( A ) Inhibition of SARS - CoV - 2 infection by ACE2 - Fc in plaque . Mixtures of ACE2 - Fc SARS - CoV - 2 were incubated for one hour before adding to Vero E6 for another 1 hr at 37 ° C . ACE2 - Fc SARS - CoV - 2 premixtures were removed , were washed once with PBS overlaid with methylcellulose with 2 % FBS for 5 - 7 days before being stained with crystal violet . Those results showed increase of plaque formation was regarded as no inhibition of plaque formation . TPCK - treated trypsin : N - tosyl - L - phenylalanine chloromethyl ketone - treated trypsin .

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(A) Inhibition of SARS-CoV-2 infection by ACE2-Fc in the plaque assay. Mixtures of ACE2-Fc and SARS-CoV-2 were incubated for one hour before adding to Vero E6 cells for another 1 hr at 37°C. The ACE2-Fc and SARS-CoV-2 premixtures were removed, and the cells were washed once with PBS and overlaid with methylcellulose with 2% FBS for 5-7 days before being stained with crystal violet. Those results showed increase of plaque formation was regarded as no inhibition of plaque formation. TPCK-treated trypsin: N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin.


Yield reduction was performed to determine inhibitory effects of ACE2 - Fc on titers culture medium extracts were harvested 24 hours post - infection for real - time PCR ( B )

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Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on virus titers The culture medium and cell extracts were harvested 24 hours post-infection for real-time PCR (B)


Yield reduction was performed to determine inhibitory effects of ACE2 - Fc on expression . culture medium extracts were harvested 24 hours post - infection for western blot ( C ) . NP / PCNA represents relative NP expression as compared to that of PCNA , which served as a loading control . NP / PCNA numbers below panel are ratios of NP / PCNA normalized to that of human IgG control group . PCNA : Proliferating antigen .

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Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on protein expression. The culture medium and cell extracts were harvested 24 hours post-infection for western blot (C). The NP/PCNA represents the relative NP expression as compared to that of PCNA, which served as a loading control. The NP/PCNA numbers below the panel are the ratios of NP/PCNA normalized to that of the human IgG control group. PCNA: Proliferating cell nuclear antigen.


( E ) lysates from pretreatment full - time experiments were harvested immunoblotted with indicated antibodies .

(E) The cell lysates from the pretreatment and full-time experiments were harvested and immunoblotted with the indicated antibodies.


( F ) SARS - CoV - 2 titer in pretreatment full - time experiments was analyzed by real - time PCR .

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(F) The SARS-CoV-2 titer in the pretreatment and full-time experiments was analyzed by real-time PCR.


( A ) ( A ) NP in lysates were determined by Western blot .

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(A) The (A) NP proteins in the cell lysates were determined by Western blot analysis.


( B , C ) in ( B ) culture medium ( C ) lysates was quantified by real - time RT - PCR . genetic information reference numbers for 5 SARS - CoV - 2 strains were summarized in Table EV1 . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * * P < 0 . 01 , * * * P < 0 . 001 . Experiments were performed at least three times with similar results .

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(B, C) The virus RNA in the (B) culture medium and (C) cell lysates was quantified by real-time RT-PCR. The genetic information and sequence reference numbers for the 5 SARS-CoV-2 strains were summarized in Table EV1. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001. Experiments were performed at least three times with similar results.


( A ) H1975 were transduced with full - Spike by Lentiviral vector . extracts were immunoblotted with indicated antibodies . O / E represents overexpress .

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(A) H1975 cells were transduced with full-length Spike by Lentiviral vector. Protein extracts were immunoblotted with the indicated antibodies. O/E represents overexpress.


( B ) effect of ACE2 - Fc activation on degranulative capacity of NK . experiments were performed with human NK derived from three independent donors . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 ( each of ACE2 - Fc vs control ) .

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(B) The effect of ACE2-Fc activation on degranulative capacity of NK cells. The experiments were performed with the primary human NK cells derived from three independent donors. Error bars represent the standard deviation (SD), n= 3. Statistical analysis was performed by unpaired two tail t-test. * P < 0.05 (each concentration of ACE2-Fc vs control).


( C ) CD107a , IFN - γ , TNF - α expression were determined by flow cytometry after NK H1975 - Spike coincubation in presence or absence of ACE2 - Fc or ACE2 control . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 . of positive in both groups were normalized to NK / H1975 - Spike only group .

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(C) The CD107a, IFN-γ, and TNF-α expression levels were determined by flow cytometry after the NK cells and H1975-Spike cells coincubation in the presence or absence of ACE2-Fc or ACE2 control. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. * P < 0.05. The percentage of positive cells in both groups were normalized to the NK/H1975-Spike only group.


( D ) differential expression of degranulation markers after treatment of ACE2 - Fc or ACE2 by flow cytometry . numbers in each plot indicate percentages of positive .

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(D) The differential expression of degranulation markers after the treatment of ACE2-Fc or ACE2 by the flow cytometry. The numbers in each plot indicate the percentages of positive cells.


Immunoblot of biotinylated isolated as in ( A ) . Parental HeLa - T - REx or expressing H3 . 1 - BirA * - HA treated with biotin for indicated times .

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Immunoblot analysis of proteins biotinylated and isolated as in (A). Parental HeLa-T-REx cells or cells expressing H3.1-BirA*-HA and treated with biotin for indicated times.


Immunoblot of TAP - H3 . 1 or - CENP - A pulldown of UBR7 - LAP ( schematic , upper ) . 293T were co - transfected with UBR7 - LAP TAP - tagged constructs , which were purified on IgG - conjugated sepharose beads .

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Immunoblot analysis of TAP-H3.1 or -CENP-A pulldown of UBR7-LAP (schematic, upper). 293T cells were co-transfected with UBR7-LAP and TAP-tagged constructs, which were purified on IgG-conjugated sepharose beads.


Immunoblot of anti - FLAG IP of 3x - FLAG tagged GFP , or FLAG - HA tagged H3 . 1 or H2B , from non - nucleosomal fraction of 293T .

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Immunoblot of anti-FLAG IP of 3x-FLAG tagged GFP, or FLAG-HA tagged H3.1 or H2B, from the non-nucleosomal fraction of 293T cells.


Representative of HeLa transfected with UBR7 - FL . is visualized via DAPI shown in magenta immunofluorescence for 3xFLAG is shown in green . within yellow dotted box in ( E ) is magnified 4x in ( E ' ) . Scale bar = 5 μm . of anti - FLAG immunofluorescence in ( E ) . Error bars represent mean + / - standard deviation . One way ANOVA was used to calculate p - values ( italics ) associated with differences between treatments . Experiment in ( E - F ) was performed twice .

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Representative images of HeLa cells transfected with UBR7-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. The area within yellow dotted box in (E) is magnified 4x in (E'). Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (E). Error bars represent mean +/- standard deviation. One way ANOVA was used to calculate p-values (italics) associated with differences between treatments. Experiment in (E-F) was performed twice.


Immunoblot of anti - FLAG pulldown of full UBR7 - FLAG ( residues 1 - 425 ) mutant UBR7 bearing deletion of UBR7 C - terminus ( ∆ C - term , residues 1 - 215 ) , UBR box ( ∆ UBR , residues 117 - 425 ) , UBR box PHD ( ∆ UBR ∆ PHD , residues 216 - 425 ) , or PHD C - terminus ( ∆ PHD ∆ C - term , residues 1 - 116 ) . 293T were co - transfected with H3 . 1 - HA UBR7 - FLAG constructs ( schematic , upper ) . Asterisk represents non - specific band . Numbers below blot indicate FLAG - normalized anti - HA immunoblot signal relative to full UBR7 - FLAG .

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Immunoblot analysis of anti-FLAG pulldown of full length UBR7-FLAG (residues 1-425) mutant UBR7 bearing deletion of UBR7 C-terminus (∆C-term, residues 1-215), UBR box (∆UBR, residues 117-425), UBR box and PHD (∆UBR∆PHD, residues 216-425), or PHD and C-terminus (∆PHD∆C-term, residues 1-116). 293T cells were co-transfected with H3.1-HA and UBR7-FLAG constructs (schematic, upper). Asterisk represents non-specific band. Numbers below blot indicate FLAG-normalized anti-HA immunoblot signal relative to full length UBR7-FLAG.


Immunoblot of anti - H3 . 1 - HA pulldown of full - UBR7 - LAP UBR7PHD fragment ( residues 117 - 215 ) ( schematic , upper ) . 293T were transfected as in ( A ) . Values below pulldown represent H3 . 1 - HA - normalized GFP - UBR7 immunoblot signal relative to UBR7 - FL . Data were acquired from a continuous , image is cropped to omit irrelevant samples . Numbers below blot indicate HA - normalized anti - GFP immunoblot signal relative to full UBR7 - LAP .

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Immunoblot analysis of anti-H3.1-HA pulldown of full-length UBR7-LAP and UBR7PHD fragment (residues 117-215) (schematic, upper). 293T cells were transfected as in (A). Values below pulldown represent H3.1-HA-normalized GFP-UBR7 immunoblot signal relative to UBR7-FL. Data were acquired from a continuous membrane, and the image is cropped to omit irrelevant samples. Numbers below blot indicate HA-normalized anti-GFP immunoblot signal relative to full length UBR7-LAP.


Anti - GFP immunoblot of streptavidin pulldown of indicated biotin - conjugated H3 peptides following incubation with 293T - GFP - UBR7 lysate after mutation of residues highlighted in ( C ) .

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Anti-GFP immunoblot analysis of streptavidin pulldown of the indicated biotin-conjugated H3 peptides following incubation with 293T-GFP-UBR7 cell lysate after mutation of residues highlighted in (C).


Immunoblot for total modified histones in anti - FLAG pulldown of constructs from soluble fractions demonstrates that UBR7 UBR box PHD are necessary for binding to post - nucleosomal H3 / H4 .

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Immunoblot analysis for total and modified histones in anti-FLAG pulldown of constructs from soluble cellular fractions demonstrates that UBR7 UBR box and PHD are necessary for binding to post-nucleosomal H3/H4.


Immunoblot of streptavidin pulldown from proximity biotinylation described in ( G ) . 4 . 5 h represent newly synthesized histones 48 h represent pre - existing histones . Wedge represents loading titration : 1x > 0 . 1x > 0 . 01x > 0 . 001x . other lanes are loaded at 1x . Immunoblot signals were normalized to anti - FLAG signal . of anti - FLAG ( H3 . 1 - APEX2 - 3xFLAG ) anti - UBR7 immunoblot represented in ( G - H ) . Lanes 8 11 were quantified . Error bar represents mean + / - standard deviation . A two - one - sample t - was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed ) .

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Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in (G). 4.5 h represent newly synthesized histones and 48 h represent pre-existing histones. Wedge represents loading titration: 1x > 0.1x > 0.01x > 0.001x. All other lanes are loaded at 1x concentration. Immunoblot signals were normalized to anti-FLAG signal.   Quantification of anti-FLAG (H3.1-APEX2-3xFLAG) and anti-UBR7 immunoblot represented in (G-H). Lanes 8 and 11 were quantified. Error bar represents mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line).


Immunoblot for total modified H3 H4 in anti - FLAG IP . Experiment was performed as in ( D ) . Dot plot error bars represent mean + / - standard deviation . Immunoblot signals were normalized to anti - H4 signal . A two - one sample t - was used to calculate p - value associated with group mean difference from a hypothetical value of 1 . 0 . Experiment was performed twice .

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Immunoblot analysis for total and modified H3 and H4 in anti-FLAG IP. Experiment was performed as in (D). Dot plot error bars represent mean +/- standard deviation. Immunoblot signals were normalized to anti-H4 signal. A two-tailed one sample t-test was used to calculate p-value associated with group mean difference from a hypothetical value of 1.0. Experiment was performed twice.


Representative of HeLa transfected with sNASP - FL . is visualized via DAPI shown in magenta immunofluorescence for 3xFLAG is shown in green . Scale bar = 5 μm . of anti - FLAG immunofluorescence in ( G ) demonstrating that sNASP - FL is highly susceptible to pre - extraction with Triton X - 100 . One way ANOVA was used to determine p - values ( italics ) associated with differences between treatments . Error bars represent mean + / - standard deviation . Experiment in ( G - H ) was performed twice .

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Representative images of HeLa cells transfected with sNASP-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (G) demonstrating that sNASP-FL is highly susceptible to pre-extraction with Triton X-100. One way ANOVA was used to determine p-values (italics) associated with differences between treatments. Error bars represent mean +/- standard deviation. Experiment in (G-H) was performed twice.


A , C . Immunoblot for modified histone in anti - FLAG pulldown of UBR7 - FL ( A ) or sNASP - FL ( C ) from non - nucleosomal fractions of untreated or treated with replication inhibitors ( hydroxyurea , aphidicolin ) or transcription inhibitors ( α - amanitin , triptolide ) . B , D . Plots represent either normalized immunoblot signals or prior to fractionation . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation , with grey dotted marking mean of No Treat condition . A two - one sample t - was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 . Experiments were performed three times . numbers were counted in two of three experiments .

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A,C. Immunoblot analysis for modified histone in anti-FLAG pulldown of UBR7-FL (A) or sNASP-FL (C) from non-nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors (hydroxyurea, aphidicolin) or transcription inhibitors (α-amanitin, triptolide). B,D. Plots represent either normalized immunoblot signals or cell counts prior to fractionation. Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation, with grey dotted line marking mean of No Treat condition. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiments were performed three times. Cells numbers were counted in two of the three experiments.


Immunoblot of H3 , UBR7 , GAPDH in non - nucleosomal fraction of parental 293T - UBR7KO . of anti - H3 immunoblot represented in ( A ) . Immunoblot signals were normalized to anti - GAPDH signal . Error bars represent mean + / - standard deviation . A two - one - sample t - was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed ) . Experiment in ( A - B ) was performed in three times .

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Immunoblot analysis of H3, UBR7, and GAPDH in the non-nucleosomal fraction of parental and 293T-UBR7KO cells.   Quantification of anti-H3 immunoblot represented in (A). Immunoblot signals were normalized to anti-GAPDH signal. Error bars represent mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (A-B) was performed in three times.


Immunoblot of anti - FLAG pulldown of sNASP - FL in parental or 293T - UBR7KO . 3xFLAG tagged sNASP - FL was immunoprecipitated from non - nucleosomal fraction eluted with FLAG peptide . Experiment was performed three times . of anti - FLAG ( sNASP - FL ) , anti - histone immunoblots represented in ( C ) . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation . A two - one sample t - was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed ) . Experiments were performed two ( sNASP , H4 ) to three ( other targets ) times .

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Immunoblot analysis of anti-FLAG pulldown of sNASP-FL in parental or 293T-UBR7KO cells. 3xFLAG tagged sNASP-FL was immunoprecipitated from non-nucleosomal fraction and eluted with FLAG peptide. Experiment was performed three times.   Quantification of anti-FLAG (sNASP-FL), and anti-histone immunoblots represented in (C). Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments were performed two (sNASP, H4) to three (all other targets) times.


Immunoblot for LAMP2A in parental 293T or expressing UBR7 - FL ( left ) or sNASP - FL ( right ) following treatment with siCTRL , siLAMP2A , or MG - 132 .

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Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7-FL (left) or sNASP-FL (right) following treatment with siCTRL, siLAMP2A, or MG-132.


of data represented in ( C , D ) . Error bars represent mean + / - standard deviation . A two - one sample t - was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed ) . Experiments in ( C - E ) were performed three times .

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Quantification of data represented in (C, D). Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments in (C-E) were performed three times.


Heatmaps of ATAC - seq , anti - GFP ( GFP - UBR7 ) ChIP - seq , anti - H3K4me3 CUT & RUN in parental UBR7KO . Plots are centered on TSS that show changes in ATAC - seq signal in UBR7KO versus parental . Sites are clustered by unsupervised k - means . Cluster color key is defined by cluster labels .

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Heatmaps of ATAC-seq, anti-GFP (GFP-UBR7) ChIP-seq, and anti-H3K4me3 CUT&RUN in parental and UBR7KO cells. Plots are centered on TSS that show changes in ATAC-seq signal in UBR7KO versus parental cells. Sites are clustered by unsupervised k-means analysis. Cluster color key is defined by cluster labels.


Low magnification views of a CD1 Otof - / - organ of Corti ( P23 ) transduced with otoferlin dual ‑ AAV ‑ TS vectors . IHCs : inner hair , OHCs : outer hair maximum projections of confocal optical

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Low magnification views of a CD1 Otof-/- organ of Corti (P23) transduced with otoferlin dual‑AAV‑TS vectors. IHCs: inner hair cells, OHCs: outer hair cells maximum intensity projections of confocal optical sections


High magnification views of CD1B6F1 Otof - / - IHCs transduced with otoferlin dual ‑ AAV ‑ TS ( P26 ) ( B ) dual ‑ AAV ‑ Hyb ( P26 ) ( C ) vectors . eGFP otoferlin immunostainings are depicted as color lookup tables in ( A - C ) with warmer colors representing higher pixel intensities ) , maximum projections of confocal optical

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High magnification views of CD1B6F1 Otof-/- IHCs transduced with otoferlin dual‑AAV‑TS (P26) (B) and dual‑AAV‑Hyb (P26) (C) vectors. Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in (A-C) with warmer colors representing higher pixel intensities ), maximum intensity projections of confocal optical sections


of N ‑ C ‑ terminal otoferlin labelled IHCs in dual ‑ AAV ‑ TS ( n = 10 mice ) dual ‑ AAV ‑ Hyb ( n = 9 mice ) injected CD1B6F1 Otof - / - mice ( P18 ‑ 30 )

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Percentage of N‑ and C‑terminal otoferlin labelled IHCs in dual‑AAV‑TS (n=10 mice) and dual‑AAV‑Hyb (n=9 mice) injected CD1B6F1 Otof-/- mice (P18‑30)


Average N - terminal C ‑ terminal otoferlin immunofluorescence in dual ‑ AAV transduced Otof - / - wild - type IHCs ( P23 - 30 ) . Otoferlin were normalized to immunofluorescence in non - transduced B6 wild - type IHCs for each antibody

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Average N-terminal and C‑terminal otoferlin immunofluorescence levels in dual‑AAV transduced Otof-/- and wild-type IHCs (P23-30). Otoferlin levels were normalized to immunofluorescence levels in non-transduced B6 wild-type IHCs for each antibody separately


High magnification views of IHCs immunolabeled for otoferlin synaptic ribbons ( CtBP2 ) from wild ‑ type ( B6 : P27 , CD1B6F1 : P29 ) , dual - AAV injected CD1B6F1 Otof - / - ( dual - AAV - TS : P26 , dual - AAV - Hyb : P28 ) , their non - injected ears . ( * ) Transduced . Maximum projections of optical confocal sections . Scale bars : 5

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High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons (CtBP2) from wild‑type (B6: P27, CD1B6F1: P29), dual-AAV injected CD1B6F1 Otof-/- (dual-AAV-TS: P26, dual-AAV-Hyb: P28), and their contralateral non-injected ears. (*) Transduced cells. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm


Synaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild ‑ type ( B6 : n = 48 IHCs , CD1B6F1 : n = 108 IHCs ) , transduced Otof - / - ( dual ‑ AAV ‑ TS : n = 59 IHCs , dual - AAV - Hyb : n = 37 IHCs ) , non - transduced Otof - / - IHCs from injected ( - AAV injected ear , n = 65 IHCs ) non - injected ( - AAV non - injected ear , n = 46 IHCs ) ears ( P25 - 29 )

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Synaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild‑type (B6: n=48 IHCs, CD1B6F1: n=108 IHCs), transduced Otof-/- (dual‑AAV‑TS: n=59 IHCs, dual-AAV-Hyb: n=37 IHCs), and non-transduced Otof-/- IHCs from injected (-AAV injected ear, n=65 IHCs) and contralateral non-injected (-AAV non-injected ear, n=46 IHCs) ears (P25-29)


synapses labelled with CtBP2 postsynaptic marker Shank1a in B6 wild - type Otof - / - P6 P14 organs of Corti . Maximum projections of optical confocal sections . Scale bars : 5 µm . D Synapse numbers quantified from IHCs in apical cochlear turns ( C ) of B6 wild - type ( P6 : n = 53 IHCs ; P14 : n = 73 IHCs ) B6 Otof - / - ( P6 : n = 62 IHCs ; P14 : n = 65 IHCs ) mice at two different developmental stages ( P6 P14 )

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IHC synapses labelled with CtBP2 and the postsynaptic marker Shank1a in B6 wild-type and Otof-/- P6 and P14 organs of Corti. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm. D Synapse numbers quantified from IHCs in apical cochlear turns (C) of B6 wild-type (P6: n= 53 IHCs; P14: n=73 IHCs) and B6 Otof-/- (P6: n=62 IHCs; P14: n=65 IHCs) mice at two different developmental stages (P6 and P14)


Ca2 + ‑ current - voltage relationship of control CD1B6F1 wild ‑ type ( n = 6 IHCs ) , dual - AAV - TS transduced ( n = 8 IHCs ) , non ‑ transduced CD1B6F1 Otof - / - ( n = 10 IHCs ) IHCs ( P14 ‑ 18 )

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Ca2+‑current-voltage relationship of control CD1B6F1 wild‑type (n=6 IHCs), dual-AAV-TS transduced (n=8 IHCs), and non‑transduced CD1B6F1 Otof-/- (n=10 IHCs) IHCs (P14‑18)


Representative Ca2 + ‑ currents ( Ica ) plasma capacitance increments ( ΔCm ) of a wild ‑ type control , transduced , non ‑ transduced Otof - / - in response to a 20 ms depolarization at maximum Ca2 + ‑ current potentials ( typically - 14 mV )

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Representative Ca2+‑currents (Ica) and IHC plasma membrane capacitance increments (ΔCm) of a wild‑type control, transduced, and non‑transduced Otof-/- IHC in response to a 20 ms depolarization pulse at maximum Ca2+‑current potentials (typically -14 mV)


Average exocytosis level measured as ΔCm ( G ) corresponding Ca2 + ‑ current integrals ( QCa2 + ) ( H ) in wild ‑ type ( CD1B6F1 : n = 6 IHCs ; B6 : n = 11 IHCs ( B6 data replotted from Strenzke et al , 2016 ) ) , dual - AAV - TS transduced Otof - / - ( n = 8 IHCs ) , non ‑ transduced ( n = 11 IHCs ) Otof - / - IHCs . Transduced IHCs that showed eGFP expression , but had almost no exocytosis are depicted as dashed lines ( not included into average )

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Average exocytosis level measured as ΔCm (G) and corresponding Ca2+‑current integrals (QCa2+) (H) in wild‑type (CD1B6F1: n=6 IHCs; B6: n=11 IHCs (B6 data replotted from Strenzke et al, 2016)), dual-AAV-TS transduced Otof-/- (n=8 IHCs), and non‑transduced (n=11 IHCs) Otof-/- IHCs. Transduced IHCs that showed eGFP expression, but had almost no exocytosis are depicted as dashed lines (not included into the average)


Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual ‑ AAV ‑ TS dual ‑ AAV ‑ Hyb injected CD1B6F1 Otof - / - . AAV2 / 6 . eGFP ( + AAV . eGFP ) dual ‑ AAV ‑ TS injected CD1B6F1 wild ‑ type , non ‑ injected control Otof - / - littermate ( - AAV ) mice served as controls . SP : summating potential ; ABR waves are indicated from I -

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Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual‑AAV‑TS and dual‑AAV‑Hyb injected CD1B6F1 Otof-/- animals. AAV2/6.eGFP (+AAV.eGFP) and dual‑AAV‑TS injected CD1B6F1 wild‑type, and non‑injected control Otof-/- littermate (-AAV) mice served as controls. SP: summating potential; ABR waves are indicated from I-V


Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks /

Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks/s


ABR click sound ( C thresholds in otoferlin dual ‑ AAV treated Otof - / - mice compared to wild ‑ type non ‑ treated Otof - / - control

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ABR click sound (C thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals


ABR tone burst ( D ) thresholds in otoferlin dual ‑ AAV treated Otof - / - mice compared to wild ‑ type non ‑ treated Otof - / - control . In D , two best are depicted with open circles . with thresholds exceeding maximum loudspeaker output ( arrows ) of 100 dB SPL for clicks 90 dB SPL for tone bursts were set to 110 dB SPL 100 dB SPL , respectively . Apical basal cochlear turns are indicated as Apex Base ,

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ABR tone burst (D) thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals. In D, the two best animals are depicted with open circles. Animals with thresholds exceeding the maximum loudspeaker output (arrows) of 100 dB SPL for clicks and 90 dB SPL for tone bursts were set to 110 dB SPL and 100 dB SPL, respectively. Apical and basal cochlear turns are indicated as Apex and Base, respectively


Summed ABR wave I - V amplitudes at different click sound intensities in otoferlin dual ‑ AAV injected , non ‑ injected Otof - / - , wild ‑ type control

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Summed ABR wave I-V amplitudes at different click sound intensities in otoferlin dual‑AAV injected, non‑injected Otof-/-, and wild‑type control mice


Summed ABR wave I ‑ V amplitudes of dual ‑ AAV ‑ TS treated CD1B6F1 Otof - / - ( n = 8 plotted against their full - otoferlin transduction

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Summed ABR wave I‑V amplitudes of individual dual‑AAV‑TS treated CD1B6F1 Otof-/- animals (n=8 animals plotted against their full-length otoferlin IHC transduction rate


A ) H & E immunohistochemistry reveal that β - catenin accumulates in tumors emerging in one - year - old miR - 424 ( 322 ) / 503 - / - mice . panel is a representative image of n = 5 independent tumors . scale bar indicates 100 micrometers ( µM ) .

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A) H&E and immunohistochemistry reveal that β-catenin accumulates in tumors emerging in one-year-old female miR-424(322)/503-/- mice. The panel is a representative image of n=5 independent tumors. The scale bar indicates 100 micrometers (µM).


B ) Bar graphic showing that human breast cancers with deletion of miR - 424 / 503 locus have higher of β - catenin ( RPPA ) . P - value is calculated on one - sided Wilcoxon rank - sum . In box - plots in B C relative expression of β - catenin in RPPA data from TCGA is shown . yellow indicates median bars indicate 75th percentile ( Q3 ) + 1 . 5 interquartile range ( IQR ) .

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B) Bar graphic showing that human breast cancers with deletion of the miR-424/503 locus have higher protein levels of β-catenin (RPPA). P-value is calculated based on one-sided Wilcoxon rank-sum test. In the box-plots in B and C the relative expression of β-catenin in the RPPA data from TCGA is shown. The yellow line indicates the median and the bars indicate the 75th percentile (Q3)+1.5 interquartile range (IQR).


C ) bar graph shows that human breast cancers with low expression of miR - 424 miR - 503 have higher of β - catenin . P - value is calculated on one - sided Wilcoxon rank - sum .

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C) The bar graph shows that human breast cancers with low expression levels of miR-424 and miR-503 have higher protein levels of β-catenin. P-value is calculated based on one-sided Wilcoxon rank-sum test.


B ) bar graphs show number of PROCR + in 1 - year - old WT miR - 424 ( 322 ) / 503 KO mice during diestrus . error bars show standard deviation .

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B) The bar graphs show the number of PROCR+ in 1-year-old female WT and miR-424(322)/503 KO mice during diestrus. The error bars show the standard deviation.


E ) panel shows lack of induction of bona fide WNT targets in miR - over expressing from panel C compared with EV control . UT indicates samples untreated with WNT3 .

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E) The panel shows the lack of induction of bona fide WNT targets in miR-over expressing cells from panel C compared with EV control. UT indicates samples untreated with WNT3.


D ) Western blot of LRP6 TBLR1 in miR - cluster - Dox - MCF - 10A model upon upregulation of miR - cluster with 100 ng / ml of doxycycline for specified number of days . numbers below blots indicate quantitation by densitometry of expression relative to β - actin . expression level of miR - 424 miR - 503 is indicated in bar graph below blots . UN indicates samples untreated with doxycycline . error bars show standard deviation .

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D) Western blot of LRP6 and TBLR1 in the miR-cluster-Dox-MCF-10A model upon upregulation of miR-cluster with 100 ng/ml of doxycycline for the specified number of days. The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β-actin. The expression level of miR-424 and miR-503 is indicated in the bar graph below the blots. UN indicates samples untreated with doxycycline. The error bars show the standard deviation.


F ) Western blot of LRP6 TBLR1 expression in WT miR - 424 / 503 - KO basal - derived organoids after 14 days in culture with without Wnt3a ( 100ng / ml ) . arrow shows correct TBLR1 band .

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F) Western blot of LRP6 and TBLR1 expression in WT and miR-424/503-KO basal cell-derived organoids after 14 days in culture with and without Wnt3a (100ng/ml). The arrow shows the correct TBLR1 band.


G ) showing that LRP6 accumulates in epithelium of miR - 424 ( 322 ) / 503 - / - mice ( 1 - year - old ) . scale bar indicates 100 micrometers ( µM ) .

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G) IHC staining showing that LRP6 accumulates in the epithelium of female miR-424(322)/503-/- mice (1-year-old). The scale bar indicates 100 micrometers (µM).


A ) Enrichment of putative LRP6 binding sites bound to AGO2 in miR - cluster - Dox - MCF - 10A after miR - 424 / 503 cluster is induced with 100ng / ml of dox for three days . A CDC25A - binding site is included as a positive control a region of B2M GAPDH are included as negative controls . error bars show standard deviation .

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A) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR-cluster-Dox-MCF-10A cells after the miR-424/503 cluster is induced with 100ng/ml of dox for three days. A CDC25A-binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls. The error bars show the standard deviation.


B ) WT - blot shows expression of LRP6 upon upregulation of miR - 424 / 503 in miR - cluster - Dox - MCF - 10A ( Dox + ) when one or both of conserved microRNA binding sites ( # 1 # 3 ) are blocked by target protection .

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B) The WT-blot shows the expression of LRP6 upon upregulation of miR-424/503 in miR-cluster-Dox-MCF-10A cells (Dox+) when one or both of the conserved microRNA binding sites (#1 and #3) are blocked by target protection.


C ) WT - blot shows expression of LRP6 β - catenin in in miR - cluster - Dox - MCF - 10A cultured in presence of Dox with without WNT3a ligand ( 100ng / ml for 6 hours ) when microRNA binding sites # 1 # 3 are blocked by target protection .

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C) The WT-blot shows the expression of LRP6 and β-catenin in cells in miR-cluster-Dox-MCF-10A cells cultured in the presence of Dox and with and without WNT3a ligand (100ng/ml for 6 hours) when the microRNA binding sites #1 and #3 are blocked by target protection.


Representative composite immune - of whole hair follicle units from non - lesional lesional scalp psoriasis . K15 ( green ) , c - JUN ( red A , C ) JUNB ( red B , D ) DAPI ( blue ) .

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Representative composite immune-fluorescent images of whole hair follicle units from non-lesional and lesional scalp psoriasis patients. K15 (green), c-JUN (red A, C) and JUNB (red B, D) and DAPI (blue).


Confocal of bulge region of human psoriatic hair follicles from non - lesional lesional regions of scalp . K15 ( green ) , c - JUN ( red E , G ) JUNB ( red F , H ) DAPI ( blue ) .

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Confocal images of the bulge region of human psoriatic hair follicles from non-lesional and lesional regions of the scalp. K15 (green), c-JUN (red E, G) and JUNB (red F, H) and DAPI (blue).


of HF - SCs ( K15 + ) that express c - JUN ( I ) JUNB ( J ) in bulge region of human psoriatic hair follicles from non - lesional lesional scalp in comparison with healthy scalp . n = 2 - 6 hair follicles in anagen per group from five psoriatic two healthy .

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Percentage of HF-SCs (K15+) that express c-JUN (I) and JUNB (J) in the bulge region of human psoriatic hair follicles from non-lesional and lesional scalp in comparison with healthy scalp. n=2-6 hair follicles in anagen per group from five psoriatic patients and two healthy patients.


Composite immunofluorescent showing GFP expression of whole - ear sections from DKO * - mT / mG mice at day 5 , 7 , 15 30 . Increased GFP expression is shown at day 7 followed by progressive decrease of GFP + keratinocytes ( dotted represents basal layer , red dotted represents outermost skin layer ) . n = 3 per time point . of GFP expression ( top ) epidermal thickness ( bottom ) of DKO * mice at different time points during psoriasis - like disease progression . n = 6 per time point . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two - relative to control group ) .

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Composite immunofluorescent images showing GFP expression of whole-ear sections from DKO*-mT/mG mice at day 5, 7, 15 and 30. Increased GFP expression is shown at day 7 followed by progressive decrease of GFP+ keratinocytes (white dotted line represents basal layer, and red dotted line represents outermost skin layer). n=3 per time point. Quantification analysis of GFP expression (top) and epidermal thickness (bottom) of DKO* mice at different time points during psoriasis-like disease progression. n=6 per time point. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to control group).


of Caspase - 3 ( cCas3 top ) Ki67 ( bottom ) in ear skin of DKO * mice at different time points during psoriasis - like disease progression . n = 3 per time point .

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Quantification analysis of cleaved Caspase-3 (cCas3 top) and Ki67 (bottom) in ear skin of DKO* mice at different time points during psoriasis-like disease progression. n=3 per time point.


Intravital confocal time - lapse imaging of non - mutant Tomato keratinocytes mutant GFP keratinocytes of DKO * - mT / mG at day 7 after first tamoxifen injection . Dotted circles emphasize areas in which mutant GFP + keratinocytes are replaced by non - mutant Tomato + keratinocytes .

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Intravital confocal time-lapse imaging of non-mutant Tomato keratinocytes and mutant GFP keratinocytes of DKO*-mT/mG at day 7 after first tamoxifen injection. Dotted circles emphasize areas in which mutant GFP+ keratinocytes are replaced by non-mutant Tomato+ keratinocytes.


of GFP + eliminated during intravital confocal time - lapse imaging at day 5 , 7 15 in DKO * - mT / mG mice ( Time - lapse 1 : 8h ) . Positive value : of GFP increased . Negative value : of GFP reduced .

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Percentage of GFP+ area eliminated during intravital confocal time-lapse imaging at day 5, 7 and 15 in DKO*-mT/mG mice (Time-lapse 1:8h). Positive value: Area of GFP increased. Negative value: Area of GFP reduced.


imaging of DKO * - mT / mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP + keratinocytes reside along hair follicles . dotted separates epidermis dermis . arrows represent GFP + hair follicles .

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Fluorescence imaging of DKO*-mT/mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP+ keratinocytes reside along the hair follicles. White dotted line separates epidermis and dermis. White arrows represent GFP+ hair follicles.


Intravital confocal time - lapse imaging of DKO * - mT / mG at day 15 after first tamoxifen injection . Mutant GFP + Keratinocytes ( arrows ) are maintained around hair follicles .

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Intravital confocal time-lapse imaging of DKO*-mT/mG at day 15 after first tamoxifen injection. Mutant GFP+ Keratinocytes (white arrows) are maintained around hair follicles.


Ear pictures of control , DKO * , DKO * K15 mice after 15 days of induction show clear signs of inflammation psoriatic - like scales in both models .

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Ear pictures of control, DKO*, and DKO*K15 mice after 15 days of induction show clear signs of inflammation and psoriatic-like scales in both models.


Representative of hematoxylin eosin ( H & E ) of ear sections from control , DKO * DKO * K15 mice .

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Representative images of hematoxylin and eosin (H&E) staining of ear sections from control, DKO* and DKO*K15 mice.


Ear thickness measurement at different time points during psoriasis - like development in control , DKO * DKO * K15 mice . n > 5 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two - relative to control group ) .

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Ear thickness measurement at different time points during psoriasis-like development in control, DKO* and DKO*K15 mice. n > 5 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to control group).


FACS of CD45 + from ear skin of control , DKO * DKO * K15 mice at different time points during psoriasis - like development . n > 5 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two - relative to controls ) .

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FACS quantification of CD45+ cells from ear skin of control, DKO* and DKO*K15 mice at different time points during psoriasis-like development. n > 5 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to controls).


of IL - 17A , IL - 6 LCN2 production in sera of control , DKO * DKO * K15 mice at day 30 of psoriasis - like disease by ELISA . n > 4 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two - relative to controls ) .

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Quantification of IL-17A, IL-6 and LCN2 production in sera of control, DKO* and DKO*K15 mice at day 30 of psoriasis-like disease by ELISA. n > 4 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to controls).


of total number of GFP + epidermal by FACS from DKO * - mT / mG DKO * K15 - mT / mG mice at different time points during psoriasis - like progression compared to Co - mT / mG mice . n = 3 - 6 per group time point .

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Quantification of total number of GFP+ epidermal cells by FACS analysis from DKO*-mT/mG and DKO*K15-mT/mG mice at different time points during psoriasis-like progression compared to Co-mT/mG mice. n = 3-6 per group and time point.


Representative immunofluorescence of ear sections from CoK15 - mT / mG DKO * K15 - mT / mG mouse models after 15 days of induction . Red ( Tomato ) , green ( GFP ) blue ( DAPI ) . Hyperplasia of Tomato + keratinocytes in psoriatic - like DKO * K15 - mT / mG mouse with clusters of GFP + keratinocytes are derived from hair follicle K15 + GFP + . n = 3 per group . Dotted lines separate epidermis dermis . of Caspase - 3 ( cCas3 ) Ki67 in ear skin of DKO * K15 - mT / mG mice at different time points during psoriasis - like disease progression .

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Representative immunofluorescence images of ear sections from CoK15-mT/mG and DKO*K15-mT/mG mouse models after 15 days of induction. Red (Tomato), green (GFP) and blue (DAPI). Hyperplasia of Tomato+ keratinocytes in psoriatic-like DKO*K15-mT/mG mouse with clusters of GFP+ keratinocytes are derived from hair follicle K15+ GFP+ stem cells. n=3 per group. Dotted lines separate epidermis and dermis. Quantification analysis of cleaved Caspase-3 (cCas3) and Ki67 in ear skin of DKO*K15-mT/mG mice at different time points during psoriasis-like disease progression.


Average number of live dead ( TOPRO - 3 + Dapi + ) keratinocytes derived from HF - SCGFP at different time points of time lapse capture during 48 hours . There is a slight increase over time of live HF - SCs from DKO * mice when compared to control , no difference in average of dead . n = 4 fields from two independent experiments . Average number of live dead ( TOPRO - 3 + DAPI + ) keratinocytes derived from basal keratinocytesGFP show a significant increase in DKO * basal live keratinocytes at 12 hours , followed by a significant increase in at 48 hours .

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Average number of live and dead (TOPRO-3+ Dapi+) primary keratinocytes derived from HF-SCGFP at different time points of time lapse capture during 48 hours. There is a slight increase over time of live HF-SCs from DKO* mice when compared to control cells, and no difference in average of dead cells. n = 4 fields from two independent experiments. Average number of live and dead (TOPRO-3+ DAPI+) primary keratinocytes derived from basal keratinocytesGFP show a significant increase in DKO* basal live keratinocytes at 12 hours, followed by a significant increase in cell death at 48 hours.


Average number of live keratinocytes from co - cultures of GFP + with Tomato + bulge HF - SCs at different time points of time lapse capture during 48 hours . Co - culture of HF - SCGFP with HF - SCTom from DKO * - mT / mG mice shows a significant increase of HF - SCTom , while HF - SCTom from DKO * - mT / mG mice show a normal growth when they are co - culture with HF - SCGFP from control mice . Average number of live keratinocytes from co - cultures of GFP + with Tomato + basal keratinocytes at different time points of time lapse capture during 48 hours . Co - culture of b - KCGFP with b - KCTom from DKO * - mT / mG mice show significant increase of b - KCTom , while these b - KCTom from DKO * - mT / mG mice show a normal growth when they are co - culture with b - KCGFP from control mice .

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Average number of live primary keratinocytes from co-cultures of GFP+ with Tomato+ bulge HF-SCs at different time points of time lapse capture during 48 hours. Co-culture of HF-SCGFP with HF-SCTom from DKO*-mT/mG mice shows a significant increase of HF-SCTom, while the HF-SCTom from DKO*-mT/mG mice show a normal growth when they are co-culture with HF-SCGFP from control mice. Average number of live primary keratinocytes from co-cultures of GFP+ with Tomato+ basal keratinocytes at different time points of time lapse capture during 48 hours. Co-culture of b-KCGFP with b-KCTom from DKO*-mT/mG mice show significant increase of b-KCTom, while these b-KCTom from DKO*-mT/mG mice show a normal growth when they are co-culture with b-KCGFP from control mice.


Average total number of live Tomato + basal keratinocytes ( from DKO * or control mice ) at different time points cultured in bottom part of trans - well system cultures with GFP + basal keratinocytes ( from DKO * or Control mice ) cultured in upper trans - well ( Tw ) . n = 5 fields from three independent experiments . Tom + basal keratinocytes significantly increased when these were cultured with mutant GFP + basal keratinocytes in trans - well in comparison with control GFP + basal keratinocytes .

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Average total number of live Tomato+ basal keratinocytes (from DKO* or control mice) at different time points cultured in the bottom part of trans-well system cultures with GFP+ basal keratinocytes (from DKO* or Control mice) cultured in the upper trans-well (Tw). n = 5 fields from three independent experiments. Tom+ basal keratinocytes significantly increased when these were cultured with mutant GFP+ basal keratinocytes in the trans-well in comparison with control GFP+ basal keratinocytes.


Heat map for specific genes relative to biological process enriched on Gene Ontology IDs for DEGs that are : i ) exclusively up - regulated in HF - SCGFP ; ii ) exclusively up - regulated in HF - SCTom ; iii ) commonly up - regulated in HF - SCGFP HF - SCTom .

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Heat map for specific genes relative to biological process enriched based on Gene Ontology IDs for DEGs that are: i) exclusively up-regulated in HF-SCGFP; ii) exclusively up-regulated in HF-SCTom; iii) commonly up-regulated in HF-SCGFP and HF-SCTom.


Gene expression of TSLP in different epidermal subpopulations sorted at mid - term ( D7 ) or late - term ( D30 ) of psoriasis - like progression in DKO * mice .

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Gene expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice.


expression of TSLP in different epidermal subpopulations sorted at mid - term ( D7 ) or late - term ( D30 ) of psoriasis - like progression in DKO * mice . n = 2 - 3 .

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protein expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice. n=2-3.


of TSLP production in sera of control , DKO * DKO * K15 mice at day 30 of psoriasis - like disease by ELISA . n = 4 per group .

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Quantification of TSLP production in sera of control, DKO* and DKO*K15 mice at day 30 of psoriasis-like disease by ELISA. n = 4 per group.


TSLP production in conditioned media quantified by ELISA three days after infection with Ad - empty or Ad - cre . n = 3 independent experiments .

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TSLP production in conditioned media quantified by ELISA three days after infection with Ad-empty or Ad-cre. n=3 independent experiments.


of EdU + Tomato + keratinocytes after blocking with anti - TSLP . Anti - TSLP neutralizes hyper - proliferation of non - mutantTom KCs in vitro after infection with Ad - cre .

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Percentage of EdU+ Tomato+ keratinocytes after blocking with anti-TSLP. Anti-TSLP neutralizes the hyper-proliferation of non-mutantTom KCs in vitro after infection with Ad-cre.


Gene expression of Tslpr after TSLP neutralization is reduced . Gene expression of IL - 7ra after TSLP neutralization is increased . Gene expression of VEGFα after TSLP neutralization is reduced .

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Gene expression of Tslpr after TSLP neutralization is reduced. Gene expression of IL-7ra after TSLP neutralization is increased. Gene expression of VEGFα after TSLP neutralization is reduced.


Representative of treated ears after 9 days of anti - TSLP or IgG treatment .

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Representative images of treated ears after 9 days of anti-TSLP or IgG treatment.


Longitudinal of ear thickness in DKO * mice at different time points during psoriasis - like disease

Longitudinal analysis of ear thickness in DKO* mice at different time points during psoriasis-like disease progression


of EdU + epidermal ( CD45 - CD49f + ) after blocking with anti - TSLP in DKO * DKO * 15

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Percentage of EdU+ epidermal cells (CD45-CD49f+) after blocking with anti-TSLP in DKO* and DKO*15 mice


Representative immune - of ear sections from control or DKO * mice treated with IgG or anti - TSLP ) stained for Ki67 . Ki67 expression was reduced in mice treated with anti - TSLP ( n = 3 ) .

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Representative immune-fluorescence images of ear sections from control or DKO* mice treated with IgG or anti-TSLP) stained for Ki67. Ki67 expression was reduced in mice treated with anti-TSLP (n =3).


Representative composite immune - of whole hair follicle units by confocal from non - lesional lesional scalp psoriasis . c - JUN ( purple ) , K - 15 ( green ) , TSLP ( red ) DAPI ( blue ) .

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Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients. c-JUN (purple), K-15 (green), TSLP (red) and DAPI (blue).


TSLP measurement in hair follicle units epidermis by ImageJ from healthy scalp , non - lesional lesional scalp psoriasis . Representation of fold change relative to control healthy scalp . n = 3 samples .

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TSLP intensity measurement in hair follicle units and epidermis by ImageJ from healthy scalp, non-lesional and lesional scalp psoriasis patients. Representation of fold change relative to control healthy scalp. n= 3 samples.


Representative composite immune - of whole hair follicle units by confocal from non - lesional lesional scalp psoriasis ( D ) magnification of specific regions from HFs ( E ) . CD200 ( green ) , c - JUN ( ) , TSLP ( red ) , DAPI ( blue ) .

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Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients (D) and magnification images of specific regions from the HFs (E). CD200 (green), c-JUN (white), TSLP (red), DAPI (blue).


b ) affinity of UVRAG - C2 domain for phospholipids was assessed using a - lipid overlay . left panel indicates identity of lipid species on PtdIns strips . - purified GST fusion of UVRAG - C2 ( wild type , WT ) , but not GST , binds PtdIns ( 3 ) P , PtdIns ( 4 ) P PtdIns ( 5 ) P . K78A / R82A mutant of C2 is defective for phospholipid - binding , whereas K87A / N88A mutant is partially impaired . Binding of FYVE PH domains of Hrs FAPP1 , respectively , to PtdIns ( 3 ) P PtdIns ( 4 ) P served as quality controls ( right ) . PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; LPA , lysophosphatidic acid ; LPC , lysophosphatidylcholine ; S1P , sphingosine 1 - phosphate ; PA , phosphatidic acid ; PS , phosphatidylserine .

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b) The affinity of the UVRAG-C2 domain for phospholipids was assessed using a protein-lipid overlay assay. The left panel indicates the identity of lipid species on PtdIns strips. Bacterial-purified GST fusion of UVRAG-C2 (wild type, WT), but not GST, binds PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. The K78A/R82A mutant of C2 is defective for phospholipid-binding, whereas the K87A/N88A mutant is partially impaired. Binding of the FYVE and PH domains of Hrs and FAPP1, respectively, to PtdIns(3)P and PtdIns(4)P served as quality controls (right). PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.


( c ) relative affinity of GST - fusion for phosphoinositides was quantified by densitometric of GST immunoblots in b from five independent experiments ( n = 5 ) . Error bars represent standard deviation ( s . d . ) . A . U . , arbitrary units .

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(c) The relative affinity of the GST-fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments (n = 5). Error bars represent standard deviation (s.d.). A.U., arbitrary units.


( d ) Binding of full - UVRAG ( untagged ) to lipid blots . same PtdIns strips ( in b ) were used immunoblotted with antibodies against IgG ( right ) UVRAG ( left ) .

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(d) Binding of full-length UVRAG (untagged) to lipid blots. The same PtdIns strips (in b) were used and immunoblotted with antibodies against IgG (right) and UVRAG (left).


( g ) Pulldown of UVRAG - C2 domain GST - fusion by liposomes bearing indicated phosphoinositides . GST - Hrs - FYVE GST - FAPP1 - PH are also shown .

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(g) Pulldown of UVRAG-C2 domain GST-fusion proteins by liposomes bearing the indicated phosphoinositides. GST-Hrs-FYVE and GST-FAPP1-PH are also shown.


( a - k ) Confocal analyses of subcellular co - localization of endogenous UVRAG with ER markers , including endogenous calnexin ( red , a ) , endogenous PDI ( red , b ) , overexpressed DSred - ER ( red , c ) ; with Golgi markers , including endogenous GM130 p115 ( red , d , e , cis - Golgi ) , overexpressed GFP - Man II ( green , f , medial - Golgi ) , endogenous TGN46 ( red , g , trans - Golgi ) , overexpressed PtdIns ( 4 ) P probe , GFP - FAPP1 - PH ( green , h ) ; with endogenous coatomer , including Sec31 ( red , i , COPII - related ) β ′ - COP ( red , j , COPI - related ) ; with endosome marker GFP - p40phox ( PX ) ( green , k ) in HeLa . insets show a high magnification of selected areas . Scale bars , 10 μm . ( l ) Confocal microscopic of co - localization of UVRAG with indicated markers ( data are mean ± s . d . , n = 150 obtained by gathering data from three independent experiments ) ; * * P0 . 01 ; * * * P0 . 001 .

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(a-k) Confocal analyses of the subcellular co-localization of endogenous UVRAG with ER markers, including endogenous calnexin (red, a), endogenous PDI (red, b), and overexpressed DSred-ER (red, c); with Golgi markers, including endogenous GM130 and p115 (red, d,e, cis-Golgi), overexpressed GFP-Man II (green, f, medial-Golgi), endogenous TGN46 (red, g, trans-Golgi), and overexpressed PtdIns(4)P probe, GFP-FAPP1-PH (green, h); with endogenous coatomer proteins, including Sec31 (red, i, COPII-related) and β′-COP (red, j, COPI-related); and with the endosome marker GFP-p40phox(PX) (green, k) in HeLa cells. The insets show a high magnification of the selected areas. Scale bars, 10 μm. (l) Confocal microscopic quantification of co-localization of UVRAG with the indicated markers (data are mean±s.d., n = 150 cells obtained by gathering data from three independent experiments); **P0.01; ***P0.001.


a ) UVRAG is released from ER in overexpressing MTMR3 . HeLa transfected with MTMR3 - mStrawberry or catalytically inactive mutant MTMR3C413S were stained with anti - UVRAG antibody processed for confocal microscopy . Asterisks indicate MTMR3 - transfected ( left ) . of transfected with diffused of UVRAG was quantified ( middle ) . Data are mean ± s . d . ; n = 240 collected from four independent experiments ; * * P0 . 01 . Western blot shows of endogenous UVRAG in presence or absence of MTMR3 ( right ) .

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a) UVRAG is released from the ER membrane in cells overexpressing MTMR3. HeLa cells transfected with MTMR3-mStrawberry or the catalytically inactive mutant MTMR3C413S were stained with anti-UVRAG antibody and processed for confocal microscopy. Asterisks indicate MTMR3-transfected cells (left). The percentage of transfected cells with diffused staining of UVRAG was quantified (middle). Data are mean±s.d.; n = 240 cells collected from four independent experiments; **P0.01. Western blot analysis shows the levels of endogenous UVRAG in the presence or absence of MTMR3 (right).


( b ) Wortmannin treatment releases UVRAG from ER . HeLa were treated with 100 nM wortmannin for 1h , processed for confocal microscopy using anti - UVRAG anti - PDI antibodies ( top panel ) . UT , untreated . Insets highlight relative localization of UVRAG at ER . Confocal co - localization is indicated ( bottom left panel ) . Data are mean ± s . d . ; n = 75 for each group from three independent experiments ; * * * P0 . 001 . Endogenous UVRAG PDI expression before after wortmannin treatment were confirmed by immunoblotting ( bottom right panel ) .

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(b) Wortmannin treatment releases UVRAG from the ER. HeLa cells were treated with 100 nM wortmannin for 1h, and processed for confocal microscopy using anti-UVRAG and anti-PDI antibodies (top panel). UT, untreated. Insets highlight the relative localization of UVRAG at the ER. Confocal co-localization is indicated (bottom left panel). Data are mean±s.d.; n = 75 cells for each group from three independent experiments; ***P0.001. Endogenous UVRAG and PDI expression before and after wortmannin treatment were confirmed by immunoblotting (bottom right panel).


( c ) Mutation of C2 domain leaves UVRAG unable to associate with ER . HeLa expressing Flag - UVRAG ( WT ) or its K78A / R82A mutant were fixed processed for confocal microscopy for co - localization with ER marker PDI ( top panel ) . Insets highlight relative distribution of UVRAG at ER . UVRAG endogenous PDI expression were confirmed by immunoblotting ( bottom panel ) . See Supplementary Fig . S9 for uncropped data . Scale bars , 10 μm .

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(c) Mutation of the C2 domain leaves UVRAG unable to associate with the ER. HeLa cells expressing Flag-UVRAG (WT) or its K78A/R82A mutant were fixed and processed for confocal microscopy for co-localization with the ER marker PDI (top panel). Insets highlight the relative distribution of UVRAG at the ER. UVRAG and endogenous PDI expression were confirmed by immunoblotting (bottom panel). See Supplementary Fig. S9 for uncropped data. Scale bars, 10 μm.


( a ) UVRAG C - terminal 270 - 442 region interacts with RINT - 1 . ( HEK293T ) were co - transfected with HA - RINT - 1 , together with Flag - UVRAG or its mutant derivatives , whole - lysates ( WCLs ) were immunoprecipitated ( IP ) with anti - Flag , followed by immunoblotting ( IB ) with an anti - HA antibody .

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(a) The UVRAG C-terminal 270-442 region interacts with RINT-1. Cells (HEK293T) were co-transfected with HA-RINT-1, together with Flag-UVRAG or its mutant derivatives, and whole-cell lysates (WCLs) were immunoprecipitated (IP) with anti-Flag, followed by immunoblotting (IB) with an anti-HA antibody.


c ) Interaction between endogenous UVRAG RINT - 1 . WCLs of HEK293T were used for immunoprecipitation with control serum ( control ) or an anti - UVRAG ( left ) or anti - RINT - 1 antibody ( right ) , followed by immunoblotting with indicated antibodies . bottom panel shows endogenous expression .

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c) Interaction between endogenous UVRAG and RINT-1. WCLs of HEK293T cells were used for immunoprecipitation with control serum (control) or an anti-UVRAG (left) or anti-RINT-1 antibody (right), followed by immunoblotting with the indicated antibodies. The bottom panel shows endogenous protein expression.


d ) ER localization of RINT - 1 , ZW10 UVRAG . HeLa were stained with anti - UVRAG ( green ) , anti - PDI ( ER marker , red ) , anti - ZW10 or anti - RINT - 1 ( blue ) , followed by confocal microscopy . Insets highlight UVRAG co - localization with RINT - 1 ZW10 at ER . Scale bars , 10 μm .

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d) ER localization of RINT-1, ZW10 and UVRAG. HeLa cells were stained with anti-UVRAG (green), anti-PDI (ER marker, red), and anti-ZW10 or anti-RINT-1 (blue), followed by confocal microscopy. Insets highlight UVRAG co-localization with RINT-1 and ZW10 at the ER. Scale bars, 10 μm.


( e ) Interaction between endogenous UVRAG RINT - 1 - ZW10 - NAG tethering . WCLs of HEK293T were used for immunoprecipitation with control serum ( IgG ) or an anti - UVRAG ( first panel ) , anti - RINT - 1 ( second panel ) , anti - ZW10 ( third panel ) anti - NAG ( fourth panel ) , followed by immunoblotting with indicated antibodies . As input , 10 % of WCLs was used . Syn 18 , syntaxin 18 ; Syn 6 , syntaxin 6 . See Supplementary Fig . S9 for uncropped data .

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(e) Interaction between endogenous UVRAG and the RINT-1-ZW10-NAG tethering complex proteins. WCLs of HEK293T cells were used for immunoprecipitation with control serum (IgG) or an anti-UVRAG (first panel), anti-RINT-1 (second panel), anti-ZW10 (third panel) and anti-NAG (fourth panel), followed by immunoblotting with the indicated antibodies. As input, 10% of the WCLs was used. Syn 18, syntaxin 18; Syn 6, syntaxin 6. See Supplementary Fig. S9 for uncropped data.


( a ) UVRAG knockdown impairs interaction of RINT - 1 - ZW10 with COPI in an autophagy - independent manner . ( HEK293T ) were transfected with control shRNA , UVRAG shRNA or Atg16L1 shRNA for 72 h . Whole - lysates ( WCLs ) were used for immunoprecipitation with anti - RINT - 1 ( first third panels ) , anti - ZW10 ( second panel ) , or anti - UVRAG ( fourth panel ) , followed by immunoblotting with indicated antibodies . Input represents 10 % whole - lysates . Note that β ′ - COP interaction was suppressed by UVRAG knockdown but unaffected by Atg16L1knockdown . See Supplementary Fig . S9 for uncropped data . (

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(a) UVRAG knockdown impairs the interaction of the RINT-1-ZW10 complex with COPI in an autophagy-independent manner. Cells (HEK293T) were transfected with control shRNA, UVRAG shRNA or Atg16L1 shRNA for 72 h. Whole-cell lysates (WCLs) were used for immunoprecipitation with anti-RINT-1 (first and third panels), anti-ZW10 (second panel), or anti-UVRAG (fourth panel), followed by immunoblotting with the indicated antibodies. Input represents 10% whole-cell lysates. Note that β′-COP interaction was suppressed by UVRAG knockdown but unaffected by Atg16L1knockdown. See Supplementary Fig. S9 for uncropped data. (


( b ) Retrograde transport of tsO45 - VSVG - KDELR - YFP from Golgi to ER is inhibited by UVRAG knockdown . HeLa serially transfected with UVRAG shRNA or control shRNA , then with VSVG - KDELR - YFP expression vector , were incubated at 32 ° C ( permissive condition ) overnight . On shifting to 40 ° C ( non - permissive condition ) , ER - like distribution pattern of VSVG - KDELR - YFP was registered . Representative confocal of distribution pattern of VSVG - KDELR at 40 ° C its co - localization with PDI are shown ( upper panel ) . Data are mean ± s . d . , n = 100 obtained from three independent experiments ; * * P0 . 01 ( bottom panel ) . Scale bars , 10 μm .

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(b) Retrograde transport of tsO45-VSVG-KDELR-YFP from the Golgi to the ER is inhibited by UVRAG knockdown. HeLa cells serially transfected with UVRAG shRNA or control shRNA, then with VSVG-KDELR-YFP expression vector, were incubated at 32°C (permissive condition) overnight. On shifting to 40°C (non-permissive condition), the ER-like distribution pattern of VSVG-KDELR-YFP was registered. Representative confocal images of the distribution pattern of VSVG-KDELR at 40°C and its co-localization with PDI are shown (upper panel). Data are mean±s.d., n = 100 cells obtained from three independent experiments; **P0.01 (bottom panel). Scale bars, 10 μm.


( c ) UVRAG interaction with bothPtdIns ( 3 ) P RINT - 1 is required for COPI - dependent retrograde transport , as shown by redistribution of VSVG - KDELR from Golgi to ER . HeLa were transfected with control shRNA or UVRAG shRNA . UVRAG - depleted were then complemented with empty vector , Flag - UVRAG , UVRAGΔ270 − 442 , UVRAGK78A / R82A or UVRAGΔC2 , along with transfection of VSVG - KDELR - YFP . ER pattern for chimaeric KDELR was quantified ( left panel ) . Data are mean ± s . d . ; n = 200 obtained from three independent experiments ; * P0 . 05 ; * * P0 . 01 . Endogenous reconstituted UVRAG expression was confirmed by immunoblotting ( right panel ) .

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

(c) UVRAG interaction with bothPtdIns(3)P and RINT-1 is required for COPI-dependent retrograde transport, as shown by the redistribution of VSVG-KDELR from the Golgi to the ER. HeLa cells were transfected with control shRNA or UVRAG shRNA. The UVRAG-depleted cells were then complemented with empty vector, Flag-UVRAG, UVRAGΔ270−442, UVRAGK78A/R82A or UVRAGΔC2, along with the transfection of VSVG-KDELR-YFP. The ER pattern for the chimaeric KDELR was quantified (left panel). Data are the mean±s.d.; n = 200 cells obtained from three independent experiments; *P0.05; **P0.01. Endogenous and reconstituted UVRAG expression was confirmed by immunoblotting (right panel).


( d ) PtdIns ( 3 ) Pdepletion inhibits Golgi - to - ER retrograde transport of VSVG - KDELR . HeLa were transfected with empty vector , wild - type MTMR3 , C413S mutant of MTMR3 , or treated with wortmannin or rapamycin , then ER pattern of VSVG - KDELR was quantified . UT , untreated . Data are mean ± s . d . ; n = 100 for each group obtained from three independent experiments ; * * P0 . 01 .

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(d) PtdIns(3)Pdepletion inhibits Golgi-to-ER retrograde transport of VSVG-KDELR. HeLa cells were transfected with empty vector, wild-type MTMR3, the C413S mutant of MTMR3, or treated with wortmannin or rapamycin, and then the ER pattern of VSVG-KDELR was quantified. UT, untreated. Data are the mean±s.d.; n = 100 cells for each group obtained from three independent experiments; **P0.01.


e ) Interaction between endogenous UVRAG ER tethering under wortmannin treatment . WCLs of HEK293T treated with wortmannin were used for immunoprecipitation with anti - UVRAG or anti - RINT - 1 antibody , followed by immunoblotting with indicated antibodies . input panel shows endogenous expression . See Supplementary Fig . S9 for uncropped data .

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e) Interaction between endogenous UVRAG and the ER tethering complex under wortmannin treatment. WCLs of HEK293T cells treated with wortmannin were used for immunoprecipitation with anti-UVRAG or anti-RINT-1 antibody, followed by immunoblotting with the indicated antibodies. The input panel shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.


( a ) Golgi on UVRAG depletion . HeLa were transfected with control or UVRAG - specific shRNA for 72 h subjected to electron microscopy ( left panel ) . When compared with control ( left ) , Golgi structure was swollen fragmented in lacking UVRAG . level of Golgi fragmentation was quantified ( right panel ) . Data represent mean ± s . d . ; n = 50 obtained by gathering data from two independent experiments ; * P0 . 05 . Scale bars , 500 nm .

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(a) Golgi morphology on UVRAG depletion. HeLa cells were transfected with control or UVRAG-specific shRNA for 72 h and subjected to electron microscopy analysis (left panel). When compared with control cells (left), the Golgi structure was swollen and fragmented in cells lacking UVRAG. The level of Golgi fragmentation was quantified (right panel). Data represent mean±s.d.; n = 50 cells obtained by gathering data from two independent experiments; *P0.05. Scale bars, 500 nm.


( b ) Depletion of UVRAG leads to cis - Golgi dispersion . HeLa were transfected with control shRNA or UVRAG - specific shRNA expressing GFP as an expression marker then stained for GM130 ( left panel ; red ; highlighted by arrows ) . Nuclei were stained with DAPI ( blue ) . dispersed distribution of GM130 in shRNA - transfected was quantified ( middle panel ) . Data are mean ± s . d . ; n = 500 obtained from five independent experiments . expression of UVRAG GM130 in treated is also shown ( right panel ) . Scale bars , 10 μm .

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(b) Depletion of UVRAG leads to cis-Golgi dispersion. HeLa cells were transfected with control shRNA or UVRAG-specific shRNA expressing GFP as an expression marker and then stained for GM130 (left panel; red; highlighted by arrows). Nuclei were stained with DAPI (blue). The dispersed distribution of GM130 in shRNA-transfected cells was quantified (middle panel). Data are the mean±s.d.; n = 500 cells obtained from five independent experiments. The expression of UVRAG and GM130 in treated cells is also shown (right panel). Scale bars, 10 μm.


( c ) effect of UVRAG on trafficking from ER to Golgi . HeLa serially transfected with UVRAG shRNA RINT - 1 shRNA or control shRNA , then with VSVG - GFP expression vector , were incubated at 40 ° C ( non - permissive condition ) overnight . were then shifted to 32 ° C ( permissive condition ) for 30 min to allow cargo transit from ER to Golgi . Before after shift , were fixed labelled with anti - PDI anti - GM130 antibodies to define ER Golgi region , respectively . Representative confocal microscopy of distribution pattern of retrograde cargo at 40 ° C 32 ° C are shown ( left panel ) . Golgi - like distribution pattern of VSVG - GFP was registered ( right panel ) . Data are mean ± s . d . ; n = 100 from three independent experiments ; * * P0 . 01 . Scale bars , 10 μm .

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

(c) The effect of UVRAG on membrane trafficking from the ER to the Golgi. HeLa cells serially transfected with UVRAG shRNA RINT-1 shRNA or control shRNA, then with VSVG-GFP expression vector, were incubated at 40°C (non-permissive condition) overnight. The cells were then shifted to 32°C (permissive condition) for 30 min to allow cargo transit from the ER to the Golgi. Before and after the temperature shift, the cells were fixed and labelled with anti-PDI and anti-GM130 antibodies to define the ER and Golgi region, respectively. Representative confocal microscopy images of the distribution pattern of the retrograde cargo at 40°C and 32°C are shown (left panel). The Golgi-like distribution pattern of VSVG-GFP was registered (right panel). Data are mean±s.d.; n = 100 cells from three independent experiments; **P0.01. Scale bars, 10 μm.


( d , e ) UVRAG interaction with PtdIns ( 3 ) P RINT - 1 is required or Golgi integrity . ( d ) HeLa were transfected with control shRNA ( first row ) or UVRAG shRNA ( second to sixth rows ) . UVRAG - depleted , as indicated by Flag expression , were then transfected with empty vector ( second row ) , UVRAGK78A / R82A - UVRAG ( third row ) , UVRAGΔ270 − 442mutant ( fourth row ) , UVRAGK78A / R82A mutant ( fifth row ) , or UVRAGΔCCD mutant ( sixth row ) , followed by confocal microscopy using anti - GM130 ( red ) for cis - Golgi , anti - Flag ( purple ) for ectopically expressed UVRAG expression , UVRAG for nuclei ( blue ) . Asterisks indicate shRNA - transfected arrows denote reconstituted with GM130 ( wild - type or mutant ) expression . ( e ) dispersed distribution of GM130 was quantified , data represent mean ± s . d . ; n = 200 from four independent experiments ; * P0 . 05 ; * * P0 . 01 .

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(d,e) UVRAG interaction with PtdIns(3)P and RINT-1 is required or Golgi integrity. (d) HeLa cells were transfected with control shRNA (first row) or UVRAG shRNA (second to sixth rows). The UVRAG-depleted cells, as indicated by Flag expression, were then transfected with empty vector (second row), UVRAGK78A/R82A-UVRAG (third row), UVRAGΔ270−442mutant (fourth row), UVRAGK78A/R82A mutant (fifth row), or UVRAGΔCCD mutant (sixth row), followed by confocal microscopy using anti-GM130 (red) for cis-Golgi staining, anti-Flag (purple) for the ectopically expressed UVRAG protein expression, and UVRAG for nuclei (blue). Asterisks indicate shRNA-transfected cells and arrows denote the cells reconstituted with GM130 (wild-type or mutant) expression. (e) The dispersed distribution of GM130 was quantified, and data represent the mean±s.d.; n = 200 cells from four independent experiments; *P0.05; **P0.01.


( a , b ) Differential interaction of UVRAG with RINT - 1 - ZW10 - NAG tethering beclin - 1 - Bif - 1 - PI ( 3 ) KC3 autophagy after treatment with rapamycin ( a ) HBSS ( b ) . ( HEK293T ) treated with rapamycin ( 50 nM ) in a or with HBSS in b were subjected to immunoprecipitation with anti - UVRAG ( first panel ) , anti - RINT - 1 ( second panel ) or anti - PI ( 3 ) KC3 ( third panel ) , followed by immunoblotting with indicated antibodies . Input ( 10 % ) shows endogenous expression . See Supplementary Fig . S9 for uncropped data .

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(a,b) Differential interaction of UVRAG with the RINT-1-ZW10-NAG tethering complex and the beclin-1-Bif-1-PI(3)KC3 autophagy complex after treatment with rapamycin (a) and HBSS (b). Cells (HEK293T) treated with rapamycin (50 nM) in a or with HBSS in b were subjected to immunoprecipitation with anti-UVRAG (first panel), anti-RINT-1 (second panel) or anti-PI(3)KC3 (third panel), followed by immunoblotting with the indicated antibodies. Input (10%) shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.


( c ) Gel filtration of UVRAG formation under normal conditions ( untreated , UT ) after rapamycin - induced autophagy ( 50 nM ) . Affinity - purified UVRAG complexes from HCT116 stably expressing Flag - UVRAG were fractionated by Superose - 6 gel filtration column eluates were analysed by western blotting for UVRAG , RINT - 1 , ZW10 , beclin 1 , PI ( 3 ) KC3 Bif - 1 . Whole - lysates ( 2 . 5 % ) were used as input ( lane - 1 ) . elution profile of each was quantified by densitometry normalized . Relative densitometry units were plotted against fraction number . Black arrows indicate positions of molecular markers . Red arrows indicate peak shift of UVRAG eluates .

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(c) Gel filtration analysis of UVRAG complex formation under normal conditions (untreated, UT) and after rapamycin-induced autophagy (50 nM). Affinity-purified UVRAG complexes from HCT116 cells stably expressing Flag-UVRAG were fractionated by Superose-6 gel filtration column and the eluates were analysed by western blotting for UVRAG, RINT-1, ZW10, beclin 1, PI(3)KC3 and Bif-1 proteins. Whole-cell lysates (2.5%) were used as the input (lane-1). The elution profile of each protein was quantified by densitometry analysis and normalized. Relative densitometry units were plotted against fraction number. Black arrows indicate the positions of the molecular weight size markers. Red arrows indicate the peak shift of the UVRAG eluates.


( a - c ) UVRAG interaction with beclin 1 PtdIns ( 3 ) P , but not with RINT - 1 , is required for autophagy - induced Atg9 dispersal . ( a ) HeLa were stably transfected with control shRNA ( first row ) or UVRAG shRNA ( second - sixth rows ) . UVRAG - depleted were then transfected with empty vector ( second row ) , Flag - UVRAG ( third row ) , UVRAGΔ270 − 442 mutant ( fourth row ) , UVRAGK78A / R82A mutant ( fifth row ) or UVRAGΔCCD mutant ( sixth row ) , incubated in complete medium ( untreated , UT , first panel ) , starvation medium ( HBSS 2 h , third panel ) , or treated with rapamycin ( 50 nM 2 h , second panel ) or SMER28 ( 50 μM , 2 h , fourth panel ) . distribution pattern of endogenous Atg9 ( green ) in UVRAG ( wild - type or mutant ) - transduced ( red ) was analysed by confocal microscopy ( a ) . Asterisks denote condensed ( non - dispersed ) Atg9 in UVRAG - depleted HeLa arrows denote complemented with wild - type or mutant UVRAG .

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(a-c) UVRAG interaction with beclin 1 and PtdIns(3)P, but not with RINT-1, is required for autophagy-induced Atg9 dispersal. (a) HeLa cells were stably transfected with control shRNA (first row) or UVRAG shRNA (second-sixth rows). The UVRAG-depleted cells were then transfected with empty vector (second row), Flag-UVRAG (third row), UVRAGΔ270−442 mutant (fourth row), UVRAGK78A/R82A mutant (fifth row) or UVRAGΔCCD mutant (sixth row), and incubated in complete medium (untreated, UT, first panel), starvation medium (HBSS 2 h, third panel), or treated with rapamycin (50 nM 2 h, second panel) or SMER28 (50 μM, 2 h, fourth panel). The distribution pattern of endogenous Atg9 (green) in the UVRAG (wild-type or mutant)-transduced cells (red) was analysed by confocal microscopy (a). Asterisks denote condensed (non-dispersed) Atg9 in UVRAG-depleted HeLa cells and arrows denote the cells complemented with wild-type or mutant UVRAG.


( b , c ) of with Atg9 translocation was quantied ( c ) expression of UVRAG actin was determined by immunoblotting ( b ) . Data represent means : d : ; n D300 from three independent experiments ; NS , not signicant ; P < 0 : 01 ; P < 0 : 001 . Scale bars , 10 m .

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(b,c) The percentage of cells with Atg9 translocation was quantied (c) and the expression of UVRAG and actin was determined by immunoblotting (b). Data represent the means:d:; n D300 cells from three independent experiments; NS, not signicant; P <0:01; P <0:001. Scale bars, 10 m.


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