated oxygen conditions (J) and subjected to the split luciferase complementation assay.K, HeLa cells overexpressing wildtype or the indicated mutant of ZBTB2 (ZF2 or ZF3-deletion) were cultured under < 0.1% oxygen conditions, and subjected to immunoprecipitation with anti-myc antibody. Coprecipitated DNA was subjected to the qRT-PCR experiment using primers against HRE regions of the STC1 promoter.L, The same experiment as I was conducted using the indicated combination of pcDNA4/ZBTB2-LgBiT (WT), pcDNA4/ZBTB2ΔZF2-LgBiT (ΔZF2), or pcDNA4/ZBTB2ΔZF3-LgBiT (ΔZF3), and pcDNA4/ZBTB2-SmBiT (WT), pcDNA4/ZBTB2ΔZF2-SmBiT (ΔZF2), or pcDNA4/ZBTB2ΔZF3-SmBiT (ΔZF3).


Figure 5B , HCT116 p53 - / - cells co - transfected with pG5H1bLuc , pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A , and either pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 8 - 11 ] ( ZBTB2 del [ 8 - 11 ] ) , pEF6 / ZBTB2 del [ 51 - 54 ] ( ZBTB2 del [ 51 - 54 ] ) , or pEF6 / myc - His B ( EV ) , as indicated , were cultured under < 0 . 1 % oxygen conditions for 24 hours and subjected to the luciferase assay . C , After transient transfection with or without the expression vectors for ZBTB2 - V5 , ZBTB2 4A - V5 , ZBTB2 - myc , ZBTB2 4A - myc , or their empty vector , as indicated , the myc - fused proteins were immunoprecipitated using an anti - myc antibody and co - precipitated ZBTB2 - V5 or ZBTB2 4A - V5 was detected ( upper ) . One - tenth of the whole cell lysate ( WCL ) was subjected to immunoblotting with the indicated antibodies ( lower ) . D , HCT116 p53 - / - ( left ) cells transiently transfected with or without either pcDNA4 / ZBTB2 - LgBiT ( ZBTB2 ) , pcDNA4 / ZBTB2 4A - LgBiT 4A ( 4A ) , or pcDNA4 / LgBiT ( - ) and either pcDNA4 / ZBTB2 - SmBiT ( ZBTB2 ) , pcDNA4 / ZBTB2 4A - SmBiT ( 4A ) , or pcDNA4 / SmBiT ( - ) , as indicated , were cultured under < 0 . 1 % oxygen and subjected to the split luciferase complementation assay . G , The same kind of luciferase assay to quantify the transactivation activity as B was carried out using either pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 4A ( 4A ) , or pEF6 / myc - His B ( EV ) . H , Growth of the indicated tumor xenografts was analyzed .

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Figure 5B, HCT116 p53-/- cells co-transfected with pG5H1bLuc, pcDNA6/Gal4 DBD-HIF-1α TAD P564A, and either pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[8-11] (ZBTB2 del[8-11]), pEF6/ZBTB2 del[51-54] (ZBTB2 del[51-54]), or pEF6/myc-His B (EV), as indicated, were cultured under < 0.1% oxygen conditions for 24 hours and subjected to the luciferase assay.C, After transient transfection with or without the expression vectors for ZBTB2-V5, ZBTB2 4A-V5, ZBTB2-myc, ZBTB2 4A-myc, or their empty vector, as indicated, the myc-fused proteins were immunoprecipitated using an anti-myc antibody and co-precipitated ZBTB2-V5 or ZBTB2 4A-V5 was detected (upper). One-tenth of the whole cell lysate (WCL) was subjected to immunoblotting with the indicated antibodies (lower).D, HCT116 p53-/- (left) cells transiently transfected with or without either pcDNA4/ZBTB2-LgBiT (ZBTB2), pcDNA4/ZBTB2 4A-LgBiT 4A (4A), or pcDNA4/LgBiT (-) and either pcDNA4/ZBTB2-SmBiT (ZBTB2), pcDNA4/ZBTB2 4A-SmBiT (4A), or pcDNA4/SmBiT (-), as indicated, were cultured under < 0.1% oxygen and subjected to the split luciferase complementation assay.G, The same kind of luciferase assay to quantify the transactivation activity as B was carried out using either pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 4A (4A), or pEF6/myc-His B (EV).H, Growth of the indicated tumor xenografts was analyzed.


Figure 1 ( D ) Western blot analysis of a strain expressing TurboID - HA - Emc6 , Bpl1 - AID * - 9myc and OsTIR1 which was grown overnight in SD media with or without auxin . The cells were then diluted into fresh SD media and grown to mid logarithmic phase ( ~ 4hours ) with or without auxin ( respectively ) . When required , biotin was added either 30min before harvesting , or for the entire 4hours . H3 ( histone H3 ) is used as a loading control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D) Western blot analysis of a strain expressing TurboID-HA-Emc6, Bpl1-AID*-9myc and OsTIR1 which was grown overnight in SD media with or without auxin. The cells were then diluted into fresh SD media and grown to mid logarithmic phase (~4hours) with or without auxin (respectively). When required, biotin was added either 30min before harvesting, or for the entire 4hours. H3 (histone H3) is used as a loading control.


Figure 2 ( B ) Volcano plots for each of the four distinct LC - MS / MS experiments highlighted in Figure 2A showing - log ( p - value ) against log2 ( fold - change ) for all proteins identified in the appropriate Emc6 / Sbh1 samples . Proteins enriched in the Emc6 samples are colour / pattern - coded as follows : dotted outline for HA - IP of BioID2 - HA - Emc6 ; blue fill for streptavidin - AP of BioID2 - HA - Emc6 ; black , solid outline for streptavidin - AP of TurboID - HA - Emc6 ; and yellow fill for streptavidin - AP of TurboID - HA - Emc6 / ABOLISH . Grey dots represent proteins that passed the p - value and fold - change criteria , but were only identified by one peptide . Only EMC complex components or proteins with EMC client features are named .

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Figure 2(B) Volcano plots for each of the four distinct LC-MS/MS experiments highlighted in Figure 2A showing -log(p-value) against log2(fold-change) for all proteins identified in the appropriate Emc6/Sbh1 samples. Proteins enriched in the Emc6 samples are colour/pattern-coded as follows: dotted outline for HA-IP of BioID2-HA-Emc6; blue fill for streptavidin-AP of BioID2-HA-Emc6; black, solid outline for streptavidin-AP of TurboID-HA-Emc6; and yellow fill for streptavidin-AP of TurboID-HA-Emc6/ABOLISH. Grey dots represent proteins that passed the p-value and fold-change criteria, but were only identified by one peptide. Only EMC complex components or proteins with EMC client features are named.


Figure 3 ( A ) Fluorescence microscopy images of control or Δemc3 strains containing either Alg1 N ' tagged with GFP , Gnp1 C ' tagged with GFP , Pdr12 N ' tagged with GFP or Pho84 C ' tagged with GFP . For the Gnp1 - GFP strain , the quantified signal depletion was ~ 10 % ( p < 0 . 0001 ) . Topological schematics feature beneath each respective image , with predicted TMDs highlighted in blue ( Weill et al , 2019 ) . All images are from two independent biological repeats and are digitally coloured using the ' fire ' lookup table ( LUT ) on Fiji . The same contrast settings were used for each image pair . ( C ) Streptavidin blots of diploid strains expressing either AviTag - Fks1 , - Gnp1 , - Pdr5 , - Pdr12 or - Stv1 ( as negative control ) together with BirA - Emc6 , grown in the conditions described in Figure 1E . For each strain , blots showing both the ' lower ' ( L ) and ' higher ' ( H ) contrast settings are shown side - by - side . The expected molecular weight in kDa for each tested protein including AviTag is written in parentheses after the protein name .

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Figure 3(A) Fluorescence microscopy images of control or Δemc3 strains containing either Alg1 N' tagged with GFP, Gnp1 C' tagged with GFP, Pdr12 N' tagged with GFP or Pho84 C' tagged with GFP. For the Gnp1-GFP strain, the quantified signal depletion was ~10% (p < 0.0001). Topological schematics feature beneath each respective image, with predicted TMDs highlighted in blue (Weill et al, 2019). All images are from two independent biological repeats and are digitally coloured using the 'fire' lookup table (LUT) on Fiji. The same contrast settings were used for each image pair.(C) Streptavidin blots of diploid strains expressing either AviTag-Fks1, -Gnp1, -Pdr5, -Pdr12 or -Stv1 (as negative control) together with BirA-Emc6, grown in the conditions described in Figure 1E. For each strain, blots showing both the 'lower' (L) and 'higher' (H) contrast settings are shown side-by-side. The expected molecular weight in kDa for each tested protein including AviTag is written in parentheses after the protein name.


Figure 1 ( B ) U2OS stably expressing FLAG - TIP60 or empty vector ( vec ) were subjected to FLAG - immunoprecipitation from nuclear lysates and analysed by Western blotting ( C ) HEK293T cells were transfected with pcDNA3 . 1 encoding FLAG - TIP60WT , FLAG - TIP60S90A or empty vector ( vec ) . The samples were subjected to FLAG affinity - purification , dephosphorylated with shrimp alkaline phosphatase ( rSAP ) and incubated as indicated with CDK9 / cyclinT1 in presence of ATP . Phosphorylation of TIP60 was analysed by a phosphoS90 - specific TIP60 antibody , and the blot was further probed with antibodies specific for FLAG and CDK9 ( D ) p53 - / - hRasG12V MEF were treated with DMSO or 1µM SNS - 032 ( SNS ) for 1h . Nuclear lysates were analysed by Western blotting ( E ) p53 - / - hRasG12V MEF were treated with DMSO , 1µM SNS - 032 ( SNS ) or 50µM DRB followed by addition of 100nM Calyculin A as indicated . Nuclear lysates were analysed by Western blotting ( F ) U2OS cells stably expressing FLAG - TIP60WT were transfected with a pool of four different CDK9 - targeting siRNAs or a control siRNA targeting Luciferase . Nuclear lysates were analysed by Western blotting

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

Figure 1 (B) U2OS stably expressing FLAG-TIP60 or empty vector (vec) were subjected to FLAG- immunoprecipitation from nuclear lysates and analysed by Western blotting  (C) HEK293T cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG- TIP60S90A or empty vector (vec). The samples were subjected to FLAG affinity-purification, dephosphorylated with shrimp alkaline phosphatase (rSAP) and incubated as indicated with CDK9/cyclinT1 in presence of ATP. Phosphorylation of TIP60 was analysed by a phosphoS90-specific TIP60 antibody, and the blot was further probed with antibodies specific for FLAG and CDK9  (D) p53-/- hRasG12V MEF were treated with DMSO or 1µM SNS-032 (SNS) for 1h. Nuclear lysates were analysed by Western blotting  (E) p53-/- hRasG12V MEF were treated with DMSO, 1µM SNS-032 (SNS) or 50µM DRB followed by addition of 100nM Calyculin A as indicated. Nuclear lysates were analysed by Western blotting  (F) U2OS cells stably expressing FLAG-TIP60WT were transfected with a pool of four different CDK9-targeting siRNAs or a control siRNA targeting Luciferase. Nuclear lysates were analysed by Western blotting


Figure 2 ( A ) Localization of TIP60 was analysed employing U2OS cells stably expressing FLAG - TIP60WT , FLAG - TIP60S90A or FLAG - TIP60S86A . Nucleoplasm ( nuc . ) and chromatin ( chr . ) fractions were generated and were subjected to Western blot analysis ( B ) The experiment as described in ( A ) was repeated twice with U2OS cells and twice with Ba / F3 cells and the blots were quantified . Each dot represents the ration of FLAG - TiIP60 to H4 in the chromatin fraction of altogether four individual experiments as shown in ( A ) ( C ) Nuclear extracts of U2OS cells stably expressing FLAG - tagged TIP60WT or TIP60S90A , or FLAG - tagged chromodomain mutants TIP60F50A , TIP60Y47A , TIP60Y44F or the empty vector ( vec ) were subjected to fractionation into nucleoplasm ( nuc . ) and chromatin ( chr . ) fraction . Both fractions were analysed by Western blotting ( D ) Nuclear extracts of U2OS cells stably expressing FLAG - tagged TIP60WT , TIP60S90A , TIP60S90E , TIP60S90D or the empty vector ( vec ) were subjected to fractionation into nucleoplasm ( nuc . ) and chromatin ( chr . ) fraction . Both fractions were analysed by Western blotting ( E ) U2OS cells stably expressing FLAG - tagged TIP60WT , TIP60S90A or empty vector ( vec ) were treated with 2 µM SNS - 032 ( SNS ) for 3 hours as indicated . The nucleus was fractionated into nucleoplasm and chromatin . Both fractions were analysed by Western

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

Figure 2 (A) Localization of TIP60 was analysed employing U2OS cells stably expressing FLAG- TIP60WT, FLAG-TIP60S90A or FLAG-TIP60S86A. Nucleoplasm (nuc.) and chromatin (chr.) fractions were generated and were subjected to Western blot analysis  (B) The experiment as described in (A) was repeated twice with U2OS cells and twice with Ba/F3 cells and the blots were quantified. Each dot represents the ration of FLAG-TiIP60 to H4 in the chromatin fraction of altogether four individual experiments as shown in (A)  (C) Nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT or TIP60S90A, or FLAG-tagged chromodomain mutants TIP60F50A, TIP60Y47A, TIP60Y44F or the empty vector (vec) were subjected to fractionation into nucleoplasm (nuc.) and chromatin (chr.) fraction. Both fractions were analysed by Western blotting  (D) Nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S90E, TIP60S90D or the empty vector (vec) were subjected to fractionation into nucleoplasm (nuc.) and chromatin (chr.) fraction. Both fractions were analysed by Western blotting (E) U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A or empty vector (vec) were treated with 2 µM SNS-032 (SNS) for 3 hours as indicated. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting


Figure 3 ( A ) FLAG - immunoprecipitations were performed from nuclear extracts of U2OS cells stably expressing FLAG - tagged TIP60WT , TIP60S90A , TIP60S86A or empty vector ( vec ) . Immunoprecipitated material and input lysate were subjected to Western blot analysis ( B ) The experiment as shown in ( A ) was repeated three times . The graphs and error bars represent mean and standard deviation of these three individual experiments ( one - way ANOVA , Fisher " s LSD test , WT vs . S86A p = 0 , 5992 , WT vs . S90A p = 0 , 0429 , S90A vs . S86A p = 0 , 0176 ( * p < 0 , 05 ; n . s . not significant , p > 0 , 05 ) ) ( C ) HEK293 cells were transfected with pcDNA3 . 1 encoding FLAG - TIP60WT , FLAG - TIP60S90A or empty vector . FLAG - immunoprecipitations were performed from nuclear extracts . Immunoprecipitated material and input lysate were subjected to Western blot analysis ( D ) U2OS cells stably expressing FLAG - TIP60WT or empty vector were treated with 2µM SNS - 032 ( SNS ) for 2 hours as indicated . FLAG - immunoprecipitations were performed from nuclear extracts . This material and input lysate were subjected to Western blot analysis ( E ) U2OS cells stably expressing FLAG - TIP60WT , FLAG - TIP60S90A or empty vector ( vec ) were subjected to ChIP using anti - FLAG antibody . Immunoprecipitated DNA and input were subjected to quantitative real - time PCR using primers annealing in the myc locus about 50bp upstream ( myc + 50 ) or about 500 bp downstream ( myc - 500 ) of the transcription start site . Promoter occupancy of FLAG - TIP60WT or FLAG - TIP60S90A was determined as percent input in relation to FLAG - TIP60WT . The graphs and error bars represent mean and standard deviation of three individual experiments ( one - way ANOVA , Bonferroni " s multiple comparison test , myc + 50 WT vs . S90A p = 0 , 0289 , myc - 500 WT vs . S90A p = 0 , 0155 ( * p < 0 , 05 ) )

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

Figure 3 (A) FLAG- immunoprecipitations were performed from nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S86A or empty vector (vec). Immunoprecipitated material and input lysate were subjected to Western blot analysis (B) The experiment as shown in (A) was repeated three times. The graphs and error bars represent mean and standard deviation of these three individual experiments (one-way ANOVA, Fisher"s LSD test, WT vs. S86A p=0,5992, WT vs. S90A p=0,0429, S90A vs. S86A p=0,0176 (*p < 0,05; n.s. not significant, p > 0,05))  (C) HEK293 cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG-TIP60S90A or empty vector. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis  (D) U2OS cells stably expressing FLAG-TIP60WT or empty vector were treated with 2µM SNS-032 (SNS) for 2 hours as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. This material and input lysate were subjected to Western blot analysis  (E) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were subjected to ChIP using anti-FLAG antibody. Immunoprecipitated DNA and input were subjected to quantitative real-time PCR using primers annealing in the myc locus about 50bp upstream (myc+50) or about 500 bp downstream (myc-500) of the transcription start site. Promoter occupancy of FLAG-TIP60WT or FLAG-TIP60S90A was determined as percent input in relation to FLAG -TIP60WT. The graphs and error bars represent mean and standard deviation of three individual experiments (one-way ANOVA, Bonferroni"s multiple comparison test, myc+50 WT vs. S90A p=0,0289, myc-500 WT vs. S90A p=0,0155 (*p < 0,05))


Figure 4 ( A ) U2OS cells stably expressing FLAG - TIP60WT , FLAG - TIP60S90A or empty vector ( vec ) were treated with 2µM SNS - 032 ( SNS ) , 2µM JQ1 ( JQ1 ) or the solvent DMSO ( d . ) for 2h as indicated . FLAG - immunoprecipitations were performed from nuclear extracts . Immunoprecipitated material and input lysate were subjected to Western blot analysis ( B ) U2OS cells stably expressing FLAG - TIP60WT were treated with 2µM JQ1 ( JQ1 ) for 2h . The nucleus was fractionated into nucleoplasm and chromatin . Both fractions were analysed by Western blotting ( C ) HEK293 cells were transiently transfected with pcDNA4 encoding Brd4 or GFP . The nucleus was fractionated into nucleoplasm and chromatin . Both fractions were analysed by Western blotting

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

Figure 4 (A) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were treated with 2µM SNS-032 (SNS), 2µM JQ1 (JQ1) or the solvent DMSO (d.) for 2h as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis (B) U2OS cells stably expressing FLAG-TIP60WT were treated with 2µM JQ1 (JQ1) for 2h. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting  (C) HEK293 cells were transiently transfected with pcDNA4 encoding Brd4 or GFP. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting


Figure 5 ( A ) U2OS cells were transfected with a siRNA pool targeting human TIP60 ( Dharmacon ) or luciferase ( Dharmacon ) . The nuclear lysates were analysed by Western blotting 24h after transfection ( B ) U2OS cells stably expressing FLAG - TIP60WT , FLAG - TIP60S90A , FLAG - TIP60S86A or empty vector ( vec ) were analysed by Western blotting 60 hours after viral transduction

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

Figure 5 (A) U2OS cells were transfected with a siRNA pool targeting human TIP60 (Dharmacon) or luciferase (Dharmacon). The nuclear lysates were analysed by Western blotting 24h after transfection  (B) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A, FLAG-TIP60S86A or empty vector (vec) were analysed by Western blotting 60 hours after viral transduction