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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

(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 ) 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 - no corrections . Data are shown as mean ± .

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

(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.


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

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

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


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

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 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

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.


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

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 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

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 plantar surface of hind paw with sumatriptan , or vehicle ± combination of wortmannin U0126 , as described in methods section . Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order . ( A - C ) of withdrawal response for mice using von Frey filaments at : 0 . 07 g ( A ) , 0 . 16 g ( B ) 0 . 4 g ( C ) . ( n = 11 - 16 per group , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One way - ANOVA with Tukey ' s multiple comparison ) . Data are shown as mean ± 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 plantar surface of hind paw with sumatriptan , or vehicle ± combination of wortmannin U0126 , as described in methods section . Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order . ( D - F ) of withdrawal response for mice using von Frey filaments at : 0 . 07 g ( D ) , 0 . 16 g ( E ) 0 . 4 g ( F ) . ( n = 12 - 16 per group , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One way - ANOVA with Tukey ' s multiple comparison ) . Data are shown as mean ± 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 . 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 did not exert any abnormal behavioral phenotype ) . While left HC solely expressed ER - tdTomato , neighboring HC expressed both , ER - tdTomato Wrb - GFP . Colocalization between both occurs in areas exhibiting pixels . Scale bars : 5

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 ) expressing an EGFP - tagged truncated Wrb fragment containing only coiled - coiled domains ( Wrbcc - EGFP , green ) . Wrbcc - EGFP distribution was diffuse found throughout HC .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR 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 ) Wrb - deficient pwi mutant fish ( wrb pwi / pwi ; D ' ) , showing strongly reduced otoferlin immunofluorescence in mutant HCs . color look - up table used represents higher pixel intensities with warmer colors .

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

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 ( arrow ) , expressing Wrb - GFP , exhibits a strongly increased otoferlin signal ( magenta ) in direct comparison to neighboring non - rescued mutant HCs . ( E ' ) Same image as in E but - coded for otoferlin .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 ( arrow ) , immunolabelled for otoferlin ( magenta ) . Wrbcc - EGFP - expressing HC shows significantly less otoferlin ( F ' ) , suggesting a dominant negative effect of Wrbcc in otoferlin biogenesis . transfected HC is encircled with a dashed .

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

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

O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR 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 . of otoferlin immunohistochemistry data shown in ( D ) from hair of control ( n = 88 HCs from three 5 - dpf larva ) , wrb pwi / pwi ( n = 80 HCs from 3 sibling larva ) normalized against mean value of control group Wrb - GFP transfected wrb pwi / pwi zebrafish inner ears . Mutant HCs showed ~ 82 % reduction in otoferlin when compared with control HCs imaged under same conditions . Otoferlin 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 same larva ) . values were normalized with average value of Wrb - EGFP negative HCs . * * * p < 0 .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O 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 ) wrb pwi / pwi ( n = 18 ) zebrafishlarva , where almost 100 % of larva responded to five successive acoustic stimuli at 3 seconds intervals . In wrb pwi / pwi , only 39 % of larva responded to first stimulus , thereafter , 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 acoustic startle reflex in mutants , while not displaying detrimental effects on overall zebrafish development . Please note 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 ( other ) different experiments were compiled in 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 B-CONTROLLED_VAR I-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 B-CONTROLLED_VAR I-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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

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 with wild - type or an ATPase - deficient mutant version of TRC40 incubated in absence or presence of ER - derived rough microsomes ( RM ) . integration ( glycosylation ) was monitored by SDS - PAGE 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

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 with wild - type or mutant TRC40 was incubated with RM in presence or absence of ATP insertion was monitored by opsin - specific immunoblot .

O O O O B-MEASURED_VAR 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. 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 with wild - type TRC40 was incubated in presence of RM , trypsin - treated rough microsomes ( TRM ) or in presence of WRBcc or CAMLcyt . insertion was monitored by opsin - specific immunoblot .

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

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

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

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

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

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

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

O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O 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. 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 ( C - D ) P20 Wrb + / + : CreA Wrbfl / fl : CreA apical turn organs of Corti following immunolabeling against otoferlin ( green ) Vglut3 ( magenta ) processed imaged under identical conditions . A ' - D ' show otoferlin stainings with an - coded lookup table . A ' ' - D ' ' present single xz projections at a central point through a representative to illustrate otoferlin subcellular distribution . Scale bars in A - D ' represent 10 μm , in A ' ' - D ' ' 2 µm . Note dramatic reduction in otoferlin alteration in its distribution as well as a change in shape position of nuclei ( for detailed of shape 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

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

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

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 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

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 cytoplasm ( cyan arrows ) . Moreover , cisternal structures resembling ( r ) ER are found unusually close to 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

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

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

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 ) Wrb + / + : CreA ( n = 15 IHCs ) mice in 2 mM extracellular [ Ca2 + ] showed no change in amplitude or voltage - dependence of Ca2 + current . Data are represented as mean ± .

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

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

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

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

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 ) corresponding Ca2 + current integrals , QCa ( bottom ) for various depolarization durations . Data are grand averages of ' means . Exocytic ΔCm was reduced in knockout from 20 ms onwards , indicating reduced sustained exocytosis . Data from OtofPga / Pga 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 B-CONTROLLED_VAR 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 + 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 ± ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

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

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

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

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 Wrb + / + : CreA SGNs indicating preserved active cochlear amplification despite disruption of Wrb in IHCs .

O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 characteristic frequency ( CF ) in Wrbfl / fl : CreA SGNs .

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

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


D - D ' ' . Mean poststimulus time histograms ( PSTH ) ± of Wrbfl / fl : CreA 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 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 cochlear nucleus ) of OtofPga / Pga 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 ) 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 B-CONTROLLED_VAR 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 with 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 B-CONTROLLED_VAR 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

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 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 a 15 ms probe tone ( CF , 30 dB above threshold ) . Bottom : in Wrbfl / fl : CreA SGNs , response to first 5 ms of masker probe tone tone ( shown as a fraction of response to first 5ms of masker ) was strongly reduced , 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 ) . Data represent means ± .

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

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 TMD mutant version . While WT otoferlin glycosylation amounted to 3 . 84 ± 0 . 53 , TMD mutant had only 2 . 13 ± 0 . 65 ( n = 3 , p < 0 . 05 ) . Data are represented as mean ± .

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

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 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 signature of auditory synaptopathy or neuropathy .

O O O O O O O O O O O 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. 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 ) compound action potential ( CAP ) recorded through transtympanic electrocochleography ( ECochG ) in response to clicks at decreasing stimulus intensities are superimposed on 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 standard errors of peak latency amplitude are also reported for each potential category . ECochG waveform resulting from CM cancellation in control begins with an abrupt negative deflection , SP , followed by a negative peak , neural CAP . In child with OTOF TMD mutation , ECochG responses begin with a rapid negative deflection that peaks at same latency as SP in control has comparable amplitude . This is followed by a low - amplitude negative potential that peaks at same CAP in controls but shows a markedly prolonged duration . In graphs time " 0 " refers to CM onset . R : right , L : left . F . Means standard errors of peak latency 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

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 recombined BrafLox − V600E allele ( Lox - V600E ) in BVE mouse tissues at 8 of without tamoxifen induction . Substantial recombination is observed in lung , while weaker recombination is also detected in liver . No recombination was detected in hematopoietic tissues ( bone marrow spleen ) even after 40 cycles of amplification ( right ) . ( Bottom ) H & amp ; amp ; E of lung sections from wild - type ( WT , 10 of ) , BVE ( 1 - 10 of ) Braf + / LSL − V600E mouse 8 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 .

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

(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 of . Scale bars , 25 μm . right bar graph summarises % Ki67 + in tumours at different ages post - partum . Four to six mice were analysed for each group as indicated , more than 3 , 500 tumour per mouse were evaluated . data represent mean ± SD .

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 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

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 Mac2 immunohistochemistry of serial lung sections from 10 - week - old BVE mice at low ( upper panels ) high ( lower panels ) magnifications . Scale bars , 100 μm ( upper panels ) or 25 μm ( lower panels ) .

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

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

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.


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

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

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).


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

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

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 without additional cytokines . grown on culture plates were imaged by phase - contrast microscopy ( left ) . Then , were trypsinised smeared on glass slides for Giemsa ( right ) . These are intermediate to large sized , round or amoeboid with vacuolation , consistent with a macrophage . Scale bars , 50 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

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.


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

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

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 tumour .

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


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

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 EMT - like morphological changes of CMT64 cultured with 25 % IMC - CM . CMT64 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 normalised to average of control as 100 % . data in bar chart represent mean + SD of five cultures utilising IMC - CM obtained from three independent sources . ( Middle ) Phase - contrast to indicate representative morphologies of CMT64 maintained without IMC - CM ( control ) or cultured with 25 % IMC - CM ( + IMC - CM ) for 2 . ( Right ) E - cadherin immunofluorescence of CMT64 cultured as above . Scale bars are 200 μm ( phase contrast ) 10 μm ( immunofluorescence ) .

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

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

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

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 intracellular signalling in AT2 cultured with IMC - CM . On left middle , AT2 from 3 - week - old BVE mice were cultured for 48 h 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 ) vimentin ( green ) confocal scanning microscopy ( CLSM ) imaging . 3 × zoomed image in middle right highlights dividing vimentin + that show internalised E - cadherin ( arrows ) . Scale bars , 50 μm . On right , AT2 from 10 - week - old BVE mice were cultured for 48 h , serum starved for 5 h treated with or without serum - free IMC - CM for 30 min . Phosphorylation of AKT , SMAD3 , MEK ERK were analysed by immunoblotting .

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

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 IMCs ( left , co - stained with Mac2 ) AT2 ( right , co - stained with E - cadherin ) detected by CLSM . Scale bars , 10 μm . CCR1 was mainly detected in vesicles within IMCs , but some CCR1 was also detected at plasma along with Mac2 ( inset in left microphotograph ) .

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

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 .

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

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 .

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

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 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 ) .

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

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 of CD11c + ( top , green ) SPC + ( bottom , red ) in lung of AdCre - infected Braf + / LSL − V600Emice treated with vehicle ( left ) or CCR1 inhibitor ( right ) . LungCD11c + ( left ) SPC + ( right ) 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 ) CD8 + ( right ) T lymphocytes were quantitated in AdCre - infected Braf + / LSL − V600E mice treated with CCR1 inhibitor or vehicle ( V ) ( n = 3 , mean + SD ) .

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

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 ) 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 .

B-MEASURED_VAR 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

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 AT2 cultures . Total of by Amido black ( bottom ) serves as a loading control showing serum albumin at 70 kDa , derived from FCS in media .

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

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

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 B-MEASURED_VAR O O O O O O O

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 lysates from freshly isolated IMCs AT2 .

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


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

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

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


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

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

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 live imaged by CLSM . Single - colour ( top ) DIC - merged ( bottom ) are indicated . Scale bar , 20 μm . ( Right ) Zoomed ( DIC - merged single - colour ) from different fields showing vesicular reticular distribution of incorporated NPC2 - Alexa488 . Scale bar , 10 μm .

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

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 .

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 B-MEASURED_VAR O O O O O O O

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 .

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

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 of lung sections from 5 - week - old BVE mice on Npc2 + / + ( + / + ) or Npc2 + / hypo ( + / hypo ) background is shown . Scale bars , 500 μm .

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

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

O O O O O B-CONTROLLED_VAR 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

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


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

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

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 + / + ( + / + ) Npc2 + / hypo ( + / hypo ) BVE mice are shown . Representative flow cytometry plots of three independent experiments are indicated .

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

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 ) , focusing on intratumour Mac2 + clusters . Scale bar , 50 μm . Enlarged of boxed areas 1 2 highlight Ki67 + Mac2 − associated with Mac2 + ( arrows ) .

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

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

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

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

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 ) LAMP2 immunofluorescence ( purple ) . Scale bars , 5 μm . Coarse structures strongly stained with filipin are surrounded by membranous of LAMP2 . Single - colour greyscale are also demonstrated in middle ( filipin ) right ( LAMP2 ) .

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

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 in Npc2 + / hypo IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . Scale bars , 25 μm . Quantitative data in bar graph were obtained by analysing 265 bNPC2 - untreated ( - bNPC2 ) 252 bNPC2 - treated ( + bNPC2 ) from three independent cultures using method described in ( A ) .

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

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

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.


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

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

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

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

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 . right bottom image highlights boxed of merged image of bNPC2 - treated IMCs showing partial co - localisation of LAMP1 CCL6 .

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

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 of intracellular 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 then treated with 200 nM bafilomycin A1 for 24 h .

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

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 SILAC - quantitative proteomics experiment in B . subtilis . 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 " " ( containing stable 12C614N2 - Lysine ) M9 minimal medium , respectively . After mixing cultures in 1 : 1 ( on OD600 ) , suspension was lysed fraction enriched for transmembrane proteome was analysed in a GeLC - MS / MS experiment . Using bioinformatic of MS data , highest - scoring candidate substrates were further evaluated .

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

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

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

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

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

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

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

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

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

(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 ) 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 ) . strains further contain a deletion in putative manganese efflux pump MntP ( ΔywlD , Table EV2 ) . Bottom panel shows that manganese is more toxic in yqgP deficient than in wild type , that reintroduction of YqgP rescues fitness during manganese stress to above - wild - type level . Top panel shows no difference between strains in absence of manganese stress . Data are shown as datapoints from three independent experiments overlaid with dashed connecting average values from each time - point , which illustrates reproducibility of .

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

(B) 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.


( C ) Top panel : growth curves of wild - type ( BTM843 ) , yqgP deficient ( BTM844 ) rescue ( BTM845 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese shock elicited by adding 75 µM MnSO4 in mid - exponential phase . Bottom panel : manganese toxicity is prevented by further adding 5 mM magnesium ( MgSO4 ) in otherwise identical conditions .

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

(C) Top panel: growth curves of wild-type (BTM843), yqgP deficient (BTM844) and rescue (BTM845) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese shock elicited by adding 75 µM MnSO4 in mid-exponential phase. Bottom panel: manganese toxicity is prevented by further adding 5 mM magnesium (MgSO4) in otherwise identical conditions.


( D ) Inhibition of YqgP by a rhomboid - specific peptidyl ketoamide inhibitor ( STS736 , i . e . compound 9 from Ticha et al . , 2017b ( ) ) abolishes YqgP induced fitness of B . subtilis under manganese stress , in a dose - dependent manner , both with endogenous YqgP ( top panel ) overexpressed YqgP ( bottom panel ) . Media growth conditions were identical to those used in panel ( B ) .

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

(D) Inhibition of YqgP by a rhomboid-specific peptidyl ketoamide inhibitor (STS736, i.e. compound 9 from Ticha et al., 2017b()) abolishes the YqgP induced fitness of B. subtilis under manganese stress, in a dose-dependent manner, both with endogenous YqgP (top panel) and overexpressed YqgP (bottom panel). Media and growth conditions were identical to those used in panel (B).


( E ) Overexpression of heterologous MgtE inhibits growth of yqgP deficient ( BTM610 , Table EV2 ) in rich LB medium supplemented with 75 μM MnSO4 . fitness is improved by overexpression of YqgP ( BTM611 , Table EV2 ) or its catalytically dead mutant YqgP . S288A ( BTM612 , Table EV2 ) . For clarity , for panels C - E , representative experiments of 2 - 3 independent biological replicates are shown .

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 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

(E) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain (BTM610, Table EV2) in rich LB medium supplemented with 75 μM MnSO4. Cell fitness is improved by overexpression of YqgP (BTM611, Table EV2) or its catalytically dead mutant YqgP.S288A (BTM612, Table EV2). For clarity, for panels C - E, representative experiments of 2-3 independent biological replicates are shown.


( B ) Detection ( left panel ) ( right panel ) of steady - state conversion of endogenous MgtE model chimeric substrate derived from Providencia stuartii TatA Stevenson et al . , 2007Ticha et al . , 2017aTicha et al . , 2017b ( , , ) ( schemes in middle ) by ectopically expressed YqgP its N - C - terminally truncated variants ( strains BS184 - 187 , Table EV2 ) in living B . subtilis grown in rich LB medium . Black arrows indicate full - substrates red arrows indicate cleavage products .

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

(B) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA Stevenson et al., 2007Ticha et al., 2017aTicha et al., 2017b(, , ) (schemes in the middle) by ectopically expressed YqgP and its N- and C-terminally truncated variants (strains BS184-187, Table EV2) in living B. subtilis grown in rich LB medium. Black arrows indicate full-length substrates and red arrows indicate cleavage products.


( C ) Similar of same strains but grown in minimal M9 medium ( with 10 µM MgSO4 ) supplemented with increasing concentrations of MnCl 2 . Black arrows denote full - substrate ( MgtE ) , red arrows denote cleavage products generated by YqgP . ( D ) Western blots of four independent experiments shown in panel ( C ) were quantified by near - infrared detection , quantified by densitometry results are displayed as fold activation of MgtE cleavage at 100 µM MnCl 2 relative to 1 µM MnCl 2 activity . Average values fours datapoints are plotted for each indicated YqgP variant .

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

(C) Similar analysis of the same strains but grown in minimal M9 medium (with 10 µM MgSO4) supplemented with increasing concentrations of MnCl2. Black arrows denote full-length substrate (MgtE), red arrows denote the cleavage products generated by YqgP. (D) Western blots of four independent experiments shown in panel (C) were quantified by near-infrared fluorescence detection, quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl2 relative to 1 µM MnCl2 activity. Average values and all fours datapoints are plotted for each indicated YqgP variant.


( A ) Detection ( left panel ) ( right panel ) of steady - state conversion of endogenous MgtE by YqgP ( BS72 , Table EV2 ) grown in minimal M9 medium containing 10 μM MgSO4 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 CaCl2 ( reference conditions ) with additions of 10 µM or 100 μM of a given divalent cation salt solution ( MnCl2 , ZnCl2 , CoCl2 , or NiCl2 ) , or 50 µM 500 μM for CaCl2 . Western blots were quantified by near - infrared detection ( left ) , quantified by densitometry displayed as relative specific activity ( graph on right ) , which is substrate conversion normalized to enzyme expression level . Black arrow denotes full - substrate ( MgtE ) , red arrow denotes N - terminal cleavage product ( s ) generated by YqgP . bands originate from same western blot identical treatment series .

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

(A) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE by YqgP (strain BS72, Table EV2) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (reference conditions) with the additions of 10 µM or 100 μM of a given divalent cation salt solution (MnCl2, ZnCl2, CoCl2, or NiCl2), or 50 µM and 500 μM for CaCl2. Western blots were quantified by near-infrared fluorescence detection (left), quantified by densitometry and displayed as relative specific activity (graph on the right), which is substrate conversion normalized to enzyme expression level. Black arrow denotes full-length substrate (MgtE), and red arrow denotes the N-terminal cleavage product(s) generated by YqgP. All bands originate from the same western blot and identical treatment series.


( B ) Isothermal titration calorimetry of purified recombinant YqgPNTD selected divalent cations .

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

(B) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations.


( C ) Cation toxicity assays their relationship to YqgP activity . Wild type B . subtilis ( BTM2 , Table EV2 ) , its variant lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) , rescue ectopically expressing YqgP ( BTM501 , Table EV2 ) were cultivated in minimal M9 medium containing 10 μM MgSO4 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 CaCl2 ( i . e . reference conditions ) , in mid - exponential phase were stressed by addition of either 70 µM MnCl2 , 500 µM ZnCl2 , 25 µM CoSO4 or 400 µM NiCl2 ( pale blue ) . YqgP activity specifically improved fitness during manganese zinc stress , while it had no effect on growth of cultivated in presence of high cobalt nickel concentrations . Representative experiments of 2 - 3 independent replicates are shown .

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

(C) Cation toxicity assays and their relationship to YqgP activity. Wild type B. subtilis (BTM2, Table EV2), its variant lacking YqgP (ΔyqgP, BTM78, Table EV2), and the rescue strain ectopically expressing YqgP (BTM501, Table EV2) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (i.e. reference conditions), and in mid-exponential phase were stressed by the addition of either 70 µM MnCl2, 500 µM ZnCl2, 25 µM CoSO4 or 400 µM NiCl2 (pale blue area). YqgP activity specifically improved cell fitness during manganese and zinc stress, while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations. Representative experiments of 2-3 independent replicates are shown.


( E Detection of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 . Black arrow marks full - MgtE , red arrow marks its N - terminal cleavage product by YqgP .

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

(E Detection of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2. Black arrow marks full-length MgtE, and red arrow marks its N-terminal cleavage product by YqgP.


F ) of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 .

O O O O O O O O O B-MEASURED_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

F) quantification of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2.


( A ) proteolytically dead mutant YqgP . S288A behanes as a pseudoprotease acts as a substrate adaptor of FtsH . Pattern of C - terminally truncated forms of endogenous MgtE formed in vivo in presence of ectopically expressed active YqgP ( BS72 , Table EV2 ) or its inactive S288A mutant ( BS73 , Table EV2 ) in rich LB medium in presence or absence of endogenous FtsH . Black arrow denotes full - MgtE substrate , red arrows denote its cleavage products generated by YqgP . While presence of faster migrating YqgP - dependent products ( full red arrows ) do not depend on presence of FtsH , slower migrating product formed in presence of YqgP . S288A ( open red arrow ) does , indicating that FtsH protease is responsible for alternative cleavage of MgtE . same arrow symbolics are valid throughout this figure .

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

(A) The proteolytically dead mutant YqgP.S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH. Pattern of C-terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP (strain BS72, Table EV2) or its inactive S288A mutant (BS73, Table EV2) in rich LB medium in the presence or absence of endogenous FtsH. Black arrow denotes the full-length MgtE substrate, and red arrows denote its cleavage products generated by YqgP. While the presence of the faster migrating YqgP-dependent products (full red arrows) do not depend on the presence of FtsH, the slower migrating product formed in the presence of YqgP.S288A (open red arrow) does, indicating that FtsH protease is responsible for the alternative cleavage of MgtE. The same arrow symbolics are valid throughout this figure.