Phase separation over time as monitored by turbidity of 50 μM MBP - N in 50 mM Tris pH 7 . 4 after addition of TEV protease A ) with varying torula yeast RNA ( at 100 mM NaCl ) or B ) varying NaCl concentrations ( at constant RNA concentration ) . Error bars represent standard deviation of three replicates .

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

Phase separation over time as monitored by turbidity of 50 μM MBP-N in 50 mM Tris pH 7.4 after addition of TEV protease A) with varying torula yeast RNA (at 100 mM NaCl) or B) varying NaCl concentrations (at constant RNA concentration). Error bars represent standard deviation of three replicates.


A ) DIC micrographs of 50 μM N and domain deletion variants in 50 mM Tris 70 mM NaCl pH 7 . 4 without and with TEV protease ( to cleave MBP from N ) and without or with 0 . 5 mg / mL desalted total torula yeast RNA . Scale bars represent 80 μm . Phase separation over time as monitored by turbidity of 50 μM full - length N or deletion variants after addition of TEV protease in the absence ( B ) or presence ( C ) of 0 . 5 mg / ml desalted total torula yeast RNA . Error bars represent standard deviation of three replicates .

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

A) DIC micrographs of 50 μM N and domain deletion variants in 50 mM Tris 70 mM NaCl pH 7.4 without and with TEV protease (to cleave MBP from N) and without or with 0.5 mg/mL desalted total torula yeast RNA. Scale bars represent 80 μm. Phase separation over time as monitored by turbidity of 50 μM full-length N or deletion variants after addition of TEV protease in the absence (B) or presence (C) of 0.5 mg/ml desalted total torula yeast RNA. Error bars represent standard deviation of three replicates.


A ) Turbidity of N is increased in the presence of torula yeast RNA and homopolymeric RNAs . Error bars represent standard deviation of three replicates . B ) DIC micrographs of MBP - N in the absence or presence of TEV ( to cleave MBP from N to initiate with indicated RNA . In the absence of TEV , MBP - N in the presence of torula yeast RNA , polyC , or polyU RNA does not undergo LLPS . However , after cleavage of MBP , N phase separates . polyA RNA increases LLPS of N . Both in the absence and presence of TEV , polyG RNA induces aggregation of MBP - N . Sample conditions : 50 µM MBP - N , 0 . 5 mg / mL RNA / polyX , 70 mM NaCl , 25 ˚ C , 50 mM Tris pH 7 . 4 . Scale bar represents 100 µm .

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

A) Turbidity of N is increased in the presence of torula yeast RNA and homopolymeric RNAs. Error bars represent standard deviation of three replicates. B) DIC micrographs of MBP-N in the absence or presence of TEV (to cleave MBP from N to initiate with indicated RNA. In the absence of TEV, MBP-N in the presence of torula yeast RNA, polyC, or polyU RNA does not undergo LLPS. However, after cleavage of MBP, N phase separates. polyA RNA increases LLPS of N. Both in the absence and presence of TEV, polyG RNA induces aggregation of MBP-N. Sample conditions:50 µM MBP-N, 0.5 mg/mL RNA/polyX, 70 mM NaCl, 25˚C, 50 mM Tris pH 7.4. Scale bar represents 100 µm.


A ) N partitions into hnRNPA2 LC droplets even with the MBP tag attached .

A) N partitions into hnRNPA2 LC droplets even with the MBP tag attached.


B ) N partitions into TDP - 43 CTD droplets even with the MBP tag attached .

B) N partitions into TDP-43 CTD droplets even with the MBP tag attached.


C ) N partitions into FUS LC droplets only after cleavage of the MBP tag .

C) N partitions into FUS LC droplets only after cleavage of the MBP tag.


D - F ) N partitions into hnRNPA2 FL ( D ) , TDP - 43 FL ( E ) , FUS FL ( F ) droplets .

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

D-F) N partitions into hnRNPA2 FL (D), TDP-43 FL (E), FUS FL (F) droplets.


( A - B ) Representative MA currents recorded in whole cell patch clamp experiments at - 60 mV in large DRG neurons in response to repetitive mechanical indentations with a blunt glass probe ; experiments were performed as described in the methods section . The applications of 25 μM baclofen ( B ) or vehicle ( A ) are indicated by the red horizontal lines . The downwards spikes represent individual MA currents induced by repetitive mechanical stimuli every 30 s for 200 ms stimulus duration .

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

(A-B) Representative MA currents recorded in whole cell patch clamp experiments at -60 mV in large DRG neurons in response to repetitive mechanical indentations with a blunt glass probe; experiments were performed as described in the methods section. The applications of 25 μM baclofen (B) or vehicle (A) are indicated by the red horizontal lines. The downwards spikes represent individual MA currents induced by repetitive mechanical stimuli every 30 s for 200 ms stimulus duration.


( C ) Quantification of normalized MA current amplitudes from DRG neurons , vehicle ( white , n = 7 ) , neurons responding to baclofen ( R , red ) ( n = 6 , * * P < 0 . 01 , Paired t - test and non - responding DRG neurons ( NR , blue , n = 6 ) . Striped column shows the average normalized current amplitudes 3 - 10 minutes after removal of baclofen in responding neurons ( Washout ) ( n = 6 ) . Data are shown as mean ± SEM and scatter plots .

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

(C) Quantification of normalized MA current amplitudes from DRG neurons, vehicle (white, n=7), neurons responding to baclofen (R, red) (n=6, **P<0.01, Paired t-test and non-responding DRG neurons (NR, blue, n=6). Striped column shows the average normalized current amplitudes 3-10 minutes after removal of baclofen in responding neurons (Washout) (n=6). Data are shown as mean ± SEM and scatter plots.


( D - E ) Representative MA currents recorded at - 60 mV in large DRG neurons in response to repetitive mechanical stimulation with a blunt glass probe displaced 2 - 6 µm in 0 . 4 µm increments every 15 s ( step protocol ) before ( black ) and after ( red ) exposure to 25 µM Baclofen . Not every trace is shown for clarity . Inset above the current traces show the mechanical step protocol indicating the displacement of the mechanical probe .

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

(D-E) Representative MA currents recorded at -60 mV in large DRG neurons in response to repetitive mechanical stimulation with a blunt glass probe displaced 2-6 µm in 0.4 µm increments every 15 s (step protocol) before (black) and after (red) exposure to 25 µM Baclofen. Not every trace is shown for clarity. Inset above the current traces show the mechanical step protocol indicating the displacement of the mechanical probe.


( F ) Quantification of normalized MA currents of responding large DRG neurons [ n = 13 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , before ( black ) and after ( red ) baclofen treatment . Dashed grey line shows average MA currents 3 - 15 minutes after washout of baclofen . Repeated measures ANOVA with student ' s t - test no corrections was used to compare baseline and baclofen treatment . Data are shown as mean ± SEM .

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

(F) Quantification of normalized MA currents of responding large DRG neurons [n=13, *P<0.05, **P<0.01, ***P<0.001, before (black) and after (red) baclofen treatment. Dashed grey line shows average MA currents 3-15 minutes after washout of baclofen. Repeated measures ANOVA with student's t-test no corrections was used to compare baseline and baclofen treatment. Data are shown as mean ± SEM.


( G ) Quantification of normalized MA currents of non - responding DRG neurons ( n = 9 ) . Data are shown as mean ± SEM .

(G) Quantification of normalized MA currents of non-responding DRG neurons (n=9). Data are shown as mean ± SEM.


( H - I ) Quantification of MA current inactivation rate ( Tau ) measured for responding ( R ) large DRG neurons ( n = 13 , * * P < 0 . 01 , Paired t - test ) and non - responding large ( NR ) DRG neurons ( n = 9 , NS , not significant ) before ( gray ) and after ( red ) baclofen application . Data are shown as mean ± SEM and scatter plots .

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

(H-I) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=13, **P<0.01, Paired t-test) and non-responding large (NR) DRG neurons (n=9, NS, not significant) before (gray) and after (red) baclofen application. Data are shown as mean ± SEM and scatter plots.


( A - B ) Representative MA currents recorded in whole cell patch clamp experiments at - 60 mV in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( for visualization ) in response to repetitive mechanical stimulation with a blunt glass probe displaced 2 - 5 . 6 µm in 0 . 4 μm increments every 15 s before ( black ) and after ( red ) exposure to 25 µM baclofen . Not every trace is shown for clarity .

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

(A-B) Representative MA currents recorded in whole cell patch clamp experiments at -60 mV in HEK293 cells transiently transfected with Piezo2 +GABAB1+ GABAB2 + GFP (for visualization) in response to repetitive mechanical stimulation with a blunt glass probe displaced 2-5.6 µm in 0.4 μm increments every 15 s before (black) and after (red) exposure to 25 µM baclofen. Not every trace is shown for clarity.


( C ) Quantification of normalized MA currents of HEK293 cells transfected with Piezo2 + GABAB1 + GABAB2 + GFP [ C , n = 13 , * P < 0 . 05 , * * P < 0 . 01 , before ( black ) and after ( red ) baclofen treatment , Repeated measures ANOVA with student ' s t - test no corrections ] . Data are shown as mean ± SEM .

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

(C) Quantification of normalized MA currents of HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP [C, n=13, *P<0.05, **P<0.01, before (black) and after (red) baclofen treatment, Repeated measures ANOVA with student's t-test no corrections]. Data are shown as mean ± SEM.


( D ) Quantification of MA current inactivation rate ( Tau ) in HEK293 cells transfected with Piezo2 + GABAB1 + GABAB2 + GFP before ( gray ) and after ( red ) baclofen application ( n = 13 , NS , not significant ; Paired t - test ) . Data are shown as mean ± SEM and scatter plots .

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

(D) Quantification of MA current inactivation rate (Tau) in HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP before (gray) and after (red) baclofen application (n=13, NS, not significant; Paired t-test). Data are shown as mean ± SEM and scatter plots.


Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GFP ( E , n = 7 ) before ( black ) and after ( red ) application of 25 µM baclofen . Data are shown as mean ± SEM .

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

Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GFP (E, n=7) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.


Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GABAB1 + GABAB2 + βARKct - mCherry ( F , n = 8 ) before ( black ) and after ( red ) application of 25 µM baclofen . Data are shown as mean ± SEM .

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

Quantification of normalized MA currents of HEK293 cells transfected Piezo2+GABAB1+ GABAB2 + βARKct-mCherry (F, n=8) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.


Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + D2RS - YFP ( A , n = 13 ) before ( black ) and after ( red ) application of quinpirole 200 nM . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM .

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

Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+D2RS-YFP (A, n=13) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.


Quantification of normalized MA Piezo2 currents in HEK293 cells transfected with Piezo2 and GFP ( B , n = 5 ) before ( black ) and after ( red ) application of quinpirole 200 nM . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM .

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

Quantification of normalized MA Piezo2 currents in HEK293 cells transfected with Piezo2 and GFP (B, n=5) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.


( C ) Quantification of Piezo2 current inactivation rate ( Tau ) in HEK cells transfected with Piezo2 + D2RS - YFP . Data are shown as mean ± SEM and scatter plots ( n = 13 )

O O O O O O O O O O O O O O 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) Quantification of Piezo2 current inactivation rate (Tau) in HEK cells transfected with Piezo2+D2RS-YFP. Data are shown as mean ± SEM and scatter plots (n=13)


( D , E ) Quantification of normalized MA currents in responding large DRG neurons ( D , n = 5 ) versus non - responding DRG neurons ( E , n = 3 ) before ( black ) and after ( red ) application of sumatriptan 10 µM to activate the Gi - coupled Htr1d receptors . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM .

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

(D,E) Quantification of normalized MA currents in responding large DRG neurons (D, n=5) versus non-responding DRG neurons (E, n=3) before (black) and after (red) application of sumatriptan 10 µM to activate the Gi-coupled Htr1d receptors. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.


( F ) Quantification of MA current inactivation rate ( Tau ) measured for responding ( R ) large DRG neurons ( n = 5 , Paired t - test ) . Data are shown as mean ± SEM and scatter plots .

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

(F) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=5, Paired t-test). Data are shown as mean ± SEM and scatter plots.


( G - H ) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng / ml Gβγ ( G , n = 7 ) or with 0 . 0001 % Lubrol ( vehicle for Gβγ ) ( H , n = 6 ) . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM .

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

(G-H) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng/ml Gβγ (G, n=7) or with 0.0001% Lubrol (vehicle for Gβγ) (H, n=6). *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.


( I , J ) Quantification of MA current inactivation rate ( Tau ) measured for large DRG neurons dialyzed with Gβγ ( n = 7 , * * P < 0 . 01 , Paired t - test ) and vehicle ( Paired t - test ) . Data are shown as mean ± SEM and scatter plots .

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

(I,J) Quantification of MA current inactivation rate (Tau) measured for large DRG neurons dialyzed with Gβγ (n=7, **P<0.01, Paired t-test) and vehicle (Paired t-test). Data are shown as mean ± SEM and scatter plots.


Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( A , n = 12 )

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

Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12)


Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( A , n = 12 ) , treated with the PI3K inhibitor Wortmannin 50 nM ( B , n = 10 ) Cells were treated with wortmannin 30 min before the experiments , and were present throughout the measurements .

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

Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the PI3K inhibitor Wortmannin 50 nM (B, n=10) Cells were treated with wortmannin 30 min before the experiments, and were present throughout the measurements.


Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( A , n = 12 ) , treated with the MAPK inhibitor U0126 10 µM ( C , n = 7 ) . Cells were treated with U0126 for 30 min before the experiments , and were present throughout the measurements .

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

Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the MAPK inhibitor U0126 10 µM (C, n=7). Cells were treated with U0126 for 30 min before the experiments, and were present throughout the measurements.


Mice were injected in the plantar surface of the hind paw with sumatriptan , or vehicle ± the combination of wortmannin and U0126 , as described in the methods section . Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order . ( A - C ) Percentage of withdrawal response for female mice using von Frey filaments at : 0 . 07 g ( A ) , 0 . 16 g ( B ) and 0 . 4 g ( C ) . ( n = 11 - 16 per group , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One way - ANOVA with Tukey ' s multiple comparison test ) . Data are shown as mean ± SEM and scatter plots .

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

Mice were injected in the plantar surface of the hind paw with sumatriptan, or vehicle ± the combination of wortmannin and U0126, as described in the methods section. Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order. (A-C) Percentage of withdrawal response for female mice using von Frey filaments at: 0.07 g (A), 0.16 g (B) and 0.4 g (C). (n= 11-16 per group, ***P<0.001, ****P<0.0001, One way- ANOVA with Tukey's multiple comparison test). Data are shown as mean ± SEM and scatter plots.


Mice were injected in the plantar surface of the hind paw with sumatriptan , or vehicle ± the combination of wortmannin and U0126 , as described in the methods section . Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order . ( D - F ) Percentage of withdrawal response for male mice using von Frey filaments at : 0 . 07 g ( D ) , 0 . 16 g ( E ) and 0 . 4 g ( F ) . ( n = 12 - 16 per group , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One way - ANOVA with Tukey ' s multiple comparison test ) . Data are shown as mean ± SEM and scatter plots .

Mice were injected in the plantar surface of the hind paw with sumatriptan, or vehicle ± the combination of wortmannin and U0126, as described in the methods section. Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order. (D-F) Percentage of withdrawal response for male mice using von Frey filaments at: 0.07 g (D), 0.16 g (E) and 0.4 g (F). (n= 12-16 per group, ***P<0.001, ****P<0.0001, One way- ANOVA with Tukey's multiple comparison test). Data are shown as mean ± SEM and scatter plots.


B . The ER marker ER - tdTomato ( magenta ) is co - localized with ectopically expressed Wrb - EGFP ( green ) in inner saccular HCs of 5 - dpf zebrafishlarvae ( ' control ' refers to either wild - type or + / pwi fish that were picked in a phenotypic screen and did not exert any abnormal behavioral phenotype ) . While the left HC solely expressed ER - tdTomato , the neighboring HC expressed both , ER - tdTomato and Wrb - GFP . Colocalization between both proteins occurs in areas exhibiting white pixels . Scale bars : 5

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The ER marker ER-tdTomato (magenta) is co-localized with ectopically expressed Wrb-EGFP (green) in inner saccular HCs of 5-dpf zebrafishlarvae ('control' refers to either wild-type or +/pwi fish that were picked in a phenotypic screen and did not exert any abnormal behavioral phenotype). While the left HC solely expressed ER-tdTomato, the neighboring HC expressed both, ER-tdTomato and Wrb-GFP. Colocalization between both proteins occurs in areas exhibiting white pixels. Scale bars: 5 µm


C . Projection of confocal sections of control inner ear HCs immunolabelled for otoferlin ( magenta ) and expressing an EGFP - tagged truncated Wrb fragment containing only the cytosolic coiled - coiled domains ( Wrbcc - EGFP , green ) . Wrbcc - EGFP distribution was diffuse and found throughout the HC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Projection of confocal sections of control inner ear HCs immunolabelled for otoferlin (magenta) and expressing an EGFP-tagged truncated Wrb fragment containing only the cytosolic coiled-coiled domains (Wrbcc-EGFP, green). Wrbcc-EGFP distribution was diffuse and found throughout the HC.


D . Projection of confocal sections of inner ear HCs of 5 - dpf control ( D ) and Wrb - deficient pwi mutant fish ( wrb pwi / pwi ; D ' ) , showing strongly reduced otoferlin immunofluorescence in the mutant HCs . The color look - up table used represents higher pixel intensities with warmer colors .

D. Projection of confocal sections of inner ear HCs of 5-dpf control (D) and Wrb-deficient pwi mutant fish (wrb pwi/pwi; D'), showing strongly reduced otoferlin immunofluorescence in the mutant HCs. The color look-up table used represents higher pixel intensities with warmer colors.


E . A representative transgenically - rescued pwi - mutant HC ( white arrow ) , expressing Wrb - GFP , exhibits a strongly increased otoferlin signal ( magenta ) in direct comparison to the neighboring non - rescued mutant HCs . ( E ' ) Same image as in E but intensity - coded for otoferlin fluorescence .

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

E. A representative transgenically-rescued pwi-mutant HC (white arrow), expressing Wrb-GFP, exhibits a strongly increased otoferlin signal (magenta) in direct comparison to the neighboring non-rescued mutant HCs. (E') Same image as in E but intensity-coded for otoferlin fluorescence.


F . A representative control HC expressing Wrbcc - EGFP ( white arrow ) , immunolabelled for otoferlin ( magenta ) . The Wrbcc - EGFP - expressing HC shows significantly less otoferlin ( F ' ) , suggesting a dominant negative effect of Wrbcc in otoferlin biogenesis . The transfected HC is encircled with a dashed line .

F. A representative control HC expressing Wrbcc-EGFP (white arrow), immunolabelled for otoferlin (magenta). The Wrbcc-EGFP-expressing HC shows significantly less otoferlin (F'), suggesting a dominant negative effect of Wrbcc in otoferlin biogenesis. The transfected HC is encircled with a dashed line.


G . Quantification otoferlin downregulation of Wrbcc - EGFP shown in F . Otoferlin immunofluorescence intensity of Wrbcc - EGFP transfected inner ear HCs n = 22 , from three 5 - dpf control larva ) showed significant decrease of ~ 43 % compared to the adjacent Wrbcc - EGFP negative wild - type HCs ( n = 22 , from the same larva ) . Fluorescent intensity values were normalized with the average value of Wrbcc - EGFP negative HCs . * * p < 0 .

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

G. Quantification otoferlin downregulation of Wrbcc-EGFP shown in F. Otoferlin immunofluorescence intensity of Wrbcc-EGFP transfected inner ear HCs n = 22, from three 5-dpf control larva) showed significant decrease of ~43% compared to the adjacent Wrbcc-EGFP negative wild-type HCs (n = 22, from the same larva). Fluorescent intensity values were normalized with the average value of Wrbcc-EGFP negative HCs. **p < 0.01


H . Quantification of otoferlin immunohistochemistry data shown in ( D ) from hair cells of control ( n = 88 HCs from three 5 - dpf larva ) , wrb pwi / pwi ( n = 80 HCs from 3 sibling larva ) normalized against the mean intensity value of the control group and Wrb - GFP transfected wrb pwi / pwi zebrafish inner ears . Mutant HCs showed ~ 82 % reduction in otoferlin fluorescence intensity when compared with control HCs imaged under the same conditions . Otoferlin fluorescent intensity of Wrb - EGFP transfected mutant HCs ( n = 24 , from three , 5 - dpf larva ) showed significant increases compared to adjacent Wrb - EGFP negative mutant HCs ( n = 24 from the same larva ) . Fluorescent intensity values were normalized with the average value of Wrb - EGFP negative HCs . * * * p < 0 .

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

H. Quantification of otoferlin immunohistochemistry data shown in (D) from hair cells of control (n = 88 HCs from three 5-dpf larva), wrb pwi/pwi (n = 80 HCs from 3 sibling larva) normalized against the mean intensity value of the control group and Wrb-GFP transfected wrb pwi/pwi zebrafish inner ears. Mutant HCs showed ~82% reduction in otoferlin fluorescence intensity when compared with control HCs imaged under the same conditions. Otoferlin fluorescent intensity of Wrb-EGFP transfected mutant HCs (n = 24, from three, 5-dpf larva) showed significant increases compared to adjacent Wrb-EGFP negative mutant HCs (n = 24 from the same larva). Fluorescent intensity values were normalized with the average value of Wrb-EGFP negative HCs. ***p<0.001


I - I ' . Acoustic startle reflex measurements of ( I ) live , intact , 5 - dpf control ( n = 20 ) and wrb pwi / pwi ( n = 18 ) zebrafishlarva , where almost 100 % of the larva responded to five successive acoustic stimuli at 3 seconds intervals . In wrb pwi / pwi animals , only 39 % of larva responded to the first stimulus , thereafter , the number of responding larva decreased rapidly to less than 6 % . ( I ' ) Acoustic startle reflexes of 5 - dpf zebrafish larva after injection with either EGFP capped mRNA ( control , n = 16 , or wrb pwi / pwi n = 17 ) , Wrb - EGFP capped mRNA ( control , n = 20 , or wrb pwi / pwi n = 20 ) or two different concentrations of otoferlin mRNA ( 1x : control , n = 13 ; wrb pwi / pwi n = 11 ; 2x : control , n = 16 ; wrb pwi / pwi n = 6 ) . In contrast to EGFP mRNA injection , Wrb - EGFP as well as otoferlin mRNAs could partially rescue the acoustic startle reflex in the mutants , while not displaying detrimental effects on overall zebrafish morphology and development . Please note the dose - dependent effect of otoferlin mRNA injection . Similar results as in 1x EGFP mRNA were also obtained from injection of 2X EGFP mRNA as control ( data not shown ) . Measurements from two ( 2x otoferlin ) or three ( all other ) different experiments were compiled in the graph .

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

I-I'. Acoustic startle reflex measurements of (I) live, intact, 5-dpf control (n = 20) and wrb pwi/pwi (n = 18) zebrafishlarva, where almost 100% of the larva responded to five successive acoustic stimuli at 3 seconds intervals. In wrb pwi/pwi animals, only 39% of larva responded to the first stimulus, thereafter, the number of responding larva decreased rapidly to less than 6%. (I') Acoustic startle reflexes of 5-dpf zebrafish larva after injection with either EGFP capped mRNA (control, n = 16, or wrb pwi/pwi n = 17), Wrb-EGFP capped mRNA (control, n = 20, or wrb pwi/pwi n = 20) or two different concentrations of otoferlin mRNA (1x: control, n = 13; wrb pwi/pwi n = 11; 2x: control, n = 16; wrb pwi/pwi n = 6). In contrast to EGFP mRNA injection, Wrb-EGFP as well as otoferlin mRNAs could partially rescue the acoustic startle reflex in the mutants, while not displaying detrimental effects on overall zebrafish morphology and development. Please note the dose-dependent effect of otoferlin mRNA injection. Similar results as in 1x EGFP mRNA were also obtained from injection of 2X EGFP mRNA as control (data not shown). Measurements from two (2x otoferlin) or three (all other) different experiments were compiled in the graph.


A . HZZ - OTOFop , carrying a C - terminal glycosylation site ( opsin tag ) was purified alone or in complex with wild - type or an ATPase - deficient mutant version of TRC40 and incubated in the absence or presence of ER - derived rough microsomes ( RM ) . Membrane integration ( glycosylation ) was monitored by SDS - PAGE and immunoblot using an anti - opsin antibody . Where indicated , EndoH was used to remove N - linked oligosaccharides .

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

A. HZZ-OTOFop, carrying a C-terminal glycosylation site (opsin tag) was purified alone or in complex with wild-type or an ATPase-deficient mutant version of TRC40 and incubated in the absence or presence of ER-derived rough microsomes (RM). Membrane integration (glycosylation) was monitored by SDS-PAGE and immunoblot using an anti-opsin antibody. Where indicated, EndoH was used to remove N-linked oligosaccharides.


B . HZZ - OTOFop in complex with wild - type or mutant TRC40 was incubated with RM in the presence or absence of ATP and membrane insertion was monitored by opsin - specific immunoblot .

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

B. HZZ-OTOFop in complex with wild-type or mutant TRC40 was incubated with RM in the presence or absence of ATP and membrane insertion was monitored by opsin-specific immunoblot.


C . HZZ - OTOFop in complex with wild - type TRC40 was incubated in the presence of RM , trypsin - treated rough microsomes ( TRM ) or in the presence of WRBcc or CAMLcyt . Membrane insertion was monitored by opsin - specific immunoblot .

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

C. HZZ-OTOFop in complex with wild-type TRC40 was incubated in the presence of RM, trypsin-treated rough microsomes (TRM) or in the presence of WRBcc or CAMLcyt. Membrane insertion was monitored by opsin-specific immunoblot.


D . Quantification of relative protein glycosylation shown in B ( n = 3 ) . Data are represented as mean ± SEM . TRC40gr , mutated version of TRC40 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( Student ' s two - sample t - test ) .

D. Quantification of relative protein glycosylation shown in B (n = 3). Data are represented as mean ± SEM. TRC40gr, mutated version of TRC40; **p<0.01; ***p<0.001 (Student's two-sample t-test).


E . Quantification of relative protein glycosylation of the data shown in C ( n = 3 ) . Data are represented as mean ± SEM . * * * p < 0 . 001 ( Student ' s two - sample t - test )

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

E. Quantification of relative protein glycosylation of the data shown in C (n = 3). Data are represented as mean ± SEM. ***p<0.001 (Student's two-sample t-test)


A . Grand averages of auditory brainstem responses ( ABR ) from Wrbfl / fl : CreA ( red traces ; SEM pink ) and Wrb + / + : CreA ( black traces ; SEM grey ) mice . ABRs were recorded for three separate age groups ( A - A ' ' , as indicated in the graph ) using click stimulation at 80 dB ( peak equivalent ) and 20 Hz stimulation rate . There was an age - progressive reduction of ABR amplitude in Wrbfl / fl : CreA mice .

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

A. Grand averages of auditory brainstem responses (ABR) from Wrbfl/fl:CreA (red traces; SEM pink) and Wrb+/+:CreA (black traces; SEM grey) mice. ABRs were recorded for three separate age groups (A-A'', as indicated in the graph) using click stimulation at 80 dB (peak equivalent) and 20 Hz stimulation rate. There was an age- progressive reduction of ABR amplitude in Wrbfl/fl:CreA mice.


B . ABRs from Wrbfl / fl : CreA mice showed a progressive threshold increase , Wrb + / + : CreA mice and Wrbfl / fl mice lacking Cre recombinase had normal thresholds . Statistical comparison of the threshold at 12 kHz was done by Kruskal - Wallis test , p values are from post - hoc Dunn ' s multiple comparison test . Measurements in which no ABR was observed at the maximal available tone burst level ( 90 dB ) or click level ( 120 dB ) scored as 100 dB threshold . Data are represented as mean ± SEM .

B. ABRs from Wrbfl/fl:CreA mice showed a progressive threshold increase, Wrb+/+:CreA mice and Wrbfl/fl mice lacking Cre recombinase had normal thresholds. Statistical comparison of the threshold at 12 kHz was done by Kruskal-Wallis test, p values are from post-hoc Dunn's multiple comparison test. Measurements in which no ABR was observed at the maximal available tone burst level (90 dB) or click level (120 dB) scored as 100 dB threshold. Data are represented as mean ± SEM.


A - D . Representative maximum projections of confocal sections of ( A - B ) P14 and ( C - D ) P20 Wrb + / + : CreA and Wrbfl / fl : CreA apical turn organs of Corti following immunolabeling against otoferlin ( green ) and Vglut3 ( magenta ) processed and imaged under identical conditions . A ' - D ' show individual otoferlin stainings with an intensity - coded lookup table . A ' ' - D ' ' present single xz projections at a central point through a representative IHC to illustrate otoferlin subcellular distribution . Scale bars in A - D ' represent 10 μm , in A ' ' - D ' ' 2 µm . Note the dramatic reduction in otoferlin fluorescence and the alteration in its distribution as well as a change in cell shape and position of nuclei ( for detailed analysis of cell shape and nuclei position , please refer to Figure EV5 ) .

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

A-D. Representative maximum projections of confocal sections of (A-B) P14 and (C-D) P20 Wrb+/+:CreA and Wrbfl/fl:CreA apical turn organs of Corti following immunolabeling against otoferlin (green) and Vglut3 (magenta) processed and imaged under identical conditions. A'-D' show individual otoferlin stainings with an intensity-coded lookup table. A''-D'' present single xz projections at a central point through a representative IHC to illustrate otoferlin subcellular distribution. Scale bars in A-D' represent 10 μm, in A''-D'' 2 µm. Note the dramatic reduction in otoferlin fluorescence and the alteration in its distribution as well as a change in cell shape and position of nuclei (for detailed analysis of cell shape and nuclei position, please refer to Figure EV5).


E - G . Semiquantitative analysis of otoferlin immunofluorescence in the apical parts of IHCs from Wrbfl / fl : CreA mice using averaged regions of interests in cell ' s maximal projections ( outlined with a dotted line in G for a single representative IHC ) normalized to the maximum values observed in the respective control IHCs . Averaged supra nuclear coronal line profiles as illustrated with a dashed line in G are shown for ( F ) P14 - 16 and ( F ' ) P18 - 21 IHCs . * * * p < 0 . 001 versus Wrb + / + :

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

E-G. Semiquantitative analysis of otoferlin immunofluorescence in the apical parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell's maximal projections (outlined with a dotted line in G for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supra nuclear coronal line profiles as illustrated with a dashed line in G are shown for (F) P14-16 and (F') P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreA


H - J . Semiquantitative analysis of otoferlin immunofluorescence in the basal parts of IHCs from Wrbfl / fl : CreA mice using averaged regions of interests in cell ' s maximal projections ( outlined with a dotted line in J for a single representative IHC ) normalized to the maximum values observed in the respective control IHCs . Averaged supranuclear coronal line profiles as illustrated with a dashed line in G are shown for ( I ) P14 - 16 and ( I ' ) P18 - 21 IHCs . * * * p < 0 . 001 versus Wrb + / + :

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

H-J. Semiquantitative analysis of otoferlin immunofluorescence in the basal parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell's maximal projections (outlined with a dotted line in J for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supranuclear coronal line profiles as illustrated with a dashed line in G are shown for (I) P14-16 and (I') P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreA


A . Representative electron micrograph of a Wrb + / + : CreA IHC ribbon synapse . Scale bar : 120 nm .

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

A. Representative electron micrograph of a Wrb+/+:CreA IHC ribbon synapse. Scale bar: 120 nm.


B - B ' . In Wrbfl / fl : CreA ( depicted are two consecutive ultrathin sections ) IHCs , accumulations of large partially amorphous vesicles were observed close to ribbon synapses ( red arrows ) , but also further away in the cytoplasm ( cyan arrows ) . Moreover , cisternal structures resembling ( r ) ER are found unusually close to the ribbon ( orange arrow in B ' ) . Scale bar : 120 nm .

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

B-B'. In Wrbfl/fl:CreA (depicted are two consecutive ultrathin sections) IHCs, accumulations of large partially amorphous vesicles were observed close to ribbon synapses (red arrows), but also further away in the cytoplasm (cyan arrows). Moreover, cisternal structures resembling (r)ER are found unusually close to the ribbon (orange arrow in B'). Scale bar: 120 nm.


E . Quantification in random ultrathin sections ( Wrb + / + : CreA n = 28 , from two animals ; Wrbfl / fl : CreA n = 44 , from three animals ) of the numbers of membrane - proximal " MP " vesicles ( in close proximity to membrane and ribbon ) ; vesicles within a 100 nm range along the active zone membrane ; vesicles at the lower " proximal " and the upper " distal " half of the ribbon as well as vesicle > 70 nm ( " large vesicles " ) in a radius of 800 nm around the ribbon . The vesicle number was significantly reduced in Wrbfl / fl : CreA IHCs at both ribbon parts but not at the presynaptic membrane . F . Average vesicle diameters of the membrane - proximal vesicles and the vesicles at both halves of the ribbon . The mean vesicle diameter increases significantly at both parts of the ribbon in Wrbfl / fl : CreA IHCs . Data are represented as mean ± SEM . Student ' s two - sample t - test * * * p < 0 .

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

E. Quantification in random ultrathin sections (Wrb+/+:CreA n = 28, from two animals; Wrbfl/fl:CreA n = 44, from three animals) of the numbers of membrane-proximal "MP" vesicles (in close proximity to membrane and ribbon); vesicles within a 100 nm range along the active zone membrane; vesicles at the lower "proximal" and the upper "distal" half of the ribbon as well as vesicle >70 nm ("large vesicles") in a radius of 800 nm around the ribbon. The vesicle number was significantly reduced in Wrbfl/fl:CreA IHCs at both ribbon parts but not at the presynaptic membrane.F. Average vesicle diameters of the membrane-proximal vesicles and the vesicles at both halves of the ribbon. The mean vesicle diameter increases significantly at both parts of the ribbon in Wrbfl/fl:CreA IHCs. Data are represented as mean ± SEM. Student's two-sample t-test ***p<0.001


A . Ca2 + current - voltage relationship of P14 - P17 IHCs of Wrbfl / fl : CreA ( n = 17 IHCs ) and Wrb + / + : CreA ( n = 15 IHCs ) mice in 2 mM extracellular [ Ca2 + ] showed no change in amplitude or voltage - dependence of the Ca2 + current . Data are represented as mean ± SEM .

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

A. Ca2+ current-voltage relationship of P14-P17 IHCs of Wrbfl/fl:CreA (n = 17 IHCs) and Wrb+/+:CreA (n = 15 IHCs) mice in 2 mM extracellular [Ca2+] showed no change in amplitude or voltage-dependence of the Ca2+ current. Data are represented as mean ± SEM.


B . Representative Ca2 + current ( top ) and Cm changes ( bottom ) in Wrbfl / fl : CreA and Wrb + / + : CreA IHCs in response to a 20 - ms step depolarizations to - 14 mV ( the potential eliciting the maximum Ca2 + current ) .

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

B. Representative Ca2+ current (top) and Cm changes (bottom) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 20-ms step depolarizations to -14 mV (the potential eliciting the maximum Ca2+ current).


C . Representative Ca2 + current ( top ) and Cm changes ( bottom ; finite impulse response filtered ) in Wrbfl / fl : CreA and Wrb + / + : CreA IHCs in response to a 100 ms step depolarizations to - 14 mV .

O O O B-MEASURED_VAR I-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

C. Representative Ca2+ current (top) and Cm changes (bottom; finite impulse response filtered) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 100 ms step depolarizations to -14 mV.


D . Exocytic ΔCm ( top ) and corresponding Ca2 + current integrals , QCa ( bottom ) for various depolarization durations . Data are grand averages of the cells ' means . Exocytic ΔCm was reduced in the knockout from 20 ms onwards , indicating reduced sustained exocytosis . Data from OtofPga / Pga animals were replotted for direct comparison from ( Pangrsic et al , 2010 ) .

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

D. Exocytic ΔCm (top) and corresponding Ca2+ current integrals, QCa (bottom) for various depolarization durations. Data are grand averages of the cells' means. Exocytic ΔCm was reduced in the knockout from 20 ms onwards, indicating reduced sustained exocytosis. Data from OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).


E . ΔCm / QCa2 + ratio indicates a lower efficacy of Ca2 + influx in driving exocytosis in Wrbfl / fl : CreA IHCs , for stimuli of 20 ms or longer . Data represents means ± SEM ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

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E. ΔCm/QCa2+ ratio indicates a lower efficacy of Ca2+ influx in driving exocytosis in Wrbfl/fl:CreA IHCs, for stimuli of 20 ms or longer. Data represents means ± SEM; *p<0.05; **p<0.01; ***p<0.001


A . All SGNs of Wrbfl / fl : CreA mice had spontaneous firing rates ( SR ) below 20 Hz ( p > 0 . 05 , Kolmogorow - Smirnov test ) .

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

A. All SGNs of Wrbfl/fl:CreA mice had spontaneous firing rates (SR) below 20 Hz (p>0.05, Kolmogorow-Smirnov test).


B . Representative tuning curves from Wrbfl / fl : CreA and Wrb + / + : CreA SGNs indicating preserved active cochlear amplification despite disruption of Wrb in IHCs .

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

B. Representative tuning curves from Wrbfl/fl:CreA and Wrb+/+:CreA SGNs indicating preserved active cochlear amplification despite disruption of Wrb in IHCs.


C . Normal thresholds at the characteristic frequency ( CF ) in Wrbfl / fl : CreA SGNs .

C. Normal thresholds at the characteristic frequency (CF) in Wrbfl/fl:CreA SGNs.


D - D ' ' . Mean poststimulus time histograms ( PSTH ) ± SEM of Wrbfl / fl : CreA and Wrb + / + : CreA SGNs in response to 50 ms tone bursts presented at CF , 30 dB above threshold at stimulus rates of 2 Hz ( D ) , 5 Hz ( D ' ) , or 10 Hz ( D ' ' ) . While the general response pattern was preserved , spike rates were drastically reduced in Wrbfl / fl : CreA SGNs , especially at higher stimulus rates . Data from auditory neurons ( SGN and cochlear nucleus ) of OtofPga / Pga animals were replotted for direct comparison from ( Pangrsic et al , 2010 ) .

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

D-D''. Mean poststimulus time histograms (PSTH) ± SEM of Wrbfl/fl:CreA and Wrb+/+:CreA SGNs in response to 50 ms tone bursts presented at CF, 30 dB above threshold at stimulus rates of 2 Hz (D), 5 Hz (D'), or 10 Hz (D''). While the general response pattern was preserved, spike rates were drastically reduced in Wrbfl/fl:CreA SGNs, especially at higher stimulus rates. Data from auditory neurons (SGN and cochlear nucleus) of OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).


E . Spike rates in response to sound onset ( maximum rate in PSTH with 0 . 5 ms binwidth ) and adapted rates ( averaged between 35 - 45 ms after stimulus onset ) were significantly lower in Wrbfl / fl : CreA SGNs ( stimulus rate 5 Hz ; p < 0 . 001 ) .

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

E. Spike rates in response to sound onset (maximum rate in PSTH with 0.5 ms binwidth) and adapted rates (averaged between 35-45 ms after stimulus onset) were significantly lower in Wrbfl/fl:CreA SGNs (stimulus rate 5 Hz; p<0.001).


F . In line with the reduction in spike rates , first spike latency ( FSL ) following stimulus onset was greatly increased in Wrbfl / fl : CreA SGNs compared to Wrb + / + : CreA ( stimulus rate 5 Hz ; p < 0 . 001 ) .

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

F. In line with the reduction in spike rates, first spike latency (FSL) following stimulus onset was greatly increased in Wrbfl/fl:CreA SGNs compared to Wrb+/+:CreA (stimulus rate 5 Hz; p<0.001).


G . Illustration of the stimulus paradigm for forward masking experiments : a 100 ms masker tone presented at CF , 30 dB above threshold was followed by a silent interval of variable duration and a 15 ms probe tone ( CF , 30 dB above threshold ) . Bottom : in Wrbfl / fl : CreA SGNs , the response to the first 5 ms of the masker probe tone tone ( shown as a fraction of the response to the first 5ms of the masker ) was strongly reduced , and the half time of recovery ( from normalized curves ) was increased from 39 . 2 ± 12 . 2 ms in Wrb + / + : CreA to 215 . 7 ± 51 . 2 ms in Wrbfl / fl : CreA SGNs ( p < 0 . 001 , Mann - Whitney U test ) . Data represent means ± SEM .

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G. Illustration of the stimulus paradigm for forward masking experiments: a 100 ms masker tone presented at CF, 30 dB above threshold was followed by a silent interval of variable duration and a 15 ms probe tone (CF, 30 dB above threshold). Bottom: in Wrbfl/fl:CreA SGNs, the response to the first 5 ms of the masker probe tone tone (shown as a fraction of the response to the first 5ms of the masker) was strongly reduced, and the half time of recovery (from normalized curves) was increased from 39.2 ± 12.2 ms in Wrb+/+:CreA to 215.7 ± 51.2 ms in Wrbfl/fl:CreA SGNs (p<0.001, Mann-Whitney U test). Data represent means ± SEM.


C . Apparent decrease in insertion efficiency for the TMD mutant version . While the WT otoferlin glycosylation amounted to 3 . 84 ± 0 . 53 , the TMD mutant had only 2 . 13 ± 0 . 65 ( n = 3 , p < 0 . 05 ) . Data are represented as mean ± SEM .

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C. Apparent decrease in insertion efficiency for the TMD mutant version. While the WT otoferlin glycosylation amounted to 3.84 ± 0.53, the TMD mutant had only 2.13 ± 0.65 (n=3, p<0.05). Data are represented as mean ± SEM.


A . Visual reinforcement audiometry performed in the free field : a mild hearing loss .

A. Visual reinforcement audiometry performed in the free field: a mild hearing loss.


B . Distortion product otoacoustic emissions ( DPOAEs ) were detected in each ear , whereasC . Auditory brainstem responses ( ABR ) were lacking , together providing the signature of auditory synaptopathy or neuropathy .

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B. Distortion product otoacoustic emissions (DPOAEs) were detected in each ear, whereasC. Auditory brainstem responses (ABR) were lacking, together providing the signature of auditory synaptopathy or neuropathy.


D - E . Cochlear microphonic potentials ( CM ) , summating potential ( SP ) and compound action potential ( CAP ) recorded through transtympanic electrocochleography ( ECochG ) in response to clicks at decreasing stimulus intensities are superimposed on the corresponding potentials recorded from one normally - hearing control ( range as measured at 120 dB 4 . 31 - 28 . 02 µV in 20 normally - hearing children ) . Means and standard errors of peak latency and amplitude are also reported for each potential category . The ECochG waveform resulting from CM cancellation in the control begins with an abrupt negative deflection , the SP , followed by a negative peak , the neural CAP . In the child with the OTOF TMD mutation , the ECochG responses begin with a rapid negative deflection that peaks at the same latency as the SP in the control and has comparable amplitude . This is followed by a low - amplitude negative potential that peaks at the same CAP in controls but shows a markedly prolonged duration . In all graphs time " 0 " refers to CM onset . R : right , L : left . F . Means and standard errors of peak latency and amplitude are reported for each potential category .

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

D-E. Cochlear microphonic potentials (CM), summating potential (SP) and compound action potential (CAP) recorded through transtympanic electrocochleography (ECochG) in response to clicks at decreasing stimulus intensities are superimposed on the corresponding potentials recorded from one normally-hearing control (range as measured at 120 dB 4.31-28.02 µV in 20 normally-hearing children). Means and standard errors of peak latency and amplitude are also reported for each potential category. The ECochG waveform resulting from CM cancellation in the control begins with an abrupt negative deflection, the SP, followed by a negative peak, the neural CAP. In the child with the OTOF TMD mutation, the ECochG responses begin with a rapid negative deflection that peaks at the same latency as the SP in the control and has comparable amplitude. This is followed by a low-amplitude negative potential that peaks at the same CAP in controls but shows a markedly prolonged duration. In all graphs time "0" refers to CM onset. R: right, L: left.F. Means and standard errors of peak latency and amplitude are reported for each potential category.


( Top ) PCR detection of the recombined BrafLox − V600E allele ( Lox - V600E ) in BVE mouse tissues at 8 weeks of age without tamoxifen induction . Substantial recombination is observed in the lung , while weaker recombination is also detected in the liver . No recombination was detected in hematopoietic tissues ( bone marrow and spleen ) even after 40 cycles of amplification ( right ) . ( Bottom ) H & amp ; amp ; E staining of lung sections from wild - type ( WT , 10 weeks of age ) , BVE ( 1 - 10 weeks of age ) and Braf + / LSL − V600E mouse 8 weeks after nasal delivery of AdCre is indicated below . V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development . Scale bars , 100 μm .

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(Top) PCR detection of the recombined BrafLox−V600E allele (Lox-V600E) in BVE mouse tissues at 8 weeks of age without tamoxifen induction. Substantial recombination is observed in the lung, while weaker recombination is also detected in the liver. No recombination was detected in hematopoietic tissues (bone marrow and spleen) even after 40 cycles of amplification (right). (Bottom) H&amp;E staining of lung sections from wild-type (WT, 10 weeks of age), BVE (1-10 weeks of age) and Braf+/LSL−V600E mouse 8 weeks after nasal delivery of AdCre is indicated below. V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development. Scale bars, 100 μm.


Ki67 immunostaining of lung sections from BVE mice at 3 - 10 weeks of age . Scale bars , 25 μm . The right bar graph summarises % Ki67 + cells in tumours at different ages post - partum . Four to six mice were analysed for each age group as indicated , and more than 3 , 500 tumour cells per mouse were evaluated . The data represent mean ± SD .

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Ki67 immunostaining of lung sections from BVE mice at 3-10 weeks of age. Scale bars, 25 μm. The right bar graph summarises %Ki67+ cells in tumours at different ages post-partum. Four to six mice were analysed for each age group as indicated, and more than 3,500 tumour cells per mouse were evaluated. The data represent mean ± SD.


Representative H & amp ; amp ; E staining and Mac2 immunohistochemistry of serial lung sections from 10 - week - old BVE mice at low ( upper panels ) and high ( lower panels ) magnifications . Scale bars , 100 μm ( upper panels ) or 25 μm ( lower panels ) .

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Representative H&amp;E staining and Mac2 immunohistochemistry of serial lung sections from 10-week-old BVE mice at low (upper panels) and high (lower panels) magnifications. Scale bars, 100 μm (upper panels) or 25 μm (lower panels).


Giemsa staining of isolated stroma IMCs . These cells show round or oval - shaped nuclei that lack indentations , and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes , suggestive of immature myeloid - lineage cells that are morphologically distinct from monocytes / DCs . Scale bar , 25 μm .

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Giemsa staining of isolated stroma IMCs. These cells show round or oval-shaped nuclei that lack indentations, and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes, suggestive of immature myeloid-lineage cells that are morphologically distinct from monocytes/DCs. Scale bar, 25 μm.


Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b , which is expressed at low levels ( overlaid with isotype control histograms shown in grey ) .

Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b, which is expressed at low levels (overlaid with isotype control histograms shown in grey).


Cell surface marker analysis ( dot plots ) of IMCs demonstrating CD11c + IMCs do not express macrophage ( F4 / 80 ) , DC ( CD24 ) or M1 - polarisation ( CD86 ) markers , but weakly express the M2 marker CD206 . Insets show isotype control staining .

Cell surface marker analysis (dot plots) of IMCs demonstrating CD11c+ IMCs do not express macrophage (F4/80), DC (CD24) or M1-polarisation (CD86) markers, but weakly express the M2 marker CD206. Insets show isotype control staining.


Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines . The cells grown on culture plates were imaged by phase - contrast microscopy ( left ) . Then , the cells were trypsinised and smeared on glass slides for Giemsa staining ( right ) . These cells are intermediate to large sized , round or amoeboid cells with cytoplasmic vacuolation , consistent with a macrophage morphology . Scale bars , 50 μm .

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Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines. The cells grown on culture plates were imaged by phase-contrast microscopy (left). Then, the cells were trypsinised and smeared on glass slides for Giemsa staining (right). These cells are intermediate to large sized, round or amoeboid cells with cytoplasmic vacuolation, consistent with a macrophage morphology. Scale bars, 50 μm.


Cell surface marker analysis of cultured IMCs showing differentiation into F4 / 80 + macrophages with CD11bhighCD11c + CD86 + CD206low surface phenotype ( overlaid with isotype control histograms shown in light grey ) .

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Cell surface marker analysis of cultured IMCs showing differentiation into F4/80+ macrophages with CD11bhighCD11c+CD86+CD206low surface phenotype (overlaid with isotype control histograms shown in light grey).


PCR detection of Braf recombination in purified IMCs , AT2 cells and tumour cell aggregates .

PCR detection of Braf recombination in purified IMCs, AT2 cells and tumour cell aggregates.


CMT64 cells ( 1 × 104 / well in 12 - well plates ) were co - cultured with 1 . 5 - 3 × 105 IMCs for 4 days . Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate . Harvested cells were subjected to counting ( left ) . Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells . The bar graph ( left ) shows CMT64 growth ( cell counting ) data pooled from two independent experiments . Phase - contrast images of CMT64 cells cultured without IMCs ( CMT64 only ) or co - cultured with IMCs ( CMT64 / IMC co - culture ) are also indicated in the middle and right . A higher magnification image of co - cultured CMT64 cells ( right ) highlights IMCs migrating into CMT64 cells ( arrow heads ) . Scale bars are 200 μm , except for the high magnification image ( right ) in which the scale bar is 100 μm .

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CMT64 cells (1 × 104/well in 12-well plates) were co-cultured with 1.5-3 × 105 IMCs for 4 days. Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate. Harvested cells were subjected to counting (left). Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells. The bar graph (left) shows CMT64 growth (cell counting) data pooled from two independent experiments. Phase-contrast images of CMT64 cells cultured without IMCs (CMT64 only) or co-cultured with IMCs (CMT64/IMC co-culture) are also indicated in the middle and right. A higher magnification image of co-cultured CMT64 cells (right) highlights IMCs migrating into CMT64 cells (arrow heads). Scale bars are 200 μm, except for the high magnification image (right) in which the scale bar is 100 μm.


( Left ) Growth and EMT - like morphological changes of CMT64 cells cultured with 25 % IMC - CM . CMT64 cells cultured for 4 days at low - density in DMEM / 10 % FCS without IMC - CM ( - IMC - CM ) or with 25 % IMC - CM ( + IMC - CM ) were counted and normalised to the average of control as 100 % . The data in the bar chart represent mean + SD of five cultures utilising IMC - CM obtained from three independent sources . ( Middle ) Phase - contrast images to indicate representative morphologies of CMT64 cells maintained without IMC - CM ( control ) or cultured with 25 % IMC - CM ( + IMC - CM ) for 2 weeks . ( Right ) E - cadherin immunofluorescence of CMT64 cells cultured as above . Scale bars are 200 μm ( phase contrast ) and 10 μm ( immunofluorescence ) .

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(Left) Growth and EMT-like morphological changes of CMT64 cells cultured with 25% IMC-CM. CMT64 cells cultured for 4 days at low-density in DMEM/10%FCS without IMC-CM (-IMC-CM) or with 25% IMC-CM (+IMC-CM) were counted and normalised to the average of control as 100%. The data in the bar chart represent mean + SD of five cultures utilising IMC-CM obtained from three independent sources. (Middle) Phase-contrast images to indicate representative morphologies of CMT64 cells maintained without IMC-CM (control) or cultured with 25% IMC-CM (+IMC-CM) for 2 weeks. (Right) E-cadherin immunofluorescence of CMT64 cells cultured as above. Scale bars are 200 μm (phase contrast) and 10 μm (immunofluorescence).


Co - culture of AT2 cells and autologous IMCs from BVE mice ( 8 - to 10 - week - old ) for 48 h using the Transwell ® culture system . In vitro BrdU incorporation ( flow cytometry , left and middle ) and EMT marker expression ( RT - PCR , right ) in co - cultured AT2 cells are indicated . Representative flow cytometry plots are indicated on the left , and the bar chart in the middle indicates % BrdU + AT2 cell numbers normalised to control cultures without IMCs ( n = 4 , mean + SD ) . Gapdh serves as a loading control for the RT - PCR on the right .

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Co-culture of AT2 cells and autologous IMCs from BVE mice (8- to 10-week-old) for 48 h using the Transwell® culture system. In vitro BrdU incorporation (flow cytometry, left and middle) and EMT marker expression (RT-PCR, right) in co-cultured AT2 cells are indicated. Representative flow cytometry plots are indicated on the left, and the bar chart in the middle indicates % BrdU+ AT2 cell numbers normalised to control cultures without IMCs (n = 4, mean + SD). Gapdh serves as a loading control for the RT-PCR on the right.


Morphological alterations and intracellular signalling in AT2 cells cultured with IMC - CM . On the left and middle , primary AT2 cells from 3 - week - old BVE mice were cultured for 48 h and then incubated in serum - free IMC - CM ( AT2 with IMC - CM ) or DMEM ( AT2 w / o IMC - CM , serving as a control ) for another 48 h , followed by immunostaining for E - cadherin ( red ) and vimentin ( green ) and confocal laser scanning microscopy ( CLSM ) imaging . The 3 × zoomed image in the middle right highlights dividing vimentin + cells that show internalised E - cadherin ( arrows ) . Scale bars , 50 μm . On the right , primary AT2 cells from 10 - week - old BVE mice were cultured for 48 h , serum starved for 5 h and treated with or without serum - free IMC - CM for 30 min . Phosphorylation of AKT , SMAD3 , MEK and ERK were analysed by immunoblotting .

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Morphological alterations and intracellular signalling in AT2 cells cultured with IMC-CM. On the left and middle, primary AT2 cells from 3-week-old BVE mice were cultured for 48 h and then incubated in serum-free IMC-CM (AT2 with IMC-CM) or DMEM (AT2 w/o IMC-CM, serving as a control) for another 48 h, followed by immunostaining for E-cadherin (red) and vimentin (green) and confocal laser scanning microscopy (CLSM) imaging. The 3× zoomed image in the middle right highlights dividing vimentin+ cells that show internalised E-cadherin (arrows). Scale bars, 50 μm. On the right, primary AT2 cells from 10-week-old BVE mice were cultured for 48 h, serum starved for 5 h and treated with or without serum-free IMC-CM for 30 min. Phosphorylation of AKT, SMAD3, MEK and ERK were analysed by immunoblotting.


CCR1 immunofluorescence of primary IMCs ( left , co - stained with Mac2 ) and AT2 cells ( right , co - stained with E - cadherin ) detected by CLSM . Scale bars , 10 μm . CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs , but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 ( inset in the left microphotograph ) .

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CCR1 immunofluorescence of primary IMCs (left, co-stained with Mac2) and AT2 cells (right, co-stained with E-cadherin) detected by CLSM. Scale bars, 10 μm. CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs, but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 (inset in the left microphotograph).


CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs . Scale bar , 50 μm .

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CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs. Scale bar, 50 μm.


CT imaging of lungs of AdCre - infected Braf + / LSL − V600E mice treated with vehicle ( top ) or CCR1 inhibitor ( bottom ) . H : heart ; * indicates a tumour region accompanied by atelectasis .

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CT imaging of lungs of AdCre-infected Braf+/LSL−V600E mice treated with vehicle (top) or CCR1 inhibitor (bottom). H: heart; * indicates a tumour region accompanied by atelectasis.


H & amp ; amp ; E staining of lung sections from vehicle ( left ) or CCR1 inhibitor ( right ) treated AdCre - infected Braf + / LSL − V600E mice . Scale bars , 250 μm ( top ) or 50 μm ( bottom ) .

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H&amp;E staining of lung sections from vehicle (left) or CCR1 inhibitor (right) treated AdCre-infected Braf+/LSL−V600E mice. Scale bars, 250 μm (top) or 50 μm (bottom).


Flow cytometry analysis of CD11c + ( top , green ) and SPC + ( bottom , red ) cells in the lung of AdCre - infected Braf + / LSL − V600Emice treated with vehicle ( left ) or CCR1 inhibitor ( right ) . LungCD11c + ( left ) and SPC + ( right ) cell numbers ( per left lobe ) were quantitated in AdCre - infected Braf + / LSL − V600Emice treated with CCR1 inhibitor or vehicle ( V ) ( n = 3 , mean + SD ) . % LungCD4 + ( left ) and CD8 + ( right ) T lymphocytes were quantitated in AdCre - infected Braf + / LSL − V600E mice treated with CCR1 inhibitor or vehicle ( V ) ( n = 3 , mean + SD ) .

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Flow cytometry analysis of CD11c+ (top, green) and SPC+ (bottom, red) cells in the lung of AdCre-infected Braf+/LSL−V600Emice treated with vehicle (left) or CCR1 inhibitor (right).LungCD11c+ (left) and SPC+ (right) cell numbers (per left lobe) were quantitated in AdCre-infected Braf+/LSL−V600Emice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).% LungCD4+ (left) and CD8+ (right) T lymphocytes were quantitated in AdCre-infected Braf+/LSL−V600E mice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).


NPC2 immunoblots of concentrated whole lung CM ( top ) and whole lung lysates ( bottom ) generated from Braf + / + ; CreER ™ ( WT ) or BVE ( VE ) mice . Whole lung lysates were obtained from 3 - to 8 - week - old mice .

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NPC2 immunoblots of concentrated whole lung CM (top) and whole lung lysates (bottom) generated from Braf+/+;CreER™ (WT) or BVE (VE) mice. Whole lung lysates were obtained from 3-to 8-week-old mice.


NPC2 immunoblots of un - concentrated CM ( containing 10 % FCS ) from fractionated IMC and AT2 cultures . Total protein staining of the membrane by Amido black ( bottom ) serves as a loading control showing serum albumin at 70 kDa , derived from FCS in the media .

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NPC2 immunoblots of un-concentrated CM (containing 10% FCS) from fractionated IMC and AT2 cultures. Total protein staining of the membrane by Amido black (bottom) serves as a loading control showing serum albumin at 70 kDa, derived from FCS in the media.


Npc2 mRNA expression in the purified IMC and AT2 populations as determined by quantitative RT - PCR . Mean + SD ( n = 3 ) of Npc2 expression normalised to Gapdh is indicated .

Npc2 mRNA expression in the purified IMC and AT2 populations as determined by quantitative RT-PCR. Mean + SD (n = 3) of Npc2 expression normalised to Gapdh is indicated.


NPC2 immunoblots of cell lysates from freshly isolated IMCs and AT2 cells .

NPC2 immunoblots of cell lysates from freshly isolated IMCs and AT2 cells.


NPC2 immunoblots of cell lysates from freshly isolated or cultured ( 48 h in serum - free DMEM ) IMCs .

NPC2 immunoblots of cell lysates from freshly isolated or cultured (48 h in serum-free DMEM) IMCs.


NPC2 immunoblots of cell lysates from primary IMCs cultured with or without 50 μg / ml bNPC2 for 48 h .

NPC2 immunoblots of cell lysates from primary IMCs cultured with or without 50 μg/ml bNPC2 for 48 h.


CLSM imaging of NPC2 - Alexa488 taken up by IMCs . ( Left ) Freshly isolated IMCs were incubated for 2 h with ( + NPC2 - Alexa488 ) or without ( - NPC2 - Alexa488 ) 82 . 5 nM NPC2 - Alexa488 , chased in NPC2 - free media for additional 2 h and live imaged by CLSM . Single - colour ( top ) and DIC - merged ( bottom ) fluorescence images are indicated . Scale bar , 20 μm . ( Right ) Zoomed images ( DIC - merged and single - colour ) from different fields showing vesicular and reticular distribution of incorporated NPC2 - Alexa488 . Scale bar , 10 μm .

CLSM imaging of NPC2-Alexa488 taken up by IMCs. (Left) Freshly isolated IMCs were incubated for 2 h with (+NPC2-Alexa488) or without (-NPC2-Alexa488) 82.5 nM NPC2-Alexa488, chased in NPC2-free media for additional 2 h and live imaged by CLSM. Single-colour (top) and DIC-merged (bottom) fluorescence images are indicated. Scale bar, 20 μm. (Right) Zoomed images (DIC-merged and single-colour) from different fields showing vesicular and reticular distribution of incorporated NPC2-Alexa488. Scale bar, 10 μm.


Npc2 mRNA expression in IMCs cultured for 48 h with or without 50 μg / ml bNPC2 as determined by qRT - PCR . Mean ± SD ( n = 3 ) of Npc2 expression normalised to Hprt1 is indicated .

Npc2 mRNA expression in IMCs cultured for 48 h with or without 50 μg/ml bNPC2 as determined by qRT-PCR. Mean ± SD (n = 3) of Npc2 expression normalised to Hprt1 is indicated.


Shortened survival of BVE mice following reduction in Npc2 gene dosage . Genotypes of mice are as follows : + / + = Braf + / LSL − V600E ; CreER + / 0 ; Npc2 + / + , + / hypo = Braf + / LSL − V600E ; CreER + / 0 , Npc2 + / hypo , hypo / hypo = Braf + / LSL − V600E ; CreER + / 0 ; Npc2hypo / hypo . Median survival time ( MST ) for each genotype is indicated .

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Shortened survival of BVE mice following reduction in Npc2 gene dosage. Genotypes of mice are as follows: +/+ = Braf+/LSL−V600E;CreER+/0;Npc2+/+, +/hypo = Braf+/LSL−V600E;CreER+/0,Npc2+/hypo, hypo/hypo = Braf+/LSL−V600E;CreER+/0;Npc2hypo/hypo. Median survival time (MST) for each genotype is indicated.


Representative H & amp ; amp ; E staining of lung sections from 5 - week - old BVE mice on the Npc2 + / + ( + / + ) or Npc2 + / hypo ( + / hypo ) background is shown . Scale bars , 500 μm .

Representative H&amp;E staining of lung sections from 5-week-old BVE mice on the Npc2+/+ (+/+) or Npc2+/hypo (+/hypo) background is shown. Scale bars, 500 μm.


Stroma IMC quantification per lung in Npc2 + / + ( + / + ) or Npc2 + / hypo ( + / hypo ) BVE mice at different ages ( 39 - 109 days p . p . ) .

Stroma IMC quantification per lung in Npc2+/+ (+/+) or Npc2+/hypo (+/hypo) BVE mice at different ages (39-109 days p.p.).


Ki67 / Mac2immunofluorescence analysis of Npc2 + / + ( + / + ) and Npc2 + / hypo ( + / hypo ) BVElung sections at 5 weeks of age . Merged images for Ki67 ( green ) , Mac2 ( red ) and DAPI ( blue ) are indicated . Arrows indicate dual positive cells for nuclearKi67 and Mac2 . Scale bars , 50 μm . The bar chart indicates % Ki67 in Mac2 + lung stroma cells ( mean + SD ) , obtained from 4 independent Npc2 + / + and Npc2 + / hypoBVE pairs .

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Ki67/Mac2immunofluorescence analysis of Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVElung sections at 5 weeks of age. Merged images for Ki67 (green), Mac2 (red) and DAPI (blue) are indicated. Arrows indicate dual positive cells for nuclearKi67 and Mac2. Scale bars, 50 μm. The bar chart indicates % Ki67 in Mac2+lung stroma cells (mean + SD), obtained from 4 independent Npc2+/+ and Npc2+/hypoBVE pairs.


In vivo BrdU incorporation of CD11c + IMCs from Npc2 + / + ( + / + ) and Npc2 + / hypo ( + / hypo ) BVE mice are shown . Representative flow cytometry plots of three independent experiments are indicated .

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In vivo BrdU incorporation of CD11c+ IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice are shown. Representative flow cytometry plots of three independent experiments are indicated.


Ki67 ( green ) / Mac2 ( red ) dual immunofluorescencestaining of lung sections from 5 - week - old Npc2 + / hypoBVEmice with nuclearDAPI ( blue ) staining , focusing on intratumour Mac2 + cell clusters . Scale bar , 50 μm . Enlarged images of the boxed areas 1 and 2 highlight Ki67 + Mac2 − cells associated with Mac2 + cells ( arrows ) .

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Ki67 (green)/Mac2 (red) dual immunofluorescencestaining of lung sections from 5-week-old Npc2+/hypoBVEmice with nuclearDAPI (blue) staining, focusing on intratumour Mac2+cell clusters. Scale bar, 50 μm. Enlarged images of the boxed areas 1 and 2 highlight Ki67+Mac2− cells associated with Mac2+ cells (arrows).


Lung SPC + / CD11c + cell numbers of Braf + / LSL − V600E ; Npc2 + / + ( + / + , n = 3 ) and Braf + / LSL − V600E ; Npc2 + / hypo ( + / hypo , n = 7 ) mice at 10 weeks post - AdCre infection .

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Lung SPC+/CD11c+cell numbers of Braf+/LSL−V600E;Npc2+/+ (+/+, n = 3) and Braf+/LSL−V600E;Npc2+/hypo (+/hypo, n = 7) mice at 10 weeks post-AdCre infection.


Intracellular un - esterified cholesterol distribution as detected by filipin staining in freshly isolated Npc2 + / + and Npc2 + / hypo IMCs . Scale bars , 25 μm . Filipin - stained cells were graded from 0 to 3 + as indicated in the microphotographs ( see Materials and Methods for criteria ) , and % of each grade in IMCs from Npc2 + / + ( + / + ) and Npc2 + / hypo ( + / hypo ) BVE mice is shown in the bar chart . The data were obtained by analysing 176 Npc2 + / + and 242 Npc2 + / hypo IMCs from three independent Npc2 + / + and Npc2 + / hypo pairs .

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

Intracellular un-esterified cholesterol distribution as detected by filipin staining in freshly isolated Npc2+/+ and Npc2+/hypo IMCs. Scale bars, 25 μm. Filipin-stained cells were graded from 0 to 3+ as indicated in the microphotographs (see Materials and Methods for criteria), and % of each grade in IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice is shown in the bar chart. The data were obtained by analysing 176 Npc2+/+ and 242 Npc2+/hypo IMCs from three independent Npc2+/+ and Npc2+/hypo pairs.


CLSM imaging of fresh Npc2 + / hypo IMCs stained with filipin ( cyan ) and LAMP2 immunofluorescence ( purple ) . Scale bars , 5 μm . Coarse structures strongly stained with filipin are surrounded by cytoplasmic membranous staining of LAMP2 . Single - colour greyscale images are also demonstrated in the middle ( filipin ) and right ( LAMP2 ) .

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CLSM imaging of fresh Npc2+/hypo IMCs stained with filipin (cyan) and LAMP2 immunofluorescence (purple). Scale bars, 5 μm. Coarse structures strongly stained with filipin are surrounded by cytoplasmic membranous staining of LAMP2. Single-colour greyscale images are also demonstrated in the middle (filipin) and right (LAMP2).


Intracellular un - esterified cholesterol distribution as detected by filipin staining in Npc2 + / hypo IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . Scale bars , 25 μm . Quantitative data in the bar graph were obtained by analysing 265 bNPC2 - untreated ( - bNPC2 ) and 252 bNPC2 - treated ( + bNPC2 ) cells from three independent cultures using the method described in ( A ) .

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Intracellular un-esterified cholesterol distribution as detected by filipin staining in Npc2+/hypo IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 25 μm. Quantitative data in the bar graph were obtained by analysing 265 bNPC2-untreated (-bNPC2) and 252 bNPC2-treated (+bNPC2) cells from three independent cultures using the method described in (A).


Immunoblot ( left ) and ELISA ( right , n = 3 ) analyses of CCL6 in IMC - CM from primary Npc2 + / + IMCs cultured with or without 50 μg / ml bNPC2 for 48 h ( immunoblots ) or 72 h ( ELISA ) . The data in the bar graph ( ELISA ) represent mean + SD .

Immunoblot (left) and ELISA (right, n = 3) analyses of CCL6 in IMC-CM from primary Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h (immunoblots) or 72 h (ELISA). The data in the bar graph (ELISA) represent mean + SD.


Quantification of secreted CCL6 into IMC - CM during the initial 24 h ( 0 - 24 h ) and subsequent 48 h ( 24 - 72 h ) of IMC culture by ELISA ( n = 3 , mean + SD ) .

Quantification of secreted CCL6 into IMC-CM during the initial 24 h (0-24 h) and subsequent 48 h (24-72 h) of IMC culture by ELISA (n = 3, mean + SD).


Immunoblot and qRT - PCR analyses of CCL6 expression in Npc2 + / + IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . The data in the bar graph ( qRT - PCR ) represent mean + SD ; n = 3 .

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Immunoblot and qRT-PCR analyses of CCL6 expression in Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. The data in the bar graph (qRT-PCR) represent mean + SD; n = 3.


CLSM imaging of CCL6 ( green ) / LAMP1 ( red ) immunofluorescence of Npc2 + / + IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . Scale bars , 10 μm . The right bottom image highlights the boxed area of the merged image of bNPC2 - treated IMCs showing partial co - localisation of LAMP1 and CCL6 .

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CLSM imaging of CCL6 (green)/LAMP1 (red) immunofluorescence of Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 10 μm. The right bottom image highlights the boxed area of the merged image of bNPC2-treated IMCs showing partial co-localisation of LAMP1 and CCL6.


Immunoblot analysis of intracellular and secreted ( CM ) CCL6 in bafilomycin - treated Npc2 + / + IMC culture . IMCs without ( left ) or with ( right ) 50 μg / ml bNPC2 pre - loading for 30 min were chased in serum - free DMEM for 3 h and then treated with 200 nM bafilomycin A1 for 24 h .

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Immunoblot analysis of intracellular and secreted (CM) CCL6 in bafilomycin-treated Npc2+/+ IMC culture. IMCs without (left) or with (right) 50 μg/ml bNPC2 pre-loading for 30 min were chased in serum-free DMEM for 3 h and then treated with 200 nM bafilomycin A1 for 24 h.


( A ) Schematic representation of the SILAC - based quantitative proteomics experiment in B . subtilis . Cells overexpressing active YqgP ( BS50 , Table EV2 ) or its catalytically dead mutant YqgP . S288A ( BS51 ) , both auxotrophic for lysine , were grown in parallel in " heavy " ( containing 13C615N2 - Lysine isotope ) or " light " ( containing stable 12C614N2 - Lysine ) M9 minimal medium , respectively . After mixing the cell cultures in the 1 : 1 ratio ( based on OD600 ) , cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC - MS / MS experiment . Using bioinformatic analysis of the MS data , highest - scoring candidate substrates were further evaluated .

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(A) Schematic representation of the SILAC-based quantitative proteomics experiment in B. subtilis. Cells overexpressing active YqgP (BS50, Table EV2) or its catalytically dead mutant YqgP.S288A (BS51), both auxotrophic for lysine, were grown in parallel in "heavy" (containing 13C615N2- Lysine isotope) or "light" (containing stable 12C614N2- Lysine) M9 minimal medium, respectively. After mixing the cell cultures in the 1:1 ratio (based on OD600), cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC-MS/MS experiment. Using bioinformatic analysis of the MS data, highest-scoring candidate substrates were further evaluated.


( E ) Results of MS analyses of affinity co - purification experiments in wild - type B . subtilis control ( strain BTM2 , Table EV2 ) and B . subtilis deficient in endogenous YqgP expressing the wild - type YqgP - sfGFP bait ( strain BTM84 , Table EV2 ) or the proteolytically inactive YqgP . S288A - sfGFP bait ( strain BBM1 , Table EV2 ) . Proteins were considered as potential interactors of YqgP if they were identified only in both positive co - purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts . The protein highlighted in red was the only overlapping hit between the two proteomics approaches .

(E) Results of MS analyses of affinity co-purification experiments in wild-type B. subtilis control (strain BTM2, Table EV2) and B. subtilis deficient in endogenous YqgP expressing the wild-type YqgP-sfGFP bait (strain BTM84, Table EV2) or the proteolytically inactive YqgP.S288A-sfGFP bait (strain BBM1, Table EV2). Proteins were considered as potential interactors of YqgP if they were identified only in both positive co-purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts. The protein highlighted in red was the only overlapping hit between the two proteomics approaches.


( A ) Steady - state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B . subtilis ( BTM2 and BTM501 , respectively , Table EV2 ) , in minimal medium at low magnesium concentration ( 10 µM ) . Strain lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) was used as a control . Proteins were detected by immunoblotting with chemiluminescence detection . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE )

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(A) Steady-state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B. subtilis (BTM2 and BTM501, respectively, Table EV2), in minimal medium at low magnesium concentration (10 µM). Strain lacking YqgP (ΔyqgP, BTM78, Table EV2) was used as a control. Proteins were detected by immunoblotting with chemiluminescence detection. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE)


( B ) The cleavage is efficiently inhibited by 1 μM STS736 , a specific peptidyl ketoamide rhomboid inhibitor Ticha et al . , 2017b ( ) . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) and ectopic YqgP by anti - YqgP antibodies .

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

(B) The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor Ticha et al., 2017b(). Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.


( C ) To map the cleavage site region within endogenous MgtE ( second lane from the left , from strain BTM501 ) , MgtE - derived reference fragments encoding first 300 , 315 , 330 amino acids as well as full - length MgtE ( black arrow ) , were in vitro transcribed and translated . Mobility of the N - terminal cleavage product ( red arrow ) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) and ectopic YqgP by anti - YqgP antibodies .

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(C) To map the cleavage site region within endogenous MgtE (second lane from the left, from strain BTM501), MgtE-derived reference fragments encoding first 300, 315, 330 amino acids as well as full-length MgtE (black arrow), were in vitro transcribed and translated. Mobility of the N-terminal cleavage product (red arrow) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.


( A ) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B . subtilis cells ( BS72 , Table EV2 ) depending on the concentrations of magnesium and manganese ions . Cells were cultivated in glucose M9 minimal medium with limiting ( 0 . 01 mM ) or high ( 1 mM ) concentration of MgSO4 , in the presence or absence of 100 μM MnCl2 , and analysed by western blotting with near - infrared detection ( upper panel ) . Black arrow denotes full - length MgtE , and red arrow denotes its N - terminal cleavage product formed by YqgP . The corresponding fluorescence signals were quantified by densitometry , and are displayed as relative specific activity , which is substrate conversion normalized to enzyme expression level ( lower panel ) .

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(A) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B. subtilis cells (BS72, Table EV2) depending on the concentrations of magnesium and manganese ions. Cells were cultivated in glucose M9 minimal medium with limiting (0.01 mM) or high (1 mM) concentration of MgSO4, in the presence or absence of 100 μM MnCl2, and analysed by western blotting with near-infrared detection (upper panel). Black arrow denotes full-length MgtE, and red arrow denotes its N-terminal cleavage product formed by YqgP. The corresponding fluorescence signals were quantified by densitometry, and are displayed as relative specific activity, which is substrate conversion normalized to enzyme expression level (lower panel).


( B ) Growth curves of wild - type ( BTM843 , Table EV2 ) , yqgP deficient ( BTM844 , Table EV2 ) and rescue ( BTM845 , Table EV2 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese stress elicited by adding 75 µM MnSO4 in mid - exponential phase ( stress - phase denoted by blueish background ) . All strains further contain a deletion in the putative manganese efflux pump MntP ( ΔywlD , Table EV2 ) . Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain , and that reintroduction of YqgP rescues fitness during manganese stress to above - wild - type level . Top panel shows no difference between the strains in the absence of manganese stress . Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time - point , which illustrates the reproducibility of the 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Growth curves of wild-type (BTM843, Table EV2), yqgP deficient (BTM844, Table EV2) and rescue (BTM845, Table EV2) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese stress elicited by adding 75 µM MnSO4 in mid-exponential phase (stress-phase denoted by blueish background). All strains further contain a deletion in the putative manganese efflux pump MntP (ΔywlD, Table EV2). Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain, and that reintroduction of YqgP rescues fitness during manganese stress to above-wild-type level. Top panel shows no difference between the strains in the absence of manganese stress. Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time-point, which illustrates the reproducibility of the assay.