( 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 .

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(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 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 .

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(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 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 .

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(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 , 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 .

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(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 ) ; and S . mansoni - infected mice ( N = 4 ) compared with non - infected control mice ( N = 5 ) , * * * P < 0 . 0001 ( fourth graph ) .

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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 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 .

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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 micrographs ) exposed to Hyp + SU5416 compared with Norm + SU5416 ( α - smooth muscle actin stain from serial sections , top row of micrographs ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 ( 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 ) .

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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 ) , 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 ) .

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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 , 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 ) .

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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 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 ) .

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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 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 ) .

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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 .

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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 .

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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 ( 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 .

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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 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 .

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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 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 ) .

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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 ) .

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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 .

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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 − / − ) .

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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 cells from PH versus non - PH mice ( N = 3 / group , * * P = 0 . 0052 ) .

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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 ( white bars , N = 11 ) ( * * * P < 0 . 0001 , * P = 0 . 0258 , * * P = 0 . 0059 ) .

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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 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 ) .

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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 ( 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 ) .

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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 ) and 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 levels 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 ) 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 ) .

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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 ) , 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 ) .

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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 cells 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 + cells even in the 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 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 ) .

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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 the 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 ( 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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

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 / + 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 ) .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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

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 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 ) .

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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 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 .

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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 ) 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 .

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

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 ) and Clrn2clarinet / clarinet ( red , P6 - 7 , n = 9 ) apical coil OHCs .

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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 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 .

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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 ) and a P8 Clrn2clarinet / clarinet ( G ) apical - coil IHC using the same experimental protocol described above .

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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 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 .

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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 ) 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 .

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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 the total K + current recorded from Clrn2clarinet / + ( P15 - 16 , n = 7 ) and 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 / + and Clrn2clarinet / clarinet apical - coil IHCs , respectively , using the 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 the total K + current recorded from the

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


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 ) .

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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).


( A ) BH3 ‐ only BIK displaces a subset of BCL ‐ 2 complexes at the ER . Purified LM from H1299 HA ‐ BCL ‐ 2b5 cells either mock infected or infected with Ad ‐ BIKb5 was subjected to cross ‐ linking with BMH and visualized by immunoblot . Asterisks ( * ) denote BCL ‐ 2 cross ‐ linked products , which are displaced by BIK . Bold arrow denotes region containing cross ‐ linked NAF ‐ 1 / BCL ‐ 2 .

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(A) BH3‐only BIK displaces a subset of BCL‐2 complexes at the ER. Purified LM from H1299 HA‐BCL‐2b5 cells either mock infected or infected with Ad‐BIKb5 was subjected to cross‐linking with BMH and visualized by immunoblot. Asterisks (*) denote BCL‐2 cross‐linked products, which are displaced by BIK. Bold arrow denotes region containing cross‐linked NAF‐1/BCL‐2.


( D ) Endogenous NAF ‐ 1 cross ‐ links with BCL ‐ 2 at the ER . LM purified from H1299 neo and HA ‐ BCL ‐ 2b5 cells were subjected to cross ‐ linking by BMH and immunoprecipitation with anti ‐ NAF ‐ 1 antibody . Precipitates were analysed by immunoblot with anti ‐ BCL ‐ 2 .

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(D) Endogenous NAF‐1 cross‐links with BCL‐2 at the ER. LM purified from H1299 neo and HA‐BCL‐2b5 cells were subjected to cross‐linking by BMH and immunoprecipitation with anti‐NAF‐1 antibody. Precipitates were analysed by immunoblot with anti‐BCL‐2.


( E ) NAF ‐ 1 is displaced from BCL ‐ 2 by BH3 ‐ only BIK . H1299 HA ‐ BCL ‐ 2b5 cells were either mock infected or infected with Ad ‐ BIK . Purified LM was treated as in ( D ) .

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(E) NAF‐1 is displaced from BCL‐2 by BH3‐only BIK. H1299 HA‐BCL‐2b5 cells were either mock infected or infected with Ad‐BIK. Purified LM was treated as in (D).


( A ) Co ‐ immunoprecipitation of endogenous NAF ‐ 1 and BCL ‐ 2 . Lysates from SK ‐ Mel5 cells were collected and immunoprecipitation was performed with anti ‐ NAF ‐ 1 antibody . The precipitate was subjected to immunoblotting with anti ‐ BCL ‐ 2 and anti ‐ NAF ‐ 1 .

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(A) Co‐immunoprecipitation of endogenous NAF‐1 and BCL‐2. Lysates from SK‐Mel5 cells were collected and immunoprecipitation was performed with anti‐NAF‐1 antibody. The precipitate was subjected to immunoblotting with anti‐BCL‐2 and anti‐NAF‐1.


( B ) H1299 cells were fixed and double stained with anti ‐ NAF ‐ 1 and anti ‐ Calnexin or anti ‐ Cytochrome c antibodies . Scale bar represents 10 μm .

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(B) H1299 cells were fixed and double stained with anti‐NAF‐1 and anti‐Calnexin or anti‐Cytochrome c antibodies. Scale bar represents 10 μm.


( C ) Co ‐ immunoprecipitation of NAF ‐ 1 ‐ Flag and HA ‐ BCL ‐ 2b5 . Lysates from H1299 HA ‐ BCL ‐ 2b5 cells infected with Ad ‐ NAF ‐ 1 ‐ Flag were collected and treated as in ( A ) .

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(C) Co‐immunoprecipitation of NAF‐1‐Flag and HA‐BCL‐2b5. Lysates from H1299 HA‐BCL‐2b5 cells infected with Ad‐NAF‐1‐Flag were collected and treated as in (A).


( D ) Mutations in the CDGSH iron ‐ binding domain of NAF ‐ 1 interfere with NAF ‐ 1 binding to BCL ‐ 2 . H1299 HA ‐ BCL ‐ 2b5 cells were infected with either Ad ‐ rtTA , Ad ‐ NAF ‐ 1 ‐ Flag , or Ad ‐ NAF ‐ 1 ‐ mut ‐ Flag ( C99S C101S C110S H114Q ) . Lysates were treated as in ( A ) . Densitometric analysis was performed using Scion Image software to quantify expression and co ‐ precipitated levels of NAF ‐ 1 ‐ Flag and NAF ‐ 1 ‐ mut ‐ Flag . Graph depicts the ratio of co ‐ precipitated protein to expression level .

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(D) Mutations in the CDGSH iron‐binding domain of NAF‐1 interfere with NAF‐1 binding to BCL‐2. H1299 HA‐BCL‐2b5 cells were infected with either Ad‐rtTA, Ad‐NAF‐1‐Flag, or Ad‐NAF‐1‐mut‐Flag (C99S C101S C110S H114Q). Lysates were treated as in (A). Densitometric analysis was performed using Scion Image software to quantify expression and co‐precipitated levels of NAF‐1‐Flag and NAF‐1‐mut‐Flag. Graph depicts the ratio of co‐precipitated protein to expression level.


( E ) A functional CDGSH iron ‐ binding domain is necessary , but not sufficient for the interaction between the cytosolic domains of NAF ‐ 1 and BCL ‐ 2 . HA ‐ BCL ‐ 2 ΔTM was in vitro translated in rabbit reticulocyte lysate and equivalent aliquots were added to each GST pull ‐ down reaction . GST ‐ fusion proteins used were GST alone , GST ‐ NAF1 ‐ C , GST ‐ NAF1 ‐ C ‐ mut ( C99S C101S C110S H114Q ) , and GST ‐ MitoNEET ‐ C . The proteins were detected using anti ‐ HA and anti ‐

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(E) A functional CDGSH iron‐binding domain is necessary, but not sufficient for the interaction between the cytosolic domains of NAF‐1 and BCL‐2. HA‐BCL‐2 ΔTM was in vitro translated in rabbit reticulocyte lysate and equivalent aliquots were added to each GST pull‐down reaction. GST‐fusion proteins used were GST alone, GST‐NAF1‐C, GST‐NAF1‐C‐mut (C99S C101S C110S H114Q), and GST‐MitoNEET‐C. The proteins were detected using anti‐HA and anti‐GST


( A ) H1299 neo and HA ‐ BCL ‐ 2b5 cells treated with control ( CTRL ) or NAF ‐ 1 shRNA were either mock infected or infected with Ad ‐ BIK in the absence or presence of 50 μM zVAD ‐ fmk . Cell lysates were analysed by immunoblot .

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(A) H1299 neo and HA‐BCL‐2b5 cells treated with control (CTRL) or NAF‐1 shRNA were either mock infected or infected with Ad‐BIK in the absence or presence of 50 μM zVAD‐fmk. Cell lysates were analysed by immunoblot.


( B ) Caspase activity was measured using the fluorescent substrate DEVD ‐ AMC . The results represent the average ± s . d . of three independent experiments .

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(B) Caspase activity was measured using the fluorescent substrate DEVD‐AMC. The results represent the average±s.d. of three independent experiments.


( C ) Prolonged BIK expression and caspase inhibition induces autophagy , which is enhanced by knockdown of NAF ‐ 1 . H1299 neo and HA ‐ BCL ‐ 2b5 cells treated with CTRL or NAF ‐ 1 shRNA were infected with Ad ‐ BIK in the presence of zVAD ‐ fmk for the indicated periods of time . Cell lysates were analysed by immunoblot . Levels of LC3 II were normalized to actin levels by densitometry analysis . Graph depicts normalized LC3 II levels of each lane .

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(C) Prolonged BIK expression and caspase inhibition induces autophagy, which is enhanced by knockdown of NAF‐1. H1299 neo and HA‐BCL‐2b5 cells treated with CTRL or NAF‐1 shRNA were infected with Ad‐BIK in the presence of zVAD‐fmk for the indicated periods of time. Cell lysates were analysed by immunoblot. Levels of LC3 II were normalized to actin levels by densitometry analysis. Graph depicts normalized LC3 II levels of each lane.


( A ) Effect of NAF ‐ 1 knockdown on starvation ‐ induced autophagy . H1299 cells infected with CTRL or NAF ‐ 1 shRNA were starved for 4 h in EBSS with DMSO ( vehicle ) or Baf A1 ( 100 nM ) . Cell lysates were analysed by immunoblot .

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(A) Effect of NAF‐1 knockdown on starvation‐induced autophagy. H1299 cells infected with CTRL or NAF‐1 shRNA were starved for 4 h in EBSS with DMSO (vehicle) or Baf A1 (100 nM). Cell lysates were analysed by immunoblot.


( B ) Representative images of GFP ‐ LC3 staining in H1299 GFP ‐ LC3 cells transfected with LUC or NAF ‐ 1 siRNA , untreated and starved . Scale bar represents 10 μm .

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(B) Representative images of GFP‐LC3 staining in H1299 GFP‐LC3 cells transfected with LUC or NAF‐1 siRNA, untreated and starved. Scale bar represents 10 μm.


( C ) Quantification of autophagy is expressed as the percentage of GFP ‐ LC3 ‐ expressing cells displaying punctate GFP ‐ LC3 . A minimum of 100 cells per sample were counted ; results represent the average ± s . d . of three independent experiments . Cell lysates were analysed by immunoblot .

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(C) Quantification of autophagy is expressed as the percentage of GFP‐LC3‐expressing cells displaying punctate GFP‐LC3. A minimum of 100 cells per sample were counted; results represent the average±s.d. of three independent experiments. Cell lysates were analysed by immunoblot.


( A ) NAF ‐ 1 contributes to the interaction between BCL ‐ 2 and Beclin 1 . H1299 HA ‐ BCL ‐ 2b5 cells were transfected with Flag ‐ Beclin 1 and either LUC or NAF ‐ 1 siRNA . Cells were lysed and subjected to immunoprecipitation with anti ‐ BCL ‐ 2 antibody . Precipitates were subjected to analysis by immunoblot using anti ‐ Beclin 1 and anti ‐ BCL ‐ 2 . All lanes are derived from the same gel and of the same exposure . Thin white lines indicate where lanes have been removed .

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(A) NAF‐1 contributes to the interaction between BCL‐2 and Beclin 1. H1299 HA‐BCL‐2b5 cells were transfected with Flag‐Beclin 1 and either LUC or NAF‐1 siRNA. Cells were lysed and subjected to immunoprecipitation with anti‐BCL‐2 antibody. Precipitates were subjected to analysis by immunoblot using anti‐Beclin 1 and anti‐BCL‐2. All lanes are derived from the same gel and of the same exposure. Thin white lines indicate where lanes have been removed.


( B ) Loss of NAF ‐ 1 prevents BCL ‐ 2b5 from antagonizing starvation ‐ induced autophagy . H1299 GFP ‐ LC3 and BCL ‐ 2b5 / GFP ‐ LC3 cells were transfected with either LUC or NAF ‐ 1 siRNA and starved for 4 h . Cells were analysed as in Figure 4C .

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(B) Loss of NAF‐1 prevents BCL‐2b5 from antagonizing starvation‐induced autophagy. H1299 GFP‐LC3 and BCL‐2b5/GFP‐LC3 cells were transfected with either LUC or NAF‐1 siRNA and starved for 4 h. Cells were analysed as in Figure 4C.


( C ) Representative electron micrographs of H1299 neo and HA ‐ BCL ‐ 2b5 cells treated with either LUC or NAF ‐ 1 siRNA , with or without subsequent starvation . Scale bar represents 10 μm . Cell lysates were analysed by immunoblot . All lanes are derived from the same gel and of the same exposure . Thin white line indicates where lanes have been removed . Autophagy levels observed by electron microscopy ( C ) were quantified and expressed as either the percentage of cells containing autophagic vacuoles ( D ) or the number of autophagosomes per cell ( E ) . A minimum of 100 cells per sample were counted ; results represent the average ± s . e . m .

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(C) Representative electron micrographs of H1299 neo and HA‐BCL‐2b5 cells treated with either LUC or NAF‐1 siRNA, with or without subsequent starvation. Scale bar represents 10 μm. Cell lysates were analysed by immunoblot. All lanes are derived from the same gel and of the same exposure. Thin white line indicates where lanes have been removed. Autophagy levels observed by electron microscopy (C) were quantified and expressed as either the percentage of cells containing autophagic vacuoles (D) or the number of autophagosomes per cell (E). A minimum of 100 cells per sample were counted; results represent the average±s.e.m.


( A ) H1299 neo and HA ‐ BCL ‐ 2b5 cells treated with either CTRL or NAF ‐ 1 shRNA were loaded with Fura ‐ 2AM , and ER Ca2 + stores were measured as the difference in cytoplasmic Ca2 + concentration before and after addition of TG ( 2 μM ) . Shown are representative traces of Fura ‐ 2AM fluorescence measured at 340 / 380 nm excitation wavelength ratio at 510 nm wavelength emission . Arrow indicates time at which TG was added , delta values indicate TG ‐ sensitive ER Ca2 + stores .

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(A) H1299 neo and HA‐BCL‐2b5 cells treated with either CTRL or NAF‐1 shRNA were loaded with Fura‐2AM, and ER Ca2+ stores were measured as the difference in cytoplasmic Ca2+ concentration before and after addition of TG (2 μM). Shown are representative traces of Fura‐2AM fluorescence measured at 340/380 nm excitation wavelength ratio at 510 nm wavelength emission. Arrow indicates time at which TG was added, delta values indicate TG‐sensitive ER Ca2+ stores.


( B ) Differences in ER Ca2 + stores are shown as the average ± s . e . m . of three independent experiments as described in ( A ) .

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(B) Differences in ER Ca2+ stores are shown as the average±s.e.m. of three independent experiments as described in (A).


( C ) Co ‐ immunoprecipitation of endogenous NAF ‐ 1 and endogenous IP3 receptor type I . Lysate from H1299 HA ‐ BCL ‐ 2b5 cells was collected and immunoprecipitation was performed with anti ‐ IP3R1 antibody . The resulting precipitate was analysed by immunoblot .

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(C) Co‐immunoprecipitation of endogenous NAF‐1 and endogenous IP3 receptor type I. Lysate from H1299 HA‐BCL‐2b5 cells was collected and immunoprecipitation was performed with anti‐IP3R1 antibody. The resulting precipitate was analysed by immunoblot.