( C - F ) WT MEFs ( C E ) Drp1 - KO MEFs ( D F ) were transduced with Su9 - Eos , along with Oma1 shRNAs ectopic Mfn1 . Su9 - Eos in small region of mitochondria ( indicated by asterisks ) was photoconverted using a 405 - nm on confocal microscope . Within 1 s after photoconversion , were obtained for both photoconverted unconverted Su9 - Eos signals . before photoconversion were also taken to detect background signals . Scale bar , 10 µm . ( E F ) To determine connectivity of mitochondria , containing photoconverted Su9 - Eos signals was divided by containing photoconverted unconverted Su9 - Eos signals ( total mitochondria ) . Values are average ± SD ( n = 30 ) .

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(C-F) WT MEFs (C and E) and Drp1-KO MEFs (D and F) were transduced with Su9-Eos, along with Oma1 shRNAs and ectopic Mfn1. Su9-Eos in the small region of mitochondria (indicated by the asterisks) was photoconverted using a 405-nm laser on confocal microscope. Within 1 s after photoconversion, images were obtained for both photoconverted and unconverted Su9-Eos signals. Images before the photoconversion were also taken to detect background signals. Scale bar, 10 µm. (E and F) To determine the connectivity of mitochondria, the area containing photoconverted Su9-Eos signals was divided by the area containing the photoconverted and unconverted Su9-Eos signals (total mitochondria). Values are average ± SD (n = 30 cells).


WT which carry shRNAs ( Oma1 or scramble ) ectopic Mfn1 , were stained with TMRE . of that maintained potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 were analyzed in each experiment .

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WT which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment.


Drp1 - KO MEFs which carry shRNAs ( Oma1 or scramble ) ectopic Mfn1 , were stained with TMRE . arrows indicate that lost mitochondrial potential in ( C ) . Scale bar , 10 µm . of that maintained potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 were analyzed in each experiment .

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Drp1-KO MEFs which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The arrows indicate cells that lost the mitochondrial membrane potential in (C). Scale bar, 10 µm. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment.


OCRs were analyzed in same set of MEFs . of basal OCRs respiratory capacity is shown . Values are average ± SD ( n = 3 ) .

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OCRs were analyzed in the same set of MEFs. Quantification of the basal OCRs and respiratory capacity is shown. Values are average ± SD (n = 3).


( A ) Western blotting of livers isolated from indicated mice at 3 months of using indicated antibodies . ( B ) of band . Values are average ± SD ( n = 3 mice for each genotype ) . Significance was calculated using ANOVA with post - hoc Tukey : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(A) Western blotting of livers isolated from the indicated mice at 3 months of age using the indicated antibodies. (B) Quantification of band intensity. Values are average ± SD (n = 3 mice for each genotype). Significance was calculated using ANOVA with post-hoc Tukey: *p<0.05, **p<0.01, ***p<0.001.


( A ) HEK293A lines stably expressing GFP , GFP - WIPI1a , GFP - WIPI2b were incubated in full medium ( F ) or starvation medium ( EBSS ) ( S ) for 2 hr before being used for GFP - TRAP pull - down .

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(A) HEK293A cell lines stably expressing GFP, GFP-WIPI1a, and GFP-WIPI2b were incubated in full medium (F) or starvation medium (EBSS) (S) for 2 hr before being used for GFP-TRAP pull-down.


( B ) WIPI2 was immunoprecipitated from HEK293A treated with DSP , at indicated concentrations , before immunoblotting .

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(B) WIPI2 was immunoprecipitated from HEK293A cells treated with DSP, at the indicated concentrations, before immunoblotting.


( C ) WT or Atg16L1Δ / Δ MEFs in fed medium or starved for 2 hr in EBSS were fixed labeled with an anti - WIPI2 antibody . Scale bars , 10 μm . ( D ) WIPI2 in ( C ) were counted , a statistical of WIPI2 was performed using an unpaired Student ' s t . ∗ p < 0 . 05 . for n = 3 .

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(C) WT or Atg16L1Δ/Δ MEFs in fed medium or starved for 2 hr in EBSS were fixed and labeled with an anti-WIPI2 antibody. Scale bars, 10 μm.(D) WIPI2 puncta in (C) were counted, and a statistical analysis of WIPI2 puncta was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3.


( E - H ) CLEM ( correlative electron microscopy ) of endogenous WIPI2 in Atg16L1Δ / Δ MEFs . ( E ) Merged phase confocal section of Atg16L1Δ / Δ labeled with anti - WIPI2 antibody . Scale bar , 10 μm . ( F ) Low - magnification of in ( E ) . Scale bar 10μm . ( G H ) High magnification of boxed regions in ( E ) ( F ) . Top box bottom box indicate panels ( G ) ( H ) , respectively . Arrows indicate open phagophores . M , mitochondria ; ER , endoplasmic reticulum . Scale bars , 1 μm ( G ) 0 . 5 μm ( H ) .

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(E-H) CLEM (correlative light and electron microscopy) of endogenous WIPI2 in Atg16L1Δ/Δ MEFs.(E) Merged phase and confocal section of Atg16L1Δ/Δ cells labeled with anti-WIPI2 antibody. Scale bar, 10 μm.(F) Low-magnification TEM of cell in (E). Scale bar 10μm.(G and H) High magnification of boxed regions in (E) and (F). Top box and bottom box indicate panels (G) and (H), respectively. Arrows indicate open phagophores. M, mitochondria; ER, endoplasmic reticulum. Scale bars, 1 μm (G) and 0.5 μm (H).


( I J ) 2GL9 ( GFP - LC3 HEK293 ; Chan et al . , 2007 ) were starved for 2 hr before visualization using indicated antibodies . Scale bars , 10 μm .

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(I and J) 2GL9 cells (GFP-LC3 HEK293 cells; Chan et al., 2007) were starved for 2 hr before visualization using indicated antibodies. Scale bars, 10 μm.


( K ) Live - imaging demonstrates that Atg16L1 translocates to WIPI2b - positive in starvation . HEK293 expressing GFP - WIPI2b mCherry - Atg16L1 were starved in EBSS imaged every 2 s using a spinning disk microscope . See also Figure S1 Movie S1 .

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(K) Live-cell imaging demonstrates that Atg16L1 translocates to WIPI2b-positive puncta in starvation. HEK293 cells expressing GFP-WIPI2b and mCherry-Atg16L1 were starved in EBSS and imaged every 2 s using a spinning disk microscope. See also Figure S1 and Movie S1.


( B ) Untransfected ( UN ) or FLAG - Atg16L1 full - ( FL ) , 79 - 623 , or 1 - 265 constructs were expressed in HEK293A stably expressing GFP - WIPI2b immunoprecipitated using GFP - TRAP . Tags were visualized by immunoblotting . I , input ; UB , unbound .

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(B) Untransfected (UN) or FLAG-Atg16L1 full-length (FL), 79-623, or 1-265 constructs were expressed in HEK293A cells stably expressing GFP-WIPI2b and immunoprecipitated using GFP-TRAP. Tags were visualized by immunoblotting. I, input; UB, unbound.


( C ) GFP , CFP - Atg5 , GFP - WIPI1a , GFP - WIPI2b were transiently expressed in HEK293A . GFP - tagged were isolated using GFP - TRAP incubated with in vitro translated 35S - labeled FLAG - Atg16L1 constructs 1 - 265 , 1 - 242 , 1 - 230 , 1 - 207 before washing by autoradiography . expression was validated by immunoblot ( bottom panel ) .

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(C) GFP, CFP-Atg5, GFP-WIPI1a, and GFP-WIPI2b were transiently expressed in HEK293A cells. GFP-tagged proteins were isolated using GFP-TRAP and incubated with in vitro translated 35S-labeled FLAG-Atg16L1 constructs 1-265, 1-242, 1-230, and 1-207 before washing and analysis by autoradiography. Protein expression was validated by immunoblot (bottom panel).


( D ) Lysates from HEK293A transiently expressing GFP , GFP - WIPI1a , or GFP - WIPI2b were mixed with lysates from HEK293A transiently expressing FLAG - Atg16L1 or Atg16L2 . complexes were immunoprecipitated using GFP - TRAP , followed by immunoblot . See also Figure S2 Table S1 .

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(D) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI1a, or GFP-WIPI2b were mixed with lysates from HEK293A cells transiently expressing FLAG-Atg16L1 or Atg16L2. Protein complexes were immunoprecipitated using GFP-TRAP, followed by immunoblot. See also Figure S2 and Table S1.


( B ) Lysates from HEK293A transfected with GFP - WIPI2b were mixed with lysates from HEK293A transfected with FLAG - Atg16L2 , FLAG - Atg16L1 , or Flag - Atg16L1 mutants , immunoprecipitated , analyzed by immunoblotting . ( C ) Statistical of FLAG - Atg16L1 binding in ( B ) was performed by Student ' s t . for n = 4 . ∗ p < 0 . 05 .

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(B) Lysates from HEK293A cells transfected with GFP-WIPI2b were mixed with lysates from HEK293A cells transfected with FLAG-Atg16L2, FLAG-Atg16L1, or Flag-Atg16L1 mutants, immunoprecipitated, and analyzed by immunoblotting.(C) Statistical analysis of FLAG-Atg16L1 binding in (B) was performed by Student's t test. SEM for n = 4. ∗p < 0.05.


( D ) lysates from HEK293A transiently expressing FLAG - Atg16L2 , FLAG - Atg16L1 , or FLAG - Atg16L1 mutants were subjected to immunoprecipitation using FLAG M2 agarose beads . complexes were analyzed using immunoblotting . ( E ) Statistical of FIP200 binding in ( D ) was performed by Student ' s t . for n = 3 . ∗ p < 0 . 05 .

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(D) Cell lysates from HEK293A cells transiently expressing FLAG-Atg16L2, FLAG-Atg16L1, or FLAG-Atg16L1 mutants were subjected to immunoprecipitation using FLAG M2 agarose beads. Protein complexes were analyzed using immunoblotting.(E) Statistical analysis of FIP200 binding in (D) was performed by Student's t test. SEM for n = 3. ∗p < 0.05.


( F ) WIPI2 was immunoprecipitated from lysates from WT or FIP200 − / − MEFS after treatment with 0 . 5 mM DSP . Bound Atg16 Atg12 - 5 were detected by immunoblotting .

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(F) WIPI2 was immunoprecipitated from lysates from WT or FIP200−/− MEFS after treatment with 0.5 mM DSP. Bound Atg16 and Atg12-5 were detected by immunoblotting.


( G ) HEK293 stably expressing GFP - WIPI2b were transiently transfected either FLAG - Atg16L1 WT , E226R E230R ( ERER ) , or D237R D239R ( DRDR ) . Bound input ( bottom ) were analyzed by immunoblotting .

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(G) HEK293 cells stably expressing GFP-WIPI2b were and transiently transfected either FLAG-Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR). Bound and input (bottom) were analyzed by immunoblotting.


( A ) Lysates from HEK293A transiently expressing GFP , GFP - WIPI1a , GFP - WIPI2a , GFP - WIPI2b , or GFP - WIPI2b FTTG mutant were used for GFP - Trap . Endogenous Atg16L1 binding was analyzed by immunoblot . ( B ) Statistical of ( A ) was performed by one - way ANOVA with Tukey ' s posttest . for n = 3 . ∗ p < 0 . 05 .

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(A) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI1a, GFP-WIPI2a, GFP-WIPI2b, or GFP-WIPI2b FTTG mutant were used for GFP-Trap. Endogenous Atg16L1 binding was analyzed by immunoblot.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's posttest. SEM for n = 3. ∗p < 0.05.


( D ) GFP - Trap from HEK293A transiently expressing GFP , GFP - WIPI1a , GFP - WIPI2b , GFP - WIPI2b R108E , GFP - WIPI2b R125E , or GFP - WIPI2b R108E R125E was mixed with in vitro translated 35S - labeled FLAG - Atg16L1 analyzed by autoradiography .

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(D) GFP-Trap from HEK293A cells transiently expressing GFP, GFP-WIPI1a, GFP-WIPI2b, GFP-WIPI2b R108E, GFP-WIPI2b R125E, or GFP-WIPI2b R108E R125E was mixed with in vitro translated 35S-labeled FLAG-Atg16L1 and analyzed by autoradiography.


( E ) Lysates from HEK293A transiently expressing GFP , GFP - WIPI2b , GFP - WIPI2b ΔCT , GFP - WIPI2a , GFP - WIPI1a , or GFP - WIPI1a ΔCT were used for GFP - Trap . Endogenous Atg16L1 binding was analyzed by immunoblot . ( F ) Statistical of ( E ) was performed by one - way ANOVA with Tukey ' s posttest . for n = 2 . ∗ p < 0 . 05 .

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(E) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI2b, GFP-WIPI2b ΔCT, GFP-WIPI2a, GFP-WIPI1a, or GFP-WIPI1a ΔCT were used for GFP-Trap. Endogenous Atg16L1 binding was analyzed by immunoblot.(F) Statistical analysis of (E) was performed by one-way ANOVA with Tukey's posttest. SEM for n = 2. ∗p < 0.05.


( G ) Lysates from HEK293A transiently expressing GFP - WIPI2b WT , GFP - WIPI2b R108E , R125E , or R108E R125E were mixed with lysates from HEK293A transiently expressing FLAG - Atg16L1 WT , E226R , E230R , or E226R E230R in possible permutations . complexes from mixed lysates were immunoprecipitated using GFP - Trap , followed by immunoblot . ( H ) Statistical of ( G ) was performed by one - way ANOVA with Tukey ' s posttest . from n = 3 . ∗ p < 0 . 05 .

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(G) Lysates from HEK293A cells transiently expressing GFP-WIPI2b WT, GFP-WIPI2b R108E, R125E, or R108E R125E were mixed with lysates from HEK293A cells transiently expressing FLAG-Atg16L1 WT, E226R, E230R, or E226R E230R in all possible permutations. Protein complexes from mixed lysates were immunoprecipitated using GFP-Trap, followed by immunoblot analysis.(H) Statistical analysis of (G) was performed by one-way ANOVA with Tukey's posttest. SEM from n = 3. ∗p < 0.05.


( I ) CLEM of HEK293 treated with siRNA to WIPI2 were transfected with GFP - WIPI2b RERE starved in EBSS . Bright - field image ( top ) , GFP - WIPI2 RERE signal ( middle ) , of selected ( bottom ) . Boxed shown in ( J ) . Scale bars represent 10 μM for bright - field confocal 5 μM for . ( J ) High - magnification of boxed in ( I ) . Arrows indicate open phagophores in vicinity of ER mitochondria ( M ) , which contain GFP - WIPI2b RERE . See also Figures S4 S5 Table S1 .

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(I) CLEM analysis of HEK293 cells treated with siRNA to WIPI2 were transfected with GFP-WIPI2b RERE and starved in EBSS. Bright-field image (top), GFP-WIPI2 RERE signal (middle), TEM of selected cell (bottom). Boxed area shown in (J). Scale bars represent 10 μM for bright-field and confocal and 5 μM for TEM.(J) High-magnification TEM of boxed area in (I). Arrows indicate open phagophores in the vicinity of ER and mitochondria (M), which contain GFP-WIPI2b RERE. See also Figures S4 and S5 and Table S1.


( A ) GFP , siRNA - resistant GFP - WIPI2b , or GFP - WIPI2b RERE was expressed in HEK293A treated for 72 hr with either RISC - free ( RF ) or WIPI2 siRNA . were left in full medium ( F ) or starved for 2 hr with EBSS ( S ) or EBSS with BafA ( B ) before immunoblot . ( B ) Statistical of ( A ) . for n = 3 . Statistical was performed by one - way ANOVA with Tukey ' s posttest . ∗ p < 0 . 05 .

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(A) GFP, siRNA-resistant GFP-WIPI2b, or GFP-WIPI2b RERE was expressed in HEK293A cells treated for 72 hr with either RISC-free (RF) or WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(B) Statistical analysis of (A). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Tukey's posttest. ∗p < 0.05.


( C ) GFP , siRNA - resistant GFP - WIPI2b , GFP - WIPI2b RERE , GFP - WIPI2b FTTG , or GFP - WIPI2b FTTG RERE was expressed in HEK293A treated for 72 hr with WIPI2 siRNA . were left in full medium ( F ) or starved for 2 hr with EBSS ( S ) or EBSS with BafA ( B ) before immunoblot . ( D ) Statistical of ( C ) was performed by one - way ANOVA with Tukey ' s posttest . for LC3 ( n = 2 ) p62 ( n = 4 ) are shown . ∗ p < 0 . 05 .

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(C) GFP, siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's posttest. The SEM for LC3 (n = 2) and p62 (n = 4) are shown. ∗p < 0.05.


( E ) siRNA - resistant GFP - WIPI2b , GFP - WIPI2b RERE , GFP - WIPI2b FTTG , or GFP - WIPI2b FTTG RERE was expressed in HEK293A treated for 72 hr with WIPI2 siRNA . were starved in EBSS for 2 hr without or with BafA , fixed , labeled , LC3 was visualized by confocal microscopy . Scale bars , 10 μm . ( F ) of WIPI2 per from ( E ) . from 10 per condition ; ∗ ∗ ∗ p < 0 . 001 using one - way ANOVA . ( G ) Statistical of LC3 from ( E ) with GFP control ( not shown in E ) . for n = 3 . Statistical was performed by one - way ANOVA with Dunn ' s posttest t . ∗ p < 0 . 05 . See also Table S1 .

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(E) siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were starved in EBSS for 2 hr without or with BafA, fixed, and labeled, and LC3 was visualized by confocal microscopy. Scale bars, 10 μm.(F) Quantification of WIPI2 puncta per cell from (E). SEM from 10 cells per condition; ∗∗∗p < 0.001 using one-way ANOVA.(G) Statistical analysis of LC3 puncta from (E) with GFP control (not shown in E). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Dunn's posttest t test. ∗p < 0.05. See also Table S1.


( A ) HEK293A transiently expressing indicated mCherry constructs were treated in either full medium ( F ) , starvation medium ( S ) , or starvation medium with wortmannin ( W ) for 2 hr before immunoblot . ( B ) Statistical of ( A ) was performed by one - way ANOVA with Tukey ' s post hoc . for n = 3 . ∗ p < 0 . 05 .

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(A) HEK293A cells transiently expressing the indicated mCherry constructs were treated in either full medium (F), starvation medium (S), or starvation medium with wortmannin (W) for 2 hr before immunoblot analysis.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05.


( C ) Complexes from HEK293A transiently expressing indicated mCherry constructs were immunoprecipitated using RFP - Trap before by immunoblotting .

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(C) Complexes from HEK293A cells transiently expressing the indicated mCherry constructs were immunoprecipitated using RFP-Trap before analysis by immunoblotting.


( D ) 2GL9 transiently expressing indicated mCherry constructs treated as in ( A ) were fixed visualized by confocal microscopy .

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(D) 2GL9 cells transiently expressing the indicated mCherry constructs treated as in (A) were fixed and visualized by confocal microscopy.


( E ) CLEM of MCF7 expressing GFP - LC3 mCherry - WIPI2b CAAX . Bright - field confocal image merged ( left ) , confocal of expressing ( middle ) , low - magnification of of interest ( right ) . Boxed indicates plasma region showing colocalization of GFP - LC3 mCherry - WIPI2b . Scale bar , 10 μm ( n = 3 ) . ( F ) High - magnification showing plasma region boxed in ( E ) . Magnified insets , in top right , show small vesicular clusters under plasma detected in mCherry - WIPI2b CAAX , GFP - LC3 expressing but not in untransfected from a control experiment .

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

(E) CLEM of MCF7 cells expressing GFP-LC3 and mCherry-WIPI2b CAAX. Bright-field and confocal image merged (left), confocal of expressing cell (middle), low-magnification TEM of cell of interest (right). Boxed area indicates plasma membrane region showing colocalization of GFP-LC3 and mCherry-WIPI2b. Scale bar, 10 μm (n = 3).(F) High-magnification TEM showing plasma membrane region boxed in (E). Magnified insets, in top right, show small vesicular clusters under the plasma membrane detected in mCherry-WIPI2b CAAX, GFP-LC3 expressing cells but not in untransfected cells from a control experiment.


( G ) HEK293A treated with either RISC - free ( RF ) , Atg16L1 siRNA , or FIP200 siRNA for 72 hr before transfection with HA - WIPI2b - CAAX were incubated in full medium ( F ) , EBSS ( S ) , or EBSS with wortmannin ( W ) for 2 hr before immunoblot . Please note that WIPI2 constructs are FTTG mutants . See also Figure S6 Table S1 .

O 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 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 O O O O O

(G) HEK293A cells treated with either RISC-free (RF), Atg16L1 siRNA, or FIP200 siRNA for 72 hr before transfection with HA-WIPI2b-CAAX were incubated in full medium (F), EBSS (S), or EBSS with wortmannin (W) for 2 hr before immunoblot analysis. Please note that all WIPI2 constructs are FTTG mutants. See also Figure S6 and Table S1.


( A ) Atg16L1Δ / Δ MEFs were transiently transfected with either mock , FLAG - tagged Atg16L1 WT , ERER , or DRDR . After 2 hr in EBSS , were fixed labeled with anti - LC3 anti - FLAG antibodies visualized by confocal microscopy . Scale bars , 20 μm . ( B ) Statistical of ( A ) was performed using one - way ANOVA with Dunn ' s posttest . ∗ p < 0 . 05 . for n = 3 .

O O O B-CONTROLLED_VAR 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 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

(A) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, ERER, or DRDR. After 2 hr in EBSS, cells were fixed and labeled with anti-LC3 and anti-FLAG antibodies and visualized by confocal microscopy. Scale bars, 20 μm.(B) Statistical analysis of (A) was performed using one-way ANOVA with Dunn's posttest. ∗p < 0.05. SEM for n = 3.


( C ) Atg16L1Δ / Δ MEFs were transiently transfected with either mock , FLAG - tagged Atg16L1 WT , E226R E230R ( ERER ) , or D237R D239R ( DRDR ) before by immunoblotting . ( D ) Statistical of ( C ) was performed by one - way ANOVA with Tukey ' s post hoc . for n = 3 . ∗ p < 0 . 05 .

O O O B-CONTROLLED_VAR 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

(C) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR) before analysis by immunoblotting.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05.


( E ) HEK293A were treated with either RISC - free control or WIPI2 siRNA before infection with Salmonella ( moi = 100 ) for 1 hr , labeled with anti - LC3 , WIPI2 , p62 antibodies , followed by confocal . Scale bars , 5 μm . ( F ) Statistical for ( E ) was performed using an unpaired Student ' s t . ∗ p < 0 . 05 . for n = 3 .

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

(E) HEK293A cells were treated with either RISC-free control or WIPI2 siRNA before infection with Salmonella (moi = 100) for 1 hr, labeled with anti-LC3, WIPI2, and p62 antibodies, and followed by confocal analysis. Scale bars, 5 μm.(F) Statistical analysis for (E) was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3.


( G ) Atg16L1Δ / Δ MEFs were transiently transfected with FLAG - Atg16L1 WT , FLAG - Atg16L1 ERER , or FLAG - Atg16L2 DRDR 24 hr before being infected with Salmonella ( moi = 25 ) for 1 hr , labeled with anti - LC3 , anti - p62 , anti - FLAG antibodies , followed by by confocal microscopy . Scale bars , 5 μm . ( H ) Statistical of ( G ) was performed by one - way ANOVA with Dunn ' s post hoc . for n = 4 . ∗ p < 0 . 05 . See also Figure S7 .

O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR 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 B-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 O O O O O O O O O O O O O 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) Atg16L1Δ/Δ MEFs were transiently transfected with FLAG-Atg16L1 WT, FLAG-Atg16L1 ERER, or FLAG-Atg16L2 DRDR 24 hr before being infected with Salmonella (moi = 25) for 1 hr, labeled with anti-LC3, anti-p62, and anti-FLAG antibodies, and followed by analysis by confocal microscopy. Scale bars, 5 μm.(H) Statistical analysis of (G) was performed by one-way ANOVA with Dunn's post hoc test. SEM for n = 4. ∗p < 0.05. See also Figure S7.


A RT - qPCR reveals that miR - 210 was increased in lungs of mice with PH triggered by various conditions : VHL − / − as compared with VHL + / + mice ( N = 4 / group ) , * * * P < 0 . 0001 ( first graph ) ; hypoxia + SU5416 ( Hyp + SU5416 ) ( N = 6 / group ) as compared with normoxia + SU5416 ( Norm + SU5416 ) ( N = 7 / group ) , * * P = 0 . 0015 ( second graph ) ; Il6 transgenic versus littermate control mice ( N = 4 / group ) , * * P = 0 . 0097 ( third graph ) ; S . mansoni - infected mice ( N = 4 ) compared with non - infected control mice ( N = 5 ) , * * * P < 0 . 0001 ( fourth graph ) .

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 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 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

A RT-qPCR reveals that miR-210 was increased in lungs of mice with PH triggered by various conditions: VHL−/− as compared with VHL+/+ mice (N = 4/group), ***P < 0.0001 (first graph); hypoxia + SU5416 (Hyp + SU5416) (N = 6/group) as compared with normoxia + SU5416 (Norm + SU5416) (N = 7/group), **P = 0.0015 (second graph); Il6 transgenic versus littermate control mice (N = 4/group), **P = 0.0097 (third graph); and S. mansoni-infected mice (N = 4) compared with non-infected control mice (N = 5), ***P < 0.0001 (fourth graph).


B From subjects in ( A ) , in situ hybridization ( ISH , purple stain ) revealed increased miR - 210 in < 100 - μm pulmonary vessels of mice suffering from PH , * P = 0 . 0493 for first graph , * * P = 0 . 0015 for second graph , * P = 0 . 0391 for third graph .

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

B From animal subjects in (A), in situ hybridization (ISH, purple stain) revealed increased miR-210 in < 100-μm pulmonary vessels of mice suffering from PH, *P = 0.0493 for first graph, **P = 0.0015 for second graph, *P = 0.0391 for third graph.


C Representative ISH stain of miR - 210 in < 100 μm pulmonary vessels of mice ( bottom ) exposed to Hyp + SU5416 compared with Norm + SU5416 ( α - smooth muscle actin stain from serial sections , top row of ) .

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

C Representative ISH stain of miR-210 in < 100 μm pulmonary vessels of mice (bottom micrographs) exposed to Hyp + SU5416 compared with Norm + SU5416 (α-smooth muscle actin stain from serial sections, top row of micrographs).


D Increased miR - 210 in < 200 - μm remodeled pulmonary vessels of suffering from PAH ( N = 19 , Supplementary Table S1 ) as compared with non - PAH donor control lung ( N = 10 ) . Serial with hematoxylin eosin is displayed in top row of ; of miRNA ISH , right graph , * P = 0 . 0167 .

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

D Increased miR-210 in < 200-μm remodeled pulmonary vessels of patients suffering from PAH (N = 19, Supplementary Table S1) as compared with non-PAH donor control lung (N = 10). Serial staining with hematoxylin and eosin is displayed in the top row of micrographs; quantification of miRNA ISH, right graph, *P = 0.0167.


E Increased of miR - 210 in plasma drawn from pulmonary circulation ( pulmonary capillary wedge position , PCWP ) of with elevated mean pulmonary arterial pressures ( mean PAP ≥ 25 mmHg ) compared with control subjects ( mPAP < 25 mmHg , N = 5 / group , demographics in Supplementary Table S2 ) , * P = 0 . 0357 .

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 O O O O O O O O O

E Increased levels of miR-210 in plasma drawn from the pulmonary circulation (pulmonary capillary wedge position, PCWP) of patients with elevated mean pulmonary arterial pressures (mean PAP ≥ 25 mmHg) compared with control subjects (mPAP < 25 mmHg, N = 5/group, demographics in Supplementary Table S2), *P = 0.0357.


F From in ( C ) immunohistochemistry ( ) revealed that miR - 210 targets ISCU1 / 2 were reciprocally down - regulated in miR - 210 - enriched remodeled pulmonary vessels - namely in PH mice exposed to Hyp + SU5416 ( F , * * * P = 0 . 0002 ) .

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

F From animals in (C) immunohistochemistry (IHC) revealed that the miR-210 targets ISCU1/2 were reciprocally down-regulated in miR-210-enriched remodeled pulmonary vessels-namely in PH mice exposed to Hyp + SU5416 (F, ***P = 0.0002).


G From humans in ( D ) , immunohistochemistry ( ) revealed that miR - 210 targets ISCU1 / 2 were reciprocally down - regulated in miR - 210 - enriched remodeled pulmonary vessels - namely in human PAH ( G , * * * P = 0 . 0008 ) .

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 O O O O O O O O O O

G From humans in (D), immunohistochemistry (IHC) revealed that the miR-210 targets ISCU1/2 were reciprocally down-regulated in miR-210-enriched remodeled pulmonary vessels-namely in human PAH patients (G, ***P = 0.0008).


H , I miR - 210 expression ( H ) was increased ( * * * P = 0 . 0003 ) , ISCU1 / 2 expression ( I ) was decreased ( * * * P < 0 . 0001 ) in PECAM + pulmonary vascular endothelial isolated from PH mice ( Hyp + SU5416 ) as compared with control ( Norm + SU5416 ) ( N = 4 / group , left bars ) .

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 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 O O

H, I miR-210 expression (H) was increased (***P = 0.0003), and ISCU1/2 expression (I) was decreased (***P < 0.0001) in PECAM+pulmonary vascular endothelial cells isolated from PH mice (Hyp + SU5416) as compared with control (Norm + SU5416) (N = 4/group, left bars).


After lentiviral delivery of GCN4 or GRX2 sensor genes to human PAECs , was measured by flow cytometry . Unlike control GCN4 sensors that homodimerized independent of Fe - S induced consistent , derived from GRX2 Fe - S - dependent sensors decreased in hypoxia , as displayed in representative flow cytometric plots ( left ) by of of positive [ ( number in M2 gate ) / ( total number ) × 100 ] . Immunoblotting revealed consistent expression of either GRX2 or GCN4 sensors ( FLAG - tagged ) in hypoxia ( Hyp ) compared with normoxia ( Norm ) ( N = 3 , * * * P < 0 . 0001 for GRX2 ; N = 3 , NS P = 0 . 1848 for GCN4 ) . In contrast to consistent from control GCN4 sensors , Fe - S - dependent GRX2 sensor was decreased by siRNA knockdown of ISCU1 / 2 ( siISCU ) as compared with control ( siCont ) ( N = 3 , * * * P < 0 . 0001 for GRX2 ; N = 3 , NS P = 0 . 1790 for GCN4 ) .

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

After lentiviral delivery of GCN4 or GRX2 sensor genes to human PAECs, cellular fluorescence was measured by flow cytometry. Unlike control GCN4 sensors that homodimerized independent of Fe-S levels and induced consistent fluorescence, fluorescence derived from the GRX2 Fe-S-dependent sensors decreased in hypoxia, as displayed in representative flow cytometric plots (left) and by quantification of percentage of positive cells [(cell number in M2 gate)/(total cell number) × 100]. Immunoblotting revealed consistent expression of either GRX2 or GCN4 sensors (FLAG-tagged) in hypoxia (Hyp) compared with normoxia (Norm) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1848 for GCN4).In contrast to consistent fluorescence from control GCN4 sensors, Fe-S-dependent GRX2 sensor fluorescence was decreased by siRNA knockdown of ISCU1/2 (siISCU) as compared with control (siCont) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1790 for GCN4).


GRX2 , but not GCN4 , sensor was decreased after transfection of miR - 210 oligonucleotide mimic ( miR - 210 ) as compared with control ( miRC ) ( N = 3 , * * * P = 0 . 0002 for GRX2 ; N = 3 , NS P = 0 . 0913 for GCN4 ) . During hypoxic exposure , GRX2 , but not GCN4 , sensor was increased after transfection of an antisense miR - 210 inhibitor ( AS210 ) as compared with control ( ASC ) ( N = 3 , * * P = 0 . 003 for GRX2 ; N = 3 , NS P = 0 . 1194 for GCN4 ) .

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

GRX2, but not GCN4, sensor fluorescence was decreased after transfection of miR-210 oligonucleotide mimic (miR-210) as compared with control (miRC) (N = 3, ***P = 0.0002 for GRX2; N = 3, NS P = 0.0913 for GCN4). During hypoxic exposure, GRX2, but not GCN4, sensor fluorescence was increased after transfection of an antisense miR-210 inhibitor (AS210) as compared with control (ASC) (N = 3, **P = 0.003 for GRX2; N = 3, NS P = 0.1194 for GCN4).


After lentiviral delivery of sensor genes , Fe - S - dependent GRX2 sensor was decreased in PECAM - positive from PH mouse lung ( Hyp + SU5416 ) as compared with control non - PH mouse lung ( Norm + SU5416 , N = 3 , * * * P < 0 . 001 ) .

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

After lentiviral delivery of sensor genes, Fe-S-dependent GRX2 sensor fluorescence was decreased in PECAM-positive cells from PH mouse lung (Hyp + SU5416) as compared with control non-PH mouse lung (Norm + SU5416, N = 3, ***P < 0.001).


By electron paramagnetic resonance ( EPR ) spectroscopy ( representative VHL − / − versus control lung ) , Fe - S cluster signal was decreased ( left graph ) in VHL − / − mice lung tissue ( VHL − / − , N = 3 ) as compared with WT control lung tissues ( WT , N = 5 ) , * * * P = 0 . 0003 .

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 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

By electron paramagnetic resonance (EPR) spectroscopy (representative VHL−/− versus control lung), Fe-S cluster signal was decreased (left graph) in VHL−/− mice lung tissue (VHL−/−, N = 3) as compared with WT control lung tissues (WT, N = 5), ***P = 0.0003.


By EPR , Fe - S - specific signal was decreased ( right graph ) in human PH - diseased lung tissue harvested at lung transplantation ( PH , N = 3 , Supplementary Table S3 ) compared with non - PH control donor lung tissues ( control , N = 3 ) , * P = 0 . 048 .

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

By EPR, Fe-S-specific signal was decreased (right graph) in human PH-diseased lung tissue harvested at lung transplantation (PH, N = 3, Supplementary Table S3) compared with non-PH control donor lung tissues (control, N = 3), *P = 0.048.


B By immunohistochemistry ( ) , ISCU1 / 2 was unchanged in < 100 μm pulmonary vessels of miR - 210 − / − mice exposed to Hyp + SU5416 ( N = 6 ) versus Norm + SU5416 ( N = 7 ) , NS P = 0 . 2833 .

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

B By immunohistochemistry (IHC), ISCU1/2 was unchanged in < 100 μm pulmonary vessels of miR-210−/−mice exposed to Hyp + SU5416 (N = 6) versus Norm + SU5416 (N = 7), NS P = 0.2833.


C Fe - S - dependent I - specific activity was decreased in WT PH mice as compared with control ( N = 7 / group , left bars , * * * P < 0 . 0001 ) , but activity was preserved in lungs of miR - 210 − / − mice in either condition ( N = 8 / group , right bars ) , NS P = 0 . 3693 .

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

C Fe-S-dependent Complex I-specific activity was decreased in WT PH mice as compared with control (N = 7/group, left bars, ***P < 0.0001), but activity was preserved in the lungs of miR-210−/−mice in either condition (N = 8/group, right bars), NS P = 0.3693.


D demonstrated that pulmonary vascular GLUT1 was increased in WT PH mice ( left bars , * * * P = 0 . 0006 ) , but GLUT1 was unchanged in miR - 210 − / − mice in either condition ( right bars , NS P = 0 . 9967 ) .

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

D IHC demonstrated that pulmonary vascular GLUT1 was increased in WT PH mice (left bars, ***P = 0.0006), but GLUT1 was unchanged in miR-210−/− mice in either condition (right bars, NS P = 0.9967).


E 3 - nitrotyrosine ( 3 - NT ) was increased in pulmonary vessels of WT PH mice but was reduced in miR - 210 − / − mice in either condition ( N = 6 / group ) ( * * * P < 0 . 0001 , NS P = 0 . 4087 ) .

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

E 3-nitrotyrosine (3-NT) was increased in pulmonary vessels of WT PH mice but was reduced in miR-210−/− mice in either condition (N = 6/group) (***P < 0.0001, NS P = 0.4087).


F In PH conditions , endothelin - 1 was decreased in miR - 210 − / − lung tissue compared with WT tissue ( N = 5 / group ) , * * P = 0 . 007 .

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

F In PH conditions, endothelin-1 was decreased in miR-210−/− lung tissue compared with WT tissue (N = 5/group), **P = 0.007.


G PCNA was increased in WT PH pulmonary vessels but was decreased in miR - 210 − / − tissue exposed to either condition ( N = 5 / group ) ( * * P = 0 . 0096 , * P = 0 . 0124 , * P = 0 . 0263 for miR - 210 − / − ) .

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

G PCNA was increased in WT PH pulmonary vessels but was decreased in miR-210−/− tissue exposed to either condition (N = 5/group) (**P = 0.0096, *P = 0.0124, *P = 0.0263 for miR-210−/−).


H PCNA was increased in PECAM + endothelial from PH versus non - PH mice ( N = 3 / group , * * P = 0 . 0052 ) .

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

H PCNA was increased in PECAM+ endothelial cells from PH versus non-PH mice (N = 3/group, **P = 0.0052).


I Unlike WT mice ( black bars ) demonstrating increased right ventricular systolic pressure ( RVSP ) after Hyp + SU5416 ( N = 10 ) versus Norm + SU5416 ( N = 11 ) , hemodynamic dysregulation was significantly alleviated in miR - 210 − / − mice ( bars , N = 11 ) ( * * * P < 0 . 0001 , * P = 0 . 0258 , * * P = 0 . 0059 ) .

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

I Unlike WT mice (black bars) demonstrating increased right ventricular systolic pressure (RVSP) after Hyp + SU5416 (N = 10) versus Norm + SU5416 (N = 11), hemodynamic dysregulation was significantly alleviated in miR-210−/− mice (white bars, N = 11) (***P < 0.0001, *P = 0.0258, **P = 0.0059).


J Compared with WT controls ( N = 9 ) , miR - 210 − / − mice ( N = 8 ) displayed a blunted increase of Fulton index ( RV / LV + S ) under PH versus baseline conditions ( expressed as a of RV / LV + S under Hyp + SU5416 versus Norm + SU5416 , * P = 0 . 031 ) .

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J Compared with WT controls (N = 9), miR-210−/− mice (N = 8) displayed a blunted increase of the Fulton index (RV/LV + S) under PH versus baseline conditions (expressed as a ratio of RV/LV + S under Hyp + SU5416 versus Norm + SU5416, *P = 0.031).


K - M Under PH ( black bars , N = 8 / group ) versus baseline conditions ( bars , N = 6 / group ) , pulmonary vascular remodeling was alleviated in miR - 210 − / − mice , as visualized via histology ( L ) , confirmed by decreased % arteriolar muscularization ( K , * * P = 0 . 001 , * * P = 0 . 0045 for miR - 210 − / − , * P = 0 . 0158 ) decreased vessel wall thickness ( M , * * * P < 0 . 0001 , * * P = 0 . 0086 ) .

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K-M Under PH (black bars, N = 8/group) versus baseline conditions (white bars, N = 6/group), pulmonary vascular remodeling was alleviated in miR-210−/− mice, as visualized via histology (L), and confirmed by decreased % arteriolar muscularization (K, **P = 0.001, **P = 0.0045 for miR-210−/−, *P = 0.0158) and decreased vessel wall thickness (M, ***P < 0.0001, **P = 0.0086).


B , C Intrapharyngeal delivery of miR - 210 mimic increased miR - 210 in whole lung ( * * * P = 0 . 0002 ) ( B ) in < 100 - μm pulmonary vessels ( * * P = 0 . 007 ) ( C ) .

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B, C Intrapharyngeal delivery of miR-210 mimic increased miR-210 in whole lung (***P = 0.0002) (B) and in < 100-μm pulmonary vessels (**P = 0.007) (C).


D miR - 210 mimic also repressed ISCU1 / 2 in those same caliber vessels ( N = 6 / group ) , * * P = 0 . 0022 .

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D miR-210 mimic also repressed ISCU1/2 levels in those same caliber vessels (N = 6/group), **P = 0.0022.


E Endothelin - 1 was increased in mouse lung tissue after delivery of miR - 210 mimic ( N = 6 / group ) , * P = 0 . 0368 .

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E Endothelin-1 was increased in mouse lung tissue after delivery of miR-210 mimic (N = 6/group), *P = 0.0368.


F - H miR - 210 mimic delivery increased RVSP ( N = 8 / group ) ( F ) vascular remodeling ( α - smooth muscle actin stain , α - SMA ) , as evidenced by increased of muscularized ( < 100 μm ) pulmonary vessels ( G ) increased medial thickening relative to vessel diameter when compared with miR - Control ( H ) ( N = 6 / group ) . * * P = 0 . 0021 for ( F ) , * * P = 0 . 0072 for ( G ) , * P = 0 . 0335 for ( H ) .

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F-H miR-210 mimic delivery increased RVSP (N = 8/group) (F) and vascular remodeling (α-smooth muscle actin stain, α-SMA), as evidenced by increased percent of muscularized (< 100 μm) pulmonary vessels (G) and increased medial thickening relative to vessel diameter when compared with miR-Control (H) (N = 6/group). **P = 0.0021 for (F), **P = 0.0072 for (G), *P = 0.0335 for (H).


B - D Intravenous delivery of anti - miR - 210 down - regulated miR - 210 expression in whole lung tissue ( N = 7 / group ) , * * * P < 0 . 0001 ( B ) , pulmonary vasculature as assessed by in situ ( N = 7 / group ) , * * * P = 0 . 0003 ( C ) , PECAM + vascular endothelial derived from whole lung tissue ( N = 4 / group ) , * * P = 0 . 0013 ( D ) .

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B-D Intravenous delivery of anti-miR-210 down-regulated miR-210 expression in whole lung tissue (N = 7/group), ***P < 0.0001 (B), the pulmonary vasculature as assessed by in situ staining (N = 7/group), ***P = 0.0003 (C), and PECAM+vascular endothelial cells derived from whole lung tissue (N = 4/group), **P = 0.0013 (D).


E Correspondingly , anti - miR - 210 delivery resulted in preservation of ISCU1 / 2 expression in < 100 - μm pulmonary vessels as compared to anti - miR - Control ( N = 5 / group ) , * * P = 0 . 0011 .

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E Correspondingly, anti-miR-210 delivery resulted in preservation of ISCU1/2 expression in < 100-μm pulmonary vessels as compared to anti-miR-Control (N = 5/group), **P = 0.0011.


F Similarly , ISCU1 / 2 expression was preserved in PECAM + endothelial derived from mice treated with anti - miR - 210 ( N = 4 / group ) , * * P = 0 . 0016 .

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F Similarly, ISCU1/2 expression was preserved in PECAM+ endothelial cells derived from mice treated with anti-miR-210 (N = 4/group), **P = 0.0016.


G Endothelin - 1 was decreased in lung tissue from mice treated with anti - miR - 210 during Hyp + SU5416 exposure ( N = 6 / group ) , * P = 0 . 0181 .

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G Endothelin-1 was decreased in lung tissue from mice treated with anti-miR-210 during Hyp + SU5416 exposure (N = 6/group), *P = 0.0181.


H As assessed by in situ immunofluorescence , PCNA was decreased in pulmonary vessels ( < 100 μm ) after anti - miR - 210 delivery , * * P = 0 . 001 .

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H As assessed by in situ immunofluorescence, PCNA was decreased in pulmonary vessels (< 100 μm) after anti-miR-210 delivery, **P = 0.001.


I - K Anti - miR - 210 delivery ( N = 10 / group ) ameliorated elevation of RVSP , * * P = 0 . 0031 ( i ) vascular remodeling , as reflected by increased of muscularized arterioles ( N = 8 / group , * * P = 0 . 0091 ) ( J ) increased medial thickness relative to vessel diameter in < 100 - μm pulmonary vessels ( N = 6 / group , * * P = 0 . 0021 ) ( K ) .

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I-K Anti-miR-210 delivery (N = 10/group) ameliorated the elevation of RVSP, **P = 0.0031 (i) and vascular remodeling, as reflected by increased percent of muscularized arterioles (N = 8/group, **P = 0.0091) (J) and increased medial thickness relative to vessel diameter in < 100-μm pulmonary vessels (N = 6/group, **P = 0.0021) (K).


M Pharmacologic inhibition of miR - 210 ( N = 7 / group ) down - regulated pulmonary expression of miR - 210 , * * * P < 0 . 0001 ( M ) .

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M Pharmacologic inhibition of miR-210 (N = 7/group) down-regulated pulmonary expression of miR-210, ***P < 0.0001 (M).


N Pharmacologic inhibition of miR - 210 ( N = 7 / group ) preserved ISCU1 / 2 expression in < 100 - μm pulmonary vessels , * * P = 0 . 0015 ( N ) .

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N Pharmacologic inhibition of miR-210 (N = 7/group) preserved ISCU1/2 expression in < 100-μm pulmonary vessels, **P = 0.0015 (N).


O - Q In contrast to anti - miR - Control ( N = 8 ) , anti - miR - 210 ameliorated elevation of RVSP ( N = 10 / group , * * * P < 0 . 0001 ) ( O ) lung remodeling ( N = 7 / group , * * P = 0 . 0022 , * P = 0 . 0167 ) ( P - Q ) .

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O-Q In contrast to anti-miR-Control (N = 8), anti-miR-210 ameliorated the elevation of RVSP (N = 10/group, ***P < 0.0001) (O) and lung remodeling (N = 7/group, **P = 0.0022, *P = 0.0167) (P-Q).


A Following schema of Fig5A in presence of hypoxia but absence of SU5416 , intravenous delivery of anti - miR - 210 ameliorated elevation of RVSP compared with anti - miR - Control ( N = 6 / group , * * * P = 0 . 001 ) .

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A Following the schema of Fig5A in the presence of hypoxia but the absence of SU5416, intravenous delivery of anti-miR-210 ameliorated the elevation of RVSP compared with anti-miR-Control (N = 6/group, ***P = 0.001).


C , D Consistent with specific delivery to vascular endothelium after intravenous administration ( Dahlman et al , 2014 ) , 7C1 - mediated delivery of anti - miR - 210 ( N = 6 / group ) decreased miR - 210 expression in PECAM + pulmonary vascular endothelial , * * P = 0 . 0013 , ( D ) but not PECAM - negative pulmonary , NS P = 0 . 3775 ( C ) .

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C, D Consistent with specific delivery to the vascular endothelium after intravenous administration (Dahlman et al, 2014), 7C1-mediated delivery of anti-miR-210 (N = 6/group) decreased miR-210 expression in PECAM+ pulmonary vascular endothelial cells, **P = 0.0013, (D) but not PECAM-negative pulmonary cells, NS P = 0.3775 (C).


E Correspondingly , endothelial - specific anti - miR - 210 ( N = 6 / group ) increased ISCU1 / 2 expression in PECAM + even in presence of chronic hypoxia , * * * P = 0 . 0003 .

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E Correspondingly, endothelial-specific anti-miR-210 (N = 6/group) increased ISCU1/2 expression in PECAM+ cells even in the presence of chronic hypoxia, ***P = 0.0003.


F - I Delivery of anti - miR - 210 ( N = 5 / group ) decreased endothelin - 1 in hypoxic mouse lung tissue , * P = 0 . 0198 ( F ) . As a result , in contrast to anti - miR - Control ( N = 8 ) , endothelial - specific anti - miR - 210 ameliorated elevation of RVSP ( N = 6 ) , * * P = 0 . 0024 ( G ) , pulmonary vascular remodeling ( N = 6 / group ) , * * P = 0 . 0021 , * P = 0 . 0117 ( H - I ) .

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F-I Delivery of anti-miR-210 (N = 5/group) decreased endothelin-1 in hypoxic mouse lung tissue, *P = 0.0198 (F). As a result, in contrast to anti-miR-Control (N = 8), endothelial-specific anti-miR-210 ameliorated the elevation of RVSP (N = 6), **P = 0.0024 (G), and pulmonary vascular remodeling (N = 6/group), **P = 0.0021, *P = 0.0117 (H-I).


B , C Pharmacologic inhibition of ISCU ( N = 8 / group ) did not influence miR - 210 expression in lungs either after Norm + SU5416 , NS P = 0 . 8634 ( B ) or Hyp + SU5416 , NS P = 0 . 9786 ( C ) .

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B, C Pharmacologic inhibition of ISCU (N = 8/group) did not influence miR-210 expression in the lungs either after Norm + SU5416, NS P = 0.8634 (B) or Hyp + SU5416, NS P = 0.9786 (C).


D As compared with control siRNA ( siCont ) where ISCU1 / 2 expression was decreased after exposure to Hyp + SU5416 ( bars ) , lung delivery of siRNA specific for ISCU1 / 2 ( siISCU ) via Staramine - mPEG nanocomplexes ( Polach et al , 2012 ) down - regulated ISCU1 / 2 in < 100 - μm pulmonary vessels in Norm + SU5416 Hyp + SU5416 ( N = 5 / group , * * * P = 0 . 0006 , * * P = 0 . 0019 for Hyp + SU5416 , * * P = 0 . 0029 for siCont ) .

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D As compared with control siRNA (siCont) where ISCU1/2 expression was decreased after exposure to Hyp + SU5416 (white bars), lung delivery of siRNA specific for ISCU1/2 (siISCU) via Staramine-mPEG nanocomplexes (Polach et al, 2012) down-regulated ISCU1/2 in < 100-μm pulmonary vessels in Norm + SU5416 and Hyp + SU5416 (N = 5/group, ***P = 0.0006, **P = 0.0019 for Hyp + SU5416, **P = 0.0029 for siCont).


E By electron paramagnetic resonance ( EPR ) spectroscopy ( representative siCont versus siISCU lung ) , Fe - S cluster signal was decreased ( quantitative graph , right ) in siISCU mouselung tissue ( N = 4 mice ) compared with siCont ( N = 5 mice ) , * P = 0 . 0143 .

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E By electron paramagnetic resonance (EPR) spectroscopy (representative siCont versus siISCU lung), Fe-S cluster signal was decreased (quantitative graph, right) in siISCU mouselung tissue (N = 4 mice) compared with siCont (N = 5 mice), *P = 0.0143.


F Endothelin - 1 was increased in lung tissue after siISCU delivery under normoxic conditions ( N = 5 / group ) , * * P = 0 . 0027 .

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F Endothelin-1 was increased in lung tissue after siISCU delivery under normoxic conditions (N = 5/group), **P = 0.0027.


G - I In absence ( left bars ) presence ( right bars ) of Hyp + SU5416 for 2 , siISCU ( black bars ) induced elevations in RVSP ( * * * P < 0 . 0001 , * * P = 0 . 0031 ) ( G ) increased pulmonary vascular remodeling in < 100 - μm pulmonary vessels ( H , I ) as compared with siCont ( N = 5 / group , * * * P = 0 . 0003 , * P = 0 . 0114 for H ; * * * P = 0 . 0006 , * * P = 0 . 0098 for I ) .

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G-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased pulmonary vascular remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I).


G - I In absence ( left bars ) presence ( right bars ) of Hyp + SU5416 for 2 , siISCU ( black bars ) induced elevations in RVSP ( * * * P < 0 . 0001 , * * P = 0 . 0031 ) ( G ) increased lung remodeling in < 100 - μm pulmonary vessels ( H , I ) as compared with siCont ( N = 5 / group , * * * P = 0 . 0003 , * P = 0 . 0114 for H ; * * * P = 0 . 0006 , * * P = 0 . 0098 for I ) .

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G-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased lung remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I).


Identification of ENU - induced hearing loss pedigree MPC169 , subsequently named clarinet . ABR phenotyping of pedigree Muta - Ped - C3PDE - 169 at 3 - months of identified 8 mice with elevated hearing thresholds ( red triangles ) compared to their normal hearing colony mates ( n = 61 , black triangles ) . Indeed , eight affected mice were found to not respond to highest stimulus ( 90 dB SPL ) at three frequencies tested , or click stimulus , so their thresholds are shown as 95 dB SPL .

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Identification of the ENU-induced hearing loss pedigree MPC169, subsequently named clarinet. ABR phenotyping of pedigree Muta-Ped-C3PDE-169 at 3-months of age identified 8 mice with elevated hearing thresholds (red triangles) compared to their normal hearing colony mates (n=61, black triangles). Indeed, all eight affected mice were found to not respond to the highest intensity stimulus (90 dB SPL) at the three frequencies tested, or the click stimulus, and so their thresholds are shown as 95 dB SPL.


Averaged ABR thresholds for Clrn2clarinet / del629 compound heterozygotes at P21 , showing significantly elevated thresholds compared to Clrn2 + / + , Clrn2clarinet / + Clrn2del629 / + control colony mates . five Clrn2clarinet / del629 mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least one frequency / click stimulus . Data shown are mean ± s . d . * * * p < 0 . 001 , one - way ANOVA ( Please see Appendix supplemental table 1 for exact P - values ) .

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Averaged ABR thresholds for Clrn2clarinet/del629 compound heterozygotes at P21, showing significantly elevated thresholds compared to Clrn2+/+, Clrn2clarinet/+ and Clrn2del629/+ control colony mates. All five Clrn2clarinet/del629 mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency/click stimulus. Data shown are mean ± s.d. ***p<0.001, one-way ANOVA (Please see Appendix supplemental table 1 for exact P-values).


Regional plot of p values for SNP association with hearing difficulty around CLRN2 gene locus . genes within region are annotated , direction of transcripts is shown by arrows . Colouring is on linkage disequilibrium ( LD ) across region with most associated SNP , rs35414371 , shown in purple .

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Regional plot of p values for SNP association with hearing difficulty around the CLRN2 gene locus. The genes within the region are annotated, and the direction of the transcripts is shown by arrows. Colouring is based on linkage disequilibrium (LD) across the region with the most associated SNP, rs35414371, shown in purple.


RT - PCR in P30 mice showing presence of Clrn2 transcripts in inner ear , eye , but not in brain or muscle . β - actin was used as a positive control .

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RT-PCR analysis in P30 mice showing the presence of Clrn2 transcripts in the inner ear, eye, but not in brain or muscle. β-actin was used as a positive control.


Clrn2 transcripts could be detected in both inner ( IHCs ) outer ( OHCs ) hair of P15 wild - type mice . Otoferlin ( Otof ) oncomodulin ( Ocm ) transcripts were used as positive controls for IHCs OHCs , respectively . Ocm transcripts were only present in OHC lysate , demonstrating that sample had not been contaminated with OHCs .

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Clrn2 transcripts could be detected in both inner (IHCs) and outer (OHCs) hair cells of P15 wild-type mice. Otoferlin (Otof) and oncomodulin (Ocm) transcripts were used as positive controls for IHCs and OHCs, respectively. Ocm transcripts were only present in the OHC lysate, demonstrating that the IHC sample had not been contaminated with OHCs.


Auditory phenotyping of clarinet mice at P16 ( C ) , P21 ( D ) ABR threshold measurements show that Clrn2clarinet / clarinet mice ( red ) exhibit a severe - to - profound hearing loss affecting frequencies tested . At 16 kHz in Clrn2clarinet / clarinet mice , mean ABR hearing thresholds vary from 55 - 65 dB SPL at P16 , 60 - 90 dB SPL at P21 , to 80 - 100 dB SPL at P28 P42 . - matched Clrn2 + / + ( black ) Clrn2clarinet / + ( grey ) controls display thresholds within expected range ( 15 - 45 dB SPL ) at frequencies time - points tested . At P16 , eight Clrn2clarinet / clarinet mice exhibited recordable ABR responses for each frequency tested click stimulus . For longitudinal ABR study , at P21 P28 three of seven Clrn2clarinet / clarinet mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least one frequency - specific / click stimulus . By P42 , five of Clrn2clarinet / clarinet mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least two frequency - specific / click stimuli . ABR data shown are mean ± s . d . * * * p < 0 . 001 , one - way ANOVA .

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Auditory phenotyping of clarinet mice at P16 (C), P21 (D) ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55-65 dB SPL at P16, 60-90 dB SPL at P21, and to 80-100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15-45 dB SPL) at all frequencies and time-points tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency-specific/click stimulus. By P42, five of the Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least two frequency-specific/click stimuli. ABR data shown are mean ± s.d. ***p<0.001, one-way ANOVA.


Auditory phenotyping of clarinet mice at P28 ( E ) P42 ( F ) . ABR threshold measurements show that Clrn2clarinet / clarinet mice ( red ) exhibit a severe - to - profound hearing loss affecting frequencies tested . At 16 kHz in Clrn2clarinet / clarinet mice , mean ABR hearing thresholds vary from 55 - 65 dB SPL at P16 , 60 - 90 dB SPL at P21 , to 80 - 100 dB SPL at P28 P42 . - matched Clrn2 + / + ( black ) Clrn2clarinet / + ( grey ) controls display thresholds within expected range ( 15 - 45 dB SPL ) at frequencies time - points tested . At P16 , eight Clrn2clarinet / clarinet mice exhibited recordable ABR responses for each frequency tested click stimulus . For longitudinal ABR study , at P21 P28 three of seven Clrn2clarinet / clarinet mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least one frequency - specific / click stimulus . By P42 , five of Clrn2clarinet / clarinet mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least two frequency - specific / click stimuli . ABR data shown are mean ± s . d . * * * p < 0 . 001 , one - way ANOVA .

Auditory phenotyping of clarinet mice at P28 (E) and P42 (F). ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55-65 dB SPL at P16, 60-90 dB SPL at P21, and to 80-100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15-45 dB SPL) at all frequencies and time-points tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency-specific/click stimulus. By P42, five of the Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least two frequency-specific/click stimuli. ABR data shown are mean ± s.d. ***p<0.001, one-way ANOVA.


Averaged DPOAE responses for clarinet mice at P28 , showing significantly reduced responses in Clrn2clarinet / clarinet mutants at frequencies tested . DPOAE data shown are mean ± s . d . * p < 0 . 02 , * * p < 0 . 01 , one - way ANOVA . Please see Appendix supplemental table 1 for exact P - values .

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Averaged DPOAE responses for clarinet mice at P28, showing significantly reduced responses in Clrn2clarinet/clarinet mutants at all frequencies tested. DPOAE data shown are mean ± s.d. *p<0.02, **p<0.01, one-way ANOVA. Please see Appendix supplemental table 1 for exact P-values.


Vestibular behavioural ( swim , contact - righting , trunk - curl , platform ) . Clrn2clarinet / clarinet mice ( red , P28 , n = 5 , P60 , n = 7 ) have no vestibular dysfunction , displaying similar performances to - matched control Clrn2clarinet / + mice ( grey , P28 , n = 3 , P60 , n = 7 ) . Being similar , values at P28 P60 were combined . Electroretinogram ( ERG ) measurements from control Clrn2clarinet / + ( grey ) mutant Clrn2clarinet / clarinet ( red ) mice . Each trace in B C is representative of an ERG response from eye of - matched Clrn2clarinet / + Clrn2clarinet / clarinet mice , showing no significant difference in a - or b - wave amplitudes . ( D - F ) lack of change in ERG amplitude responses in Clrn2clarinet / clarinet mice ( aged 3 or 6 - 7 months ) , regardless of conditions : scotopic ( D , E ) , or photopic ( F ) , indicate normal photoreceptor kinetics no change in sensitivity of photoreceptor .

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Vestibular behavioural tests (swim tests, contact-righting, trunk-curl, and platform). The Clrn2clarinet/clarinet mice (red, P28, n=5, and P60, n=7) have no vestibular dysfunction, displaying similar performances to age-matched control Clrn2clarinet/+ mice (grey, P28, n=3, and P60, n=7). Being similar, the values at P28 and P60 were combined. Electroretinogram (ERG) measurements from control Clrn2clarinet/+ (grey) and mutant Clrn2clarinet/clarinet (red) mice. Each trace in B and C is representative of an ERG response from the eye of age-matched Clrn2clarinet/+ and Clrn2clarinet/clarinet mice, showing no significant difference in a- or b-wave amplitudes. (D-F) The lack of change in ERG amplitude responses in Clrn2clarinet/clarinet mice (aged 3 or 6-7 months), regardless of the test conditions: scotopic (D,E), or photopic (F), indicate normal photoreceptor kinetics and no change in the sensitivity of photoreceptor cells.


Electroretinogram ( ERG ) measurements from control Clrn2clarinet / + ( grey ) mutant Clrn2clarinet / clarinet ( red ) mice . Each trace in B C is representative of an ERG response from eye of - matched Clrn2clarinet / + Clrn2clarinet / clarinet mice , showing no significant difference in a - or b - wave amplitudes . ( D - F ) lack of change in ERG amplitude responses in Clrn2clarinet / clarinet mice ( aged 3 or 6 - 7 months ) , regardless of conditions : scotopic ( D , E ) , or photopic ( F ) , indicate normal photoreceptor kinetics no change in sensitivity of photoreceptor .

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Electroretinogram (ERG) measurements from control Clrn2clarinet/+ (grey) and mutant Clrn2clarinet/clarinet (red) mice. Each trace in B and C is representative of an ERG response from the eye of age-matched Clrn2clarinet/+ and Clrn2clarinet/clarinet mice, showing no significant difference in a- or b-wave amplitudes. (D-F) The lack of change in ERG amplitude responses in Clrn2clarinet/clarinet mice (aged 3 or 6-7 months), regardless of the test conditions: scotopic (D,E), or photopic (F), indicate normal photoreceptor kinetics and no change in the sensitivity of photoreceptor cells.


Confocal microscopy of whole - mount preparations of mid - basal cochlear sensory epithelia from Clrn2clarinet / + ( A ) Clrn2clarinet / clarinet ( B ) P6 mice immunostained for actin . Despite lack of clarin - 2 , developing sensory epithelium of mutants is similar to that of heterozygous controls .

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Confocal microscopy images of whole-mount preparations of mid-basal cochlear sensory epithelia from Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) P6 mice immunostained for actin. Despite lack of clarin-2, the developing sensory epithelium of mutants is similar to that of heterozygous controls.


Confocal microscopy of whole - mount preparations of cochlear sensory epithelia from Clrn2clarinet / + ( C ) , Clrn1 - / - ( D ) , Clrn2clarinet / clarinet ( E ) P6 mice immunostained for stereocilin ( green ) actin ( red ) . Unlike fragmented immunostaining in Clrn1 - / - mice ( mirroring fragmentation of hair bundle ) , stereocilin immunostaining in Clrn2clarinet / clarinet mice reflects normally V - shaped bundles of OHCs , similar to Clrn2clarinet / + OHCs .

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Confocal microscopy images of whole-mount preparations of cochlear sensory epithelia from Clrn2clarinet/+ (C), Clrn1-/- (D), and Clrn2clarinet/clarinet (E) P6 mice immunostained for stereocilin (green) and actin (red). Unlike the fragmented immunostaining in Clrn1-/- mice (mirroring the fragmentation of the hair bundle), stereocilin immunostaining in Clrn2clarinet/clarinet mice reflects the normally V-shaped bundles of OHCs, similar to Clrn2clarinet/+ OHCs.


Representative scanning electron of sensory epithelium of Clrn2clarinet / + ( F ) Clrn2clainet / clarinet ( G ) P8 mice , showing no apparent differences in gross patterning of IHCs OHCs . Arrowheads in a , b ( ) , f g ( black ) illustrate convex shape of apical circumference of OHCs .

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Representative scanning electron micrographs of the sensory epithelium of Clrn2clarinet/+ (F) and Clrn2clainet/clarinet (G) P8 mice, showing no apparent differences in the gross patterning of IHCs and OHCs. Arrowheads in a,b (white),f and g (black) illustrate the convex shape of the apical circumference of OHCs.


Pseudo - coloured scanning electron of outer inner hair bundles from clarinet mice at P8 P16 , showing that gross of OHC bundles is similar between Clrn2 + / + Clrn2clarinet / clarinet mice . Representative from mid region of cochlear spiral are shown close - up views illustrate three full rows , tallest ( red ) , middle ( blue ) short ( yellow ) , of stereocilia in OHC hair bundles . At P8 P16 ( upper panels in A B ) , three stereocilia rows are observed in Clrn2clarinet / clarinet OHCs ( A ) IHCs ( B ) , although shortest middle row of stereocilia appear less prolate compared to controls . ( C , D ) Distribution of stereocilia measures across genotypes at P8 P16 .

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Pseudo-coloured scanning electron micrographs of individual outer and inner hair cell bundles from clarinet mice at P8 and P16, showing that gross morphology of OHC and IHC bundles is similar between Clrn2+/+ and Clrn2clarinet/clarinet mice. Representative images from the mid region of the cochlear spiral are shown and close-up views illustrate the three full rows, tallest (red), middle (blue) and short (yellow), of stereocilia in IHC and OHC hair bundles. At P8 and P16 (upper panels in A and B), the three stereocilia rows are observed in Clrn2clarinet/clarinet OHCs (A) and IHCs (B), although the shortest and middle row of IHC stereocilia appear less prolate compared to controls. (C,D) Distribution of individual stereocilia measures across genotypes at P8 and P16.


OHC stereocilia measurements at both P8 P16 ( C ) show a significant difference in height of Clrn2 + / + stereocilia compared to Clrn2clarinet / clarinet in : e . g . for shortest row at P8 : Clrn2 + / + : 0 . 6188 ± 0 . 0944 p . µm ( n = 27 ) ; Clrn2clarinet / clarinet : 0 . 2948 ± 0 . 1029 p . µm ( n = 27 ) ( p < 0 . 0001 , Mann - Whitney ranks comparison ) , at P16 : Clrn2 + / + : 0 . 579 ± 0 . 06179 p . µm ( n = 27 ) ; Clrn2clarinet / clarinet : 0 . 07282 ± 0 . 1626 p . µm ( n = 27 ) ( p < 0 . 0001 , Mann - Whitney ranks comparison ) . Note amount of value points equal to zero at P16 . ( D ) Conversely , stereocilia measurements at P8 P16 did not show a difference between Clrn2 + t / + Clrn2clarinet / clarinet in : for shortest row at P8 : Clrn2 + / + : 0 . 8808 ± 0 . 1753 p . µm ( n = 27 ) ; Clrn2clarinet / clarinet : 0 . 9313 ± 0 . 1217 p . µm ( n = 27 ) ( p = 0 . 2244 , unpaired t - ) ; at P16 : Clrn2 + / + : 0 . 6784 ± 0 . 1171 p . µm ( n = 23 ) ; Clrn2clarinet / clarinet : 0 . 6063 ± 0 . 1855 p . µm ( n = 26 ) ( p = 0 . 2436 , Mann - Whitney ranks comparison ) .

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The OHC stereocilia measurements at both P8 and P16 (C) show a significant difference in height of Clrn2+/+ stereocilia compared to Clrn2clarinet/clarinet in: e.g. for the shortest row at P8: Clrn2+/+: 0.6188 ± 0.0944 p.µm (n=27); Clrn2clarinet/clarinet: 0.2948 ± 0.1029 p.µm (n=27) (p<0.0001, Mann-Whitney ranks comparison), and at P16: Clrn2+/+: 0.579 ± 0.06179 p.µm (n=27); Clrn2clarinet/clarinet: 0.07282 ± 0.1626 p.µm (n=27) (p<0.0001, Mann-Whitney ranks comparison). Note the amount of value points equal to zero at P16. (D) Conversely, IHC stereocilia measurements at P8 and P16 did not show a difference between Clrn2+t/+ and Clrn2clarinet/clarinet in: for the shortest row at P8: Clrn2+/+: 0.8808 ± 0.1753 p.µm (n=27); Clrn2clarinet/clarinet: 0.9313 ± 0.1217 p.µm (n=27) (p=0.2244, unpaired t-test); and at P16: Clrn2+/+: 0.6784 ± 0.1171 p.µm (n=23); Clrn2clarinet/clarinet: 0.6063 ± 0.1855 p.µm (n=26) (p=0.2436, Mann-Whitney ranks comparison).


Sensory hair bundle patterning measurements in clarinet mice Pseudo - coloured scanning electron of OHC ( left panels ) ( right panels ) hair bundles from clarinet mice at P28 . Clrn2clarinet / clarinet mutants have only two rows of OHC stereocilia , middle row of stereocilia is less uniform in height compared to controls . middle short rows of stereocilia in Clrn2clarinet / clarinet bundles appear fewer in number , heterogeneous in height . Scale - 2 µm .

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Sensory hair cell bundle patterning and measurements in clarinet mice Pseudo-coloured scanning electron micrographs of individual OHC (left panels) and IHC (right panels) hair bundles from clarinet mice at P28. Clrn2clarinet/clarinet mutants have only two rows of OHC stereocilia, and the middle row of stereocilia is less uniform in height compared to controls. The middle and short rows of stereocilia in Clrn2clarinet/clarinet IHC bundles appear fewer in number, and heterogeneous in height. Scale - 2 µm.


Schematic representation of auditory sensory organ , illustrating positioning of electrode used for injectoporation of GFP - tagged clarin - 2 construct into cochlear supporting hair . ( B - F ) Top side views of representative of supporting ( asterisk ) hair expressing clarin - 2 . In supporting , GFP - tagged clarin - 2 ( green ) was distributed diffusely throughout cytoplasm ( A , B ) . By contrast , in injectoporated hair , majority of clarin - 2 was observed in apical stereocilia ( C - E ) . Scale - 2 µm .

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Schematic representation of the auditory sensory organ, illustrating the positioning of the electrode used for injectoporation of GFP-tagged clarin-2 construct into cochlear supporting and hair cells. (B-F) Top and side views of representative images of supporting cells (asterisk) and hair cells expressing clarin-2. In supporting cells, GFP-tagged clarin-2 (green) was distributed diffusely throughout the cytoplasm (A,B). By contrast, in all injectoporated hair cells, the majority of clarin-2 was observed in the apical stereocilia (C-E). Scale - 2 µm.


Confocal of OHC hair bundles of Clrn2clarinet / clarinet mice heterozygous at P6 immunostained for PDZD7 ( A , green ) whirlin ( B , green ) actin ( red in both figures ) . PDZD7 immunostaining is normally restricted to base of stereocilia in both Clrn2clarinet / + Clrn2clarinet / clarinet P6 mice ( A ) . Whirlin immunostaining is properly located at stereocilia tips of OHC hair bundles ( B ) .

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Confocal images of IHC and OHC hair bundles of Clrn2clarinet/clarinet mice and heterozygous littermates at P6 immunostained for PDZD7 (A, green) and whirlin (B, green) and actin (red in both figures). The PDZD7 immunostaining is normally restricted to the base of stereocilia in both Clrn2clarinet/+ and Clrn2clarinet/clarinet P6 mice (A). Whirlin immunostaining is properly located at the stereocilia tips of IHC and OHC hair bundles (B).


Harmonin - b immunostaining in hair bundles . In Clrn2clarinet / clarinet mice ( D ) , harmonin - b immunoreactive ( green ) were still observable on stereocilia , but unlike in - matched heterozygote ( C ) , were located much closer to tip of stereocilia . two diagrams in ( C ) ( D ) illustrate position of harmonin - b immunostaining ( green ) corresponding to site of upper attachment point of tip link ( UAPTL ) , facing tip link . bright green signal outside stereociliary bundles are non - specific . change of harmonin - b localization along stereocilium in Clrn2clarinet / clarinet mice is illustrated further by scan ( E ) ( F ) analyses . insets in ( E ) show of stereocilia used for scan signal . harmonin - b immunoreactive were located within 850 ± 28 nm from tip of tallest stereocilia in Clrn2clarine / + mice ( n = 31 hair bundles from 5 mice ) ( F ) , within 575 ± 23 nm in Clrn2clarinet / clarinet mice ( n = 35 hair bundles from 5 mice ) .

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Harmonin-b immunostaining in IHC hair bundles. In Clrn2clarinet/clarinet mice (D), the harmonin-b immunoreactive puncta (green) were still observable on the stereocilia, but unlike in age-matched heterozygote littermates (C), were located much closer to the tip of stereocilia. The two diagrams in (C) and (D) illustrate the position of harmonin-b immunostaining (green) corresponding to the site of the upper attachment point of the tip link (UAPTL), facing the tip link. The bright green signal outside stereociliary bundles are non-specific. The change of harmonin-b localization along the stereocilium in Clrn2clarinet/clarinet mice is illustrated further by line scan (E) and quantification (F) analyses. The insets in (E) show images of individual stereocilia used for the line scan signal analysis. The harmonin-b immunoreactive puncta were located within 850 ± 28 nm distance from the tip of the tallest stereocilia in Clrn2clarine/+ mice (n=31 hair bundles from 5 mice) (F), and within 575 ± 23 nm in Clrn2clarinet/clarinet mice (n=35 hair bundles from 5 mice).


Cochlear whole - mounts from P21 clarinet mice , labelled with pre - synaptic ribbon marker Ribeye ( red ) post - synaptic density marker GluR2 ( green ) , showing a similar number of total matched Ribeye - positive GluR2 - postive in Clrn2clarinet / clarinet mutant cochleae compared to Clrn2clarinet / + , which is reflected in counted per five hair ( H ) n = 4 per genotype .

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Cochlear whole-mounts from P21 clarinet mice, labelled with the IHC pre-synaptic ribbon marker Ribeye (red) and the post-synaptic density marker GluR2 (green), showing a similar number of total and matched Ribeye-positive and GluR2-postive puncta in Clrn2clarinet/clarinet mutant cochleae compared to Clrn2clarinet/+ littermates, which is reflected in puncta counted per five hair cells (H) n = 4 per genotype.


Saturating MET currents recorded from P6 Clrn2clarinet / + ( A ) Clrn2clarinet / clarinet ( B ) apical - coil OHCs by applying sinusoidal force stimuli of 50 Hz to hair bundles at − 121 mV + 99 mV . driver voltage ( DV ) signal of ± 40 V to fluid jet is shown above traces ( positive deflections of DV are excitatory ) . holding potential was − 81 mV . Extracellular Ca2 + was 1 . 3 mM . Arrows arrowheads indicate closure of MET currents ( i . e . resting MET current ) elicited during inhibitory bundle displacements at hyperpolarized depolarized potentials , respectively . Dashed lines indicate holding current , which is current at holding potential .

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

Saturating MET currents recorded from P6 Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) apical-coil OHCs by applying sinusoidal force stimuli of 50 Hz to the hair bundles at −121 mV and +99 mV. The driver voltage (DV) signal of ± 40 V to the fluid jet is shown above the traces (positive deflections of the DV are excitatory). The holding potential was −81 mV. Extracellular Ca2+ concentration was 1.3 mM. Arrows and arrowheads indicate the closure of the MET currents (i.e. resting MET current) elicited during inhibitory bundle displacements at hyperpolarized and depolarized membrane potentials, respectively. Dashed lines indicate the holding current, which is the current at the holding membrane potential.


Average peak - to - peak current - voltage curves recorded from Clrn2clarinet / + ( grey , P6 , n = 7 ) Clrn2clarinet / clarinet ( red , P6 - 7 , n = 9 ) apical coil OHCs .

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

Average peak-to-peak current-voltage curves recorded from Clrn2clarinet/+ (grey, P6, n=7) and Clrn2clarinet/clarinet (red, P6-7, n=9) apical coil OHCs.


Resting open probability ( Po ) of MET current at potential of − 121 mV + 99 mV from apical - ( D ) basal - coil ( E ) OHCs . Number of OHCs investigated is shown above columns .

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Resting open probability (Po) of the MET current at the membrane potential of −121 mV and + 99 mV from apical- (D) and basal-coil (E) OHCs. Number of OHCs investigated is shown above the columns.


Saturating MET currents recorded from a P7 Clrn2clarinet / + ( F ) a P8 Clrn2clarinet / clarinet ( G ) apical - coil using same experimental protocol described above .

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

Saturating MET currents recorded from a P7 Clrn2clarinet/+ (F) and a P8 Clrn2clarinet/clarinet (G) apical-coil IHC using the same experimental protocol described above.


Average Po of MET current measured in apical coil IHCs at potential of − 121 mV + 99 mV from Clrn2clarinet / + ( P7 , n = 3 ) Clrn2clarinet / clarinet ( P7 - 8 , n = 6 ) apical - coil IHCs .

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

Average Po of the MET current measured in apical coil IHCs at the membrane potential of −121 mV and + 99 mV from Clrn2clarinet/+ (P7, n=3) and Clrn2clarinet/clarinet (P7-8, n=6) apical-coil IHCs.


Potassium currents recorded from mature Clrn2clarinet / + ( A , P15 ) Clrn2clarinet / clarinet ( B , P16 ) apical - coil OHCs . Currents were elicited by depolarizing voltage steps ( 10 mV nominal increments ) from - 144 mV to more depolarized values from holding potential of - 84 mV . Note that current characteristic of mature OHCs , of IK , n , measured in isolation as deactivating tail currents ( difference between instantaneous steady - state inward currents ) for voltage steps from holding potential to − 124 mV , was 545 ± 115 pA , ( n = 5 ) in Clrn2clarinet / + 595 ± 67 pA , ( n = 7 ) in Clrn2clarinet / clarinet OHCs .

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

Potassium currents recorded from mature Clrn2clarinet/+ (A, P15) and Clrn2clarinet/clarinet (B, P16) apical-coil OHCs. Currents were elicited by depolarizing voltage steps (10 mV nominal increments) from -144 mV to more depolarized values from the holding potential of -84 mV. Note that the current characteristic of mature OHCs, The size of IK,n, measured in isolation as the deactivating tail currents (difference between instantaneous and steady-state inward currents) for voltage steps from the holding potential to −124 mV, was 545 ± 115 pA, (n=5) in Clrn2clarinet/+ and 595 ± 67 pA, (n=7) in Clrn2clarinet/clarinet OHCs.


Average peak current - voltage relation for total K + current recorded from Clrn2clarinet / + ( P15 - 16 , n = 7 ) Clrn2clarinet / clarinet ( P15 - 16 , n = 5 ) OHCs .

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Average peak current-voltage relation for the total K+ current recorded from Clrn2clarinet/+ (P15-16, n=7) and Clrn2clarinet/clarinet (P15-16, n=5) OHCs.


Potassium currents recorded from P22 mature Clrn2clarinet / + Clrn2clarinet / clarinet apical - coil IHCs , respectively , using same voltage protocol described above . Holding potential of - 64 mV .

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Potassium currents recorded from P22 mature Clrn2clarinet/+ and Clrn2clarinet/clarinet apical-coil IHCs, respectively, using the same voltage protocol described above. Holding potential of -64 mV.


Peak current - voltage relation for total K + current recorded from

Peak current-voltage relation for the total K+ current recorded from the IHCs


Voltage responses from P22 Clrn2clarinet / + ( G ) Clrn2clarinet / clarinet ( H , I ) IHCs . Note that Ca2 + - dependent action potentials could be induced in mature IHCs ( I ) .

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Voltage responses from P22 Clrn2clarinet/+ (G) and Clrn2clarinet/clarinet (H,I) IHCs. Note that Ca2+-dependent action potentials could be induced in mature IHCs (I).