D , E Quantitative RT - PCR and western blot analyses of the expression of osteogenic markers . Cells were cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by t

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D, E Quantitative RT-PCR and western blot analyses of the expression of osteogenic markers. Cells were cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test


F H & E staining of the MSCs - mediated ectopic bone formation . Scale bar , 50 μm

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F H&E staining of the MSCs-mediated ectopic bone formation. Scale bar, 50 μm


G Quantitative analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; n = 6 . * : p < 0 . 05 by t

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G Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=6. *: p<0.05 by t test


A Scatter plot of RNA - seq expression analysis . 508 genes were upregulated and 391 genes were downregulated . Human MSCs were transfected with USP34 or control siRNAs and cultured in osteogenic medium for 2 days . Two biological replicates per group

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A Scatter plot of RNA-seq expression analysis. 508 genes were upregulated and 391 genes were downregulated. Human MSCs were transfected with USP34 or control siRNAs and cultured in osteogenic medium for 2 days. Two biological replicates per group


C Heatmap of representative genes associated with BMP2 signaling pathway


D Relative activity of BRE luciferase assay . 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± SEM ; n = 3 . * * * : p < 0 . 001 by t

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D Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. ***: p<0.001 by t test


E Immunoblot analysis of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f and Usp34f / f control MSCs were starved overnight and then stimulated with 100 ng / ml BMP2 for 24

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E Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hours


F Quantitative RT - PCR analysis of BMP2 - induce Sp7 transcription . Cells were depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 and * * : p < 0 . 01 by t

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F Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Cells were depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by t test


G Representative images of ALP and ARS staining . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 10 and 21 days ,

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G Representative images of ALP and ARS staining. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 10 and 21 days, respectively


H , I Quantitative analyses of the ALP activity ( n = 4 ) and calcium mineralization ( n = 5 ) . Results are shown as mean ± SEM ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by t

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H, I Quantitative analyses of the ALP activity (n=4) and calcium mineralization (n=5). Results are shown as mean ± SEM; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test


J , K Representative microCT images and quantification of newly formed bone in response to BMP2 . Results are shown as mean ± SEM ; n = 8 . * : p < 0 . 05 by t test . Scale bar , 2500 μm

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J, K Representative microCT images and quantification of newly formed bone in response to BMP2. Results are shown as mean ± SEM; n=8. *: p<0.05 by t test. Scale bar, 2500 μm


L H & E staining of BMP2 - induced newly formed bone . Scale bar , 50

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L H&E staining of BMP2-induced newly formed bone. Scale bar, 50 μm


M Quantitative analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by t

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M Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test


N H & E staining of calvarial organ cultures stimulated with or without 100 ng / ml BMP2 . Red dotted lines indicate the interface between original and newly formed bone . Scale bar , 50

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N H&E staining of calvarial organ cultures stimulated with or without 100 ng/ml BMP2. Red dotted lines indicate the interface between original and newly formed bone. Scale bar, 50 μm


O Quantitative analysis of newly formed bone volume versus original bone volume ( % ) . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by t

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O Quantitative analysis of newly formed bone volume versus original bone volume (%). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test


A Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged Smad1 in 293T

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A Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged Smad1 in 293T cells


B Immunoblot analysis showing decreased FLAG - Smad1 after USP34 knockdown

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B Immunoblot analysis showing decreased FLAG-Smad1 after USP34 knockdown


C Immunoblot of Smad1 - linked polyUb . 293T cells were co - transfected with FLAG - Smad1 overexpression plasmid and USP34 siRNA , and treated with 10 µM MG132 for 4 hours before collection

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C Immunoblot of Smad1-linked polyUb. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 10 µM MG132 for 4 hours before collection


D Immunoblot analysis of the degradation of FLAG - Smad1 protein . 293T cells were co - transfected with FLAG - Smad1 overexpression plasmid and USP34 siRNA , and treated with 50 µg / ml cycloheximide ( CHX ) before collection at designated time points . Right : relative quantification of FLAG - Smad1 protein levels at different time

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D Immunoblot analysis of the degradation of FLAG-Smad1 protein. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 50 µg/ml cycloheximide (CHX) before collection at designated time points. Right: relative quantification of FLAG-Smad1 protein levels at different time points


E Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged RUNX2 in 293T

E Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged RUNX2 in 293T cells


F Co - immunoprecipitation of USP34 with endogenous RUNX2 in mouse

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F Co-immunoprecipitation of USP34 with endogenous RUNX2 in mouse MSCs


G Immunoblot of RUNX2 - linked polyUb . Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection . Prx1 - Cre ( + ) indicates MSCs isolated from Prx1 - Cre ; Usp34f / f mice , and Prx1 - Cre ( - ) indicates those from Usp34f / f control

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G Immunoblot of RUNX2-linked polyUb. Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection. Prx1-Cre(+) indicates MSCs isolated from Prx1-Cre;Usp34f/f mice, and Prx1-Cre(-) indicates those from Usp34f/f control mice


H Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs . Prx1 - Cre ; Usp34f / f and Usp34f / f control MSCs were stimulated with BMP2 , treated with 50 µg / ml CHX , and collected at designated time points . Right : relative quantification of RUNX2 protein levels at different time

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H Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were stimulated with BMP2, treated with 50 µg/ml CHX, and collected at designated time points. Right: relative quantification of RUNX2 protein levels at different time points


I Representative images of ALP and ARS staining . Usp34f / f and Prx1 - Cre ; Usp34f / f MSCs were transduced with GFP , Smad1 , Runx2 or Smad1 + Runx2 adenoviruses , and then cultured in osteogenic medium for 5 and 10 days ,

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I Representative images of ALP and ARS staining. Usp34f/f and Prx1-Cre;Usp34f/f MSCs were transduced with GFP, Smad1, Runx2 or Smad1+Runx2 adenoviruses, and then cultured in osteogenic medium for 5 and 10 days, respectively


J , K Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

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J, K Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


L , M H & E staining and quantitative analysis of MSCs - mediated ectopic bone formation . Scale bar , 50 μm . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

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L, M H&E staining and quantitative analysis of MSCs-mediated ectopic bone formation. Scale bar, 50 μm. Results are shown as mean ± SEM; n=8; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test


A Relative activity of BRE luciferase assay . 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± SEM ; n = 3 . * : p < 0 . 05 by ANOVA with Tukey ' s post hoc

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A Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. *: p<0.05 by ANOVA with Tukey's post hoc test


B Immunoblot analysis of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , starved overnight and then stimulated with 100 ng / ml BMP2 for 24

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B Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hours


C Quantitative RT - PCR analysis of BMP2 - induce Sp7 transcription . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , and depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

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C Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, and depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test


D ALP staining and quantitative measurement of ALP activity . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 5 days . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

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D ALP staining and quantitative measurement of ALP activity. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 5 days. Results are shown as mean ± SEM; n=8; **: p<0.01 by ANOVA with Tukey's post hoc test


E Representative images of ARS staining and quantification . Cells were cultured with normal medium supplemented with 100 ng / ml BMP2 for 14 days . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc test

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E Representative images of ARS staining and quantification. Cells were cultured with normal medium supplemented with 100 ng/ml BMP2 for 14 days. Results are shown as mean ± SEM; n=8; *: p<0.05, and **: p<0.01 by ANOVA with Tukey's post hoc test


F Representative images of ALP and ARS staining . Mouse MSCs were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 and 10 days ,

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F Representative images of ALP and ARS staining. Mouse MSCs were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 and 10 days, respectively


G , H Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc test

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G, H Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


I , J Quantitative RT - PC analyses of the expression of osteogenic markers . Cells were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

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I, J Quantitative RT-PC analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


western blot analyses of the expression of osteogenic markers . Cells were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

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western blot analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


B Phospholipid transport / synthesis , as measured by 3H - serine incorporation , in human fibroblasts ( mean ± SE ; n = 9 , with 5 replicates / experiment ) and in primary mouse hippocampal neurons ( mean ± SE ; n = 3 , with 4 or 5 replicates / experiment ) . * , p < 0 . 05 vs E3 .

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B Phospholipid transport/synthesis, as measured by 3H-serine incorporation, in human fibroblasts (mean±SE; n=9, with 5 replicates/experiment) and in primary mouse hippocampal neurons (mean±SE; n=3, with 4 or 5 replicates/experiment). *, p<0.05 vs E3.


C Comparison of 3H - serine incorporation in ApoE ACM - treated WT - and Mfn2 - KO MEFs ( mean ± SE ; n = 3 , with 3 replicates / experiment ) .

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C Comparison of 3H-serine incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 3 replicates/experiment).


D Duramycin sensitivity in ApoE ACM - treated fibroblasts . Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4 - treated cells , which was blocked upon the addition of 15 μM exogenous PtdEtn ( mean ± SD ; n = 3 , with 3 replicates / experiment ) .

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D Duramycin sensitivity in ApoE ACM-treated fibroblasts. Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4-treated cells, which was blocked upon the addition of 15 μM exogenous PtdEtn (mean±SD; n=3, with 3 replicates/experiment).


B Fractions from the discontinuous gradient were blotted with anti - ApoE ( WUE - 4 ) antibody ; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [ 16 ] .

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B Fractions from the discontinuous gradient were blotted with anti-ApoE (WUE-4) antibody; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [16].


C An ApoE - containing fraction ( fraction 5 [ + ] ) , an ApoE - negative fraction ( fraction 3 [ - ] ) , and recombinant lipid - free ApoE protein were applied to human fibroblasts . Note increase in phospholipid synthesis using an ApoE4 - containing lipoprotein fraction , but not with either an ApoE4 - negative fraction or with lipoprotein - free recombinant ApoE3 or ApoE4 protein ( mean ± SE ; n = 3 , with 3 replicates / experiment ) .

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C An ApoE-containing fraction (fraction 5 [+]), an ApoE-negative fraction (fraction 3 [-]), and recombinant lipid-free ApoE protein were applied to human fibroblasts. Note increase in phospholipid synthesis using an ApoE4-containing lipoprotein fraction, but not with either an ApoE4-negative fraction or with lipoprotein-free recombinant ApoE3 or ApoE4 protein (mean ± SE; n=3, with 3 replicates/experiment).


A Cholesteryl ester synthesis , as measured by 3H - oleate incorporation . Note increased CE in ApoE4 - treated cells , whereas there was essentially no difference in triglyceride ( TAG ) production ( mean ± SE ; n = 3 . with 3 replicates / experiment ) . † , p = 0 . 06 vs E3 .

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A Cholesteryl ester synthesis, as measured by 3H-oleate incorporation. Note increased CE in ApoE4-treated cells, whereas there was essentially no difference in triglyceride (TAG) production (mean±SE; n=3. with 3 replicates/experiment). †, p=0.06 vs E3.


B Comparison of 3H - oleate incorporation in ApoE ACM - treated WT - and Mfn2 - KO MEFs ( mean ± SE ; n = 3 , with 5 replicates / experiment ) .

B Comparison of 3H-oleate incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 5 replicates/experiment).


C ApoE ACM - treated fibroblasts were stained with LipidTox green neutral lipid stain , and lipid droplets were visualized ( example at left ) and counted ( right ) . Note significantly more lipid droplets in ApoE4 - treated cells ( mean ± SD , n = 3 ) .

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C ApoE ACM-treated fibroblasts were stained with LipidTox green neutral lipid stain, and lipid droplets were visualized (example at left) and counted (right). Note significantly more lipid droplets in ApoE4-treated cells (mean ± SD, n=3).


HeLa cells were transfected with plasmids to visualize ER ( in green ) and mitochondria ( in red ) , and then were treated with ApoE3 - and ApoE4 - ACM for 24 hours . Shown is a box - and - whisker plot comparing co - localization in ApoE3 ACM ( n = 15 images analyzed [ gray circles , average of co - localization in ~ 5 cells in each image field ] ) compared to that in ApoE4 ACM ( n = 18 images ) .

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HeLa cells were transfected with plasmids to visualize ER (in green) and mitochondria (in red), and then were treated with ApoE3- and ApoE4-ACM for 24 hours. Shown is a box-and-whisker plot comparing co-localization in ApoE3 ACM (n = 15 images analyzed [gray circles, average of co-localization in ~5 cells in each image field]) compared to that in ApoE4 ACM (n = 18 images).


A Expression of transcripts coding for PI3K subunit genes in pDCs ( n = 12 biological replicates ) . In the boxplot , the middle , the bottom , and the top of the box represent the median , the 1st quantile and the 3rd quantile , respectively ; the height of the box is given by the difference between the 3rd and the 1st quantiles ( IQR , interquartile range ) ; the whiskers length is 1 . 5 times the interquartile range . Data points beyond the whiskers represent values more extreme than 1 . 5 times the interquartile range above or below the 3rd or the 1st quantile , respectively .

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A Expression of transcripts coding for PI3K subunit genes in pDCs (n =12 biological replicates). In the boxplot, the middle, the bottom, and the top of the box represent the median, the 1st quantile and the 3rd quantile, respectively; the height of the box is given by the difference between the 3rd and the 1st quantiles (IQR, interquartile range); the whiskers length is 1.5 times the interquartile range. Data points beyond the whiskers represent values more extreme than 1.5 times the interquartile range above or below the 3rd or the 1st quantile, respectively.


C Kyn levels in culture supernatants of pDCs incubated for 24 h with medium alone ( Ctrl ) or IFN - γ after 1 − h pretreatment with PI3K inhibitors .

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C Kyn levels in culture supernatants of pDCs incubated for 24 h with medium alone (Ctrl) or IFN-γ after 1−h pretreatment with PI3K inhibitors.


D Real - time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs stimulated for 18 h with TGF - β , in the presence or absence ( Ctrl , control ) of catalytic PI3K inhibitors , normalized to the expression of Gapdh and presented relative to results in untreated cells ( dotted line , 1 - fold ) .

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D Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs stimulated for 18 h with TGF-β, in the presence or absence (Ctrl, control) of catalytic PI3K inhibitors, normalized to the expression of Gapdh and presented relative to results in untreated cells (dotted line, 1-fold).


E In vivo suppression of the activity of HY - pulsed CD8 − DCs in combination with a minority fraction ( 5 % ) of pDCs conditioned in vitro with TGF - β ( or medium ) for 24 h in the presence or absence ( Ctrl ) of catalytic inhibitors as in C . Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight ( experimental versus control footpads ) .

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E In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs conditioned in vitro with TGF-β (or medium) for 24 h in the presence or absence (Ctrl) of catalytic inhibitors as in C. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).


F Real - time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs transfected with p110α - , β - , δ - specific , or negative control ( nc ) siRNA and stimulated for 18 h with TGF - β , normalized to the expression of Gapdh and presented relative to results in unstimulated cells ( dotted line , 1 - fold ) .

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F Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs transfected with p110α-, β-, δ-specific, or negative control (nc) siRNA and stimulated for 18 h with TGF-β, normalized to the expression of Gapdh and presented relative to results in unstimulated cells (dotted line, 1-fold).


G In vivo suppression of the activity of HY - pulsed CD8 − DCs in combination with a minority fraction ( 5 % ) of pDCs transfected with siRNA as in ( F ) and conditioned in vitro with TGF - β ( or medium ) for 24 h . Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight ( experimental versus control footpads ) .

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G In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs transfected with siRNA as in (F) and conditioned in vitro with TGF-β (or medium) for 24 h. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).


C Pull - down ( PD ) assay of lysates from P1 . HTR cells with unphosphorylated ( YENM ) and tyrosine - phosphorylated IDO1 peptides ( pYENM , pITIM1 , and pITIM2 ) , analyzed by sequential immunoblotting with anti − p85 , − SHP - 1 , and - SHP - 2 antibodies .

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C Pull-down (PD) assay of lysates from P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated IDO1 peptides (pYENM, pITIM1, and pITIM2), analyzed by sequential immunoblotting with anti−p85, −SHP-1, and -SHP-2 antibodies.


D Immunoblotting with anti - IDO1 and - p85 antibodies of IDO1 immunoprecipitates obtained from P1 . HTR cells transfected with IDO1 . WT or IDO1 . Y149F . Mock - transfected P1 . HTR cells were used as a control .

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D Immunoblotting with anti-IDO1 and -p85 antibodies of IDO1 immunoprecipitates obtained from P1.HTR cells transfected with IDO1.WT or IDO1.Y149F. Mock-transfected P1.HTR cells were used as a control.


E Tyrosine phosphatase activity in anti - IDO1 immunoprecipitates from lysates of P1 . HTR transfectants as in D .

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E Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from lysates of P1.HTR transfectants as in D.


F Kyn release over 24 h by P1 . HTR transfectants as in D .

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F Kyn release over 24 h by P1.HTR transfectants as in D.


A Confocal immunofluorescence microscopy images of P1 . HTR cells either untransfected ( Untr ) or transfected with IDO1 . WT ( WT ) or vector alone ( Mock ) and stained with antibodies recognizing IDO1 ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 20 µm .

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A Confocal immunofluorescence microscopy images of P1.HTR cells either untransfected (Untr) or transfected with IDO1.WT (WT) or vector alone (Mock) and stained with antibodies recognizing IDO1 (green). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.


B Confocal immunofluorescence microscopy images of P1 . HTR cells transfected with IDO1 . WT , co - stained with antibodies recognizing IDO1 ( green ) and markers of intracellular organelles and structures ( as indicated ; red ) . Nuclei were stained with DAPI ( blue ) . The merge imagine is shown for all stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm .

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B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT, co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (as indicated; 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 Immunoblotting analysis of EE purified from P1 . IDO1 . WT cells . Mock - transfected cells as in A were used as control .

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C Immunoblotting analysis of EE purified from P1.IDO1.WT cells. Mock-transfected cells as in A were used as control.


D Distribution of the IDO1 . WT protein expressed by P1 . HTR transfected cells in isopycnic sucrose gradients . Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated .

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D Distribution of the IDO1.WT protein expressed by P1.HTR transfected cells in isopycnic sucrose gradients. Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated.


A 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 .

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A 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 .

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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 .

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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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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).


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 .

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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 .

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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 ) .

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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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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).


A 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 .

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A 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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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).


( 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 .

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(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 .

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(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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(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.


( 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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(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.


( B ) STXBP4 forms a complex with several Hippo PY motif - containing proteins . Immunoprecipitation was performed with STXBP4 antibody .

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(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 .

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(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 .

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(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 .

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(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 )

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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 ) .

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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 .

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(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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(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.


( B ) STXBP4 forms a protein complex with α - catenin and a group of Hippo pathway proteins .

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(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 .

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(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 .

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(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 .

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(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 .

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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 .

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(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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(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.


STXBP4 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 .

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STXBP4 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 .

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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 .

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(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 .

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(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 .

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(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 .

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(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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(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.


A , RT - qPCR ( n = 3 - 12 biological replicates ) analyses of CGN expression in different mouse organs at P21 .

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A, 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 .

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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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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.


B . MDCK cells expressing WT or mutant CGN were immunolabeled with CGN or Flag antibodies .

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B. 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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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.


A - 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 .

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A-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 .

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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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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.


C . Whole mount immunofluorescence of CGN expression in organ of Corti from P21 CgndelG mouse .

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C. 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 .

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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 ) .

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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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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.


F . 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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F. 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.


Whole mount immunofluorescence with LMO7 antibody on 2 - month old CgndelG mice at 32 kHz cochlear

O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD B-ORGANISM O O O B-TISSUE

Whole mount immunofluorescence with LMO7 antibody on 2-month old CgndelG mice at 32 kHz cochlear region


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

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD B-ORGANISM O O O B-TISSUE

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


A . 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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A. 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).


A 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 .

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A 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 .

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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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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.


B 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 .

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B 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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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.


A 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 .

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A 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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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.


A 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 .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

A 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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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.


A 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 .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

A 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 .

O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O 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-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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.


B 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 .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

B 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.


D 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 .

O B-GENEPROD O O O B-CELL_LINE O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O 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-SMALL_MOLECULE O O O B-ORGANISM B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O

D 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.


B 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 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O

B 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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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.


A 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

A 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 .

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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 .

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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 .

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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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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.


B Hemolysis by AMSIN . Meucin - 18 was used as a positive control . Mean ± SD of three technical replicates is displayed .

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B 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

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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 significance


D 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

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D 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 significance


E 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 .

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E 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

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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 significance


J 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 .

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J 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.