( A ) ADAR2 is most strongly expressed in vascular tissues . Graph showing ADAR2 gene expression data derived from GTEx for few representative human tissues . Note the highest expression of ADAR2 in the tibial artery followed by colon and esophagus . Boxes represent the 25th and the 75th percentile with median represented by the black line in the box . The whiskers depict the minimum and the maximum value .

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(A) ADAR2 is most strongly expressed in vascular tissues. Graph showing ADAR2 gene expression data derived from GTEx for few representative human tissues. Note the highest expression of ADAR2 in the tibial artery followed by colon and esophagus. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.


( B ) FLNA gene expression in few representative human tissues from > 500 different donors using GTEx data . Tibial artery , colon and esophagus show highest gene expression similar to ADAR2 expression . Boxes represent the 25th and the 75th percentile with median represented by the black line in the box . The whiskers depict the minimum and the maximum value .

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(B) FLNA gene expression in few representative human tissues from >500 different donors using GTEx data. Tibial artery, colon and esophagus show highest gene expression similar to ADAR2 expression. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.


( C ) Bar graph shows FLNA RNA editing ( % ) among few representative human tissues . Note very high editing levels in the arterial system ( red bars ) .

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(C) Bar graph shows FLNA RNA editing (%) among few representative human tissues. Note very high editing levels in the arterial system (red bars).


( D ) Scatter plot shows the log fold change in editing levels of several candidates between ventricles of healthy donors and heart samples of cardiomyopathy patients . FLNA marked in the graph is one significant discriminator . Y axis is the - log 10 of the p value for the difference between healthy and sick . The colors ( red vs green ) reflect the threshold cutoffs randomly assigned to distinguish the sites , which demonstrate large , highly significant differences between the two groups . Fold change cutoff of 0 . 9 was used .

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(D) Scatter plot shows the log fold change in editing levels of several candidates between ventricles of healthy donors and heart samples of cardiomyopathy patients. FLNA marked in the graph is one significant discriminator. Y axis is the -log 10 of the p value for the difference between healthy and sick. The colors (red vs green) reflect the threshold cutoffs randomly assigned to distinguish the sites, which demonstrate large, highly significant differences between the two groups. Fold change cutoff of 0.9 was used.


( E ) Scatter plot shows the FLNA RNA editing in control and diseased human tibial arteries and aorta . At least 4 control and 7 diseased human donors were used in each case . P value < 0 . 05 measured by t test was considered significant .

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(E) Scatter plot shows the FLNA RNA editing in control and diseased human tibial arteries and aorta. At least 4 control and 7 diseased human donors were used in each case. P value <0.05 measured by t test was considered significant.


( A ) Scheme showing the FLNA wild - type ( wt ) allele , the targeting vector and the FLNAΔECS neo - allele . The loxP flanked PGK - neo cassette in the targeting vector replaced the editing complementary sequence ( ECS ) using homologous recombination , which was then deleted using Cre recombinase . Right , southern blotting analysis screened for positive clones shown . The positions of loxP sites , restriction enzyme ( EcoRI ) and Southern blotting probe are also indicated .

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(A) Scheme showing the FLNA wild-type (wt) allele, the targeting vector and the FLNAΔECS neo-allele. The loxP flanked PGK-neo cassette in the targeting vector replaced the editing complementary sequence (ECS) using homologous recombination, which was then deleted using Cre recombinase. Right, southern blotting analysis screened for positive clones shown. The positions of loxP sites, restriction enzyme (EcoRI) and Southern blotting probe are also indicated.


( B ) Sequencing electropherograms show the average FLNA editing levels in wt and FLNAΔECS tissues ( Stomach , colon , heart , lung and tibial artery ) . Editing levels were checked in tissues from 3 independent mice and the value below the chromatogram depict the average value of 3 replicates . Data information : Arrows indicate the FLNA editing site .

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(B) Sequencing electropherograms show the average FLNA editing levels in wt and FLNAΔECS tissues (Stomach, colon, heart, lung and tibial artery). Editing levels were checked in tissues from 3 independent mice and the value below the chromatogram depict the average value of 3 replicates. Data information: Arrows indicate the FLNA editing site.


( C ) FLNA mRNA expression levels measured by qPCR in wt and FLNAΔECS colon tissue showed no difference . Data information : data shown as mean ± SD from three independent experiments . * * P < 0 .

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(C) FLNA mRNA expression levels measured by qPCR in wt and FLNAΔECS colon tissue showed no difference. Data information: data shown as mean ± SD from three independent experiments. ** P<0.05


( D ) FLNA protein levels measured by western blotting are identical in wt and FLNAΔECS stomach tissue . Y - axis represents FLNA expression normalized to tubulin levels . Data information : data shown as mean ± SD from three independent experiments . * * P < 0 .

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(D) FLNA protein levels measured by western blotting are identical in wt and FLNAΔECS stomach tissue. Y-axis represents FLNA expression normalized to tubulin levels. Data information: data shown as mean ± SD from three independent experiments. ** P<0.05


( E ) FLNA editing levels ( % ) in wt whole dorsal aorta , tunica media and tunica adventitia . Tunica media consisting of smooth muscle cells show the highest FLNA RNA editing . Data information : Arrows indicate the FLNA editing site . data shown as mean ± SD from three independent experiments . * * P < 0 .

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(E) FLNA editing levels (%) in wt whole dorsal aorta, tunica media and tunica adventitia. Tunica media consisting of smooth muscle cells show the highest FLNA RNA editing. Data information: Arrows indicate the FLNA editing site. data shown as mean ± SD from three independent experiments. ** P<0.05


A , B ) Treatment of aortic rings with thromboxane A2 receptor agonist U46619 leads to ( A ) hypercontraction and ( B ) increased contraction force in FLNAΔECS aortae in myograph chambers . Emax was higher in FLNAΔECS aortae without any difference in EC50 as compared to wt aortae . For each condition , 10 - 12 aortic rings from at least 4 wild - type ( wt ) and 4 FLNAΔECS mice were used . Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t test )

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A,B) Treatment of aortic rings with thromboxane A2 receptor agonist U46619 leads to (A) hypercontraction and (B) increased contraction force in FLNAΔECS aortae in myograph chambers. Emax was higher in FLNAΔECS aortae without any difference in EC50 as compared to wt aortae. For each condition, 10-12 aortic rings from at least 4 wild-type (wt) and 4 FLNAΔECS mice were used. Data shown as mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001 (Student's t test)


( C ) Graph shows the normalized cell contraction measured by Xcelligence Real time cell analyzer in wt ( red ) and FLNAΔECS ( green ) primary vascular smooth muscle cells ( vSMC ) indicating hypercontraction in FLNAΔECS cells in response to 10μM U46619 . Data shown as mean ± SD from three independent experiments .

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(C) Graph shows the normalized cell contraction measured by Xcelligence Real time cell analyzer in wt (red) and FLNAΔECS (green) primary vascular smooth muscle cells (vSMC) indicating hypercontraction in FLNAΔECS cells in response to 10μM U46619. Data shown as mean ± SD from three independent experiments.


( D ) Quantification of cell index measurements plotted as percentage of contraction following different concentrations of U46619 . Data shown as mean ± SD from three independent experiments . P value < 0 . 05 ( Student ' s t test ) was considered significant . The difference between wt and FLNAΔECS was significant at every concentration of U46619 .

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(D) Quantification of cell index measurements plotted as percentage of contraction following different concentrations of U46619. Data shown as mean ± SD from three independent experiments. P value <0.05 (Student's t test) was considered significant. The difference between wt and FLNAΔECS was significant at every concentration of U46619.


( A ) Myosin light chain phosphorylation detected by a phospho - specific antibody ( pMLC ) and normalized to total myosin light chain ( MLC ) . Tubulin served as a loading control . Quantification shows a significant increase in pMLC in FLNAΔECS vSMCs . Data information data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t test )

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(A) Myosin light chain phosphorylation detected by a phospho-specific antibody (pMLC) and normalized to total myosin light chain (MLC). Tubulin served as a loading control. Quantification shows a significant increase in pMLC in FLNAΔECS vSMCs. Data information data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


( B ) Rhotekin Pull down activation assay was performed on wt and FLNAΔECS vSMC lysates and activated ( GTP ) bound RhoA was compared between the 2 genotypes . FLNAΔECS vSMCs show more GTP - loaded RhoA than wt vSMCs . Tubulin was used as a loading control . Data information : data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t test )

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(B) Rhotekin Pull down activation assay was performed on wt and FLNAΔECS vSMC lysates and activated (GTP) bound RhoA was compared between the 2 genotypes. FLNAΔECS vSMCs show more GTP-loaded RhoA than wt vSMCs. Tubulin was used as a loading control. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


( C ) Thromoboxane A2 stimulation with U46619 increased phosphorylation of myosin light chain phosphatase ( pMYPT1 ) in FLNAΔECS vSMCs as shown by western blotting on vSMC lysates with phosphor - specific MYPT1 antibody . Data information : data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t test )

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(C) Thromoboxane A2 stimulation with U46619 increased phosphorylation of myosin light chain phosphatase (pMYPT1) in FLNAΔECS vSMCs as shown by western blotting on vSMC lysates with phosphor-specific MYPT1 antibody. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


( D ) CPI17 phosphorylation levels were measured in wt and FLNAΔECS vSMCs in untreated cells and in the presence of ROCK and PKC inhibitor using pCPI17 antibody . Data information : data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t test )

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(D) CPI17 phosphorylation levels were measured in wt and FLNAΔECS vSMCs in untreated cells and in the presence of ROCK and PKC inhibitor using pCPI17 antibody. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


A - C ) Telemetry measurements were done on mice continuously for 72 hrs and reading was recorded every minute to analyse blood pressure in wt and FLNAΔECS mice . Scatter plots show diastolic blood pressure over 72 hrs ( A ) , diastolic blood pressure during inactive phase ( B ) and diastolic pressure during active phase ( C ) . Note : Significant increase in diastolic blood pressure in FLNAΔECS mice during the inactive phase . 9 mice ( 5 males marked as square + 4 females marked as round ) were analyzed for each genotype . Data shown as mean ± SEM . P value < 0 . 05 ( Student ' s t test ) was considered significant . Boxes represent the 25th and the 75th percentile with median represented by the black line in the box . The whiskers depict the minimum and the maximum value .

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A-C) Telemetry measurements were done on mice continuously for 72 hrs and reading was recorded every minute to analyse blood pressure in wt and FLNAΔECS mice. Scatter plots show diastolic blood pressure over 72 hrs (A), diastolic blood pressure during inactive phase (B) and diastolic pressure during active phase (C). Note: Significant increase in diastolic blood pressure in FLNAΔECS mice during the inactive phase. 9 mice (5 males marked as square + 4 females marked as round) were analyzed for each genotype. Data shown as mean ± SEM. P value <0.05 (Student's t test) was considered significant. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.


( A ) Dorsal aortae were fixed , cross - sectioned and then stained with Elastica van Gieson stain to visualize collagen around the blood vessel . 6 independent mice ( 5 - 6 months ) of each genotype were used for the analysis . Scale bar : 50 μm . ( B ) Plot shows individual data points ( black - wt , red - mutant ) of adventitial area in both the genotypes . Measurements show increased adventitial area in FLNAΔECS aortae . P value < 0 . 05 was considered significant ( Student ' s t test )

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(A) Dorsal aortae were fixed, cross-sectioned and then stained with Elastica van Gieson stain to visualize collagen around the blood vessel. 6 independent mice (5-6 months) of each genotype were used for the analysis. Scale bar: 50 μm. (B) Plot shows individual data points (black-wt, red-mutant) of adventitial area in both the genotypes. Measurements show increased adventitial area in FLNAΔECS aortae. P value <0.05 was considered significant (Student's t test)


( C ) Representative heart sections of a 5 - 6 months old mice stained with Masson Trichrome shows increased collagen ( blue ) around coronary vessels ( marked by arrowheads ) Scale bar : 50 μm . ( D ) Quantification shows a significant increase in perivascular fibrosis in FLNAΔECS hearts . Four sections from each heart were measured . Four mice analyzed for each genotype . P value < 0 . 05 considered significant . ( mixed model approach )

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(C) Representative heart sections of a 5-6 months old mice stained with Masson Trichrome shows increased collagen (blue) around coronary vessels (marked by arrowheads) Scale bar: 50 μm. (D) Quantification shows a significant increase in perivascular fibrosis in FLNAΔECS hearts. Four sections from each heart were measured. Four mice analyzed for each genotype. P value <0.05 considered significant. (mixed model approach)


( E ) Representative longitudinal heart sections stained with H & E staining ( RV : right ventricle , LV : left ventricle ) . Black lines represent thickness of interventricular ( IVS ) and lateral left ventricular wall ( LLVH ) . Scale bar : 1mm . ( F ) Scatter graph shows increased ventricular wall thickness in FlnAΔECS mice hearts . Six male mice ( 6months old ) analyzed for each genotype . P value < 0 . 05 was considered significant . ( Student ' s t test )

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(E) Representative longitudinal heart sections stained with H&E staining (RV: right ventricle, LV: left ventricle). Black lines represent thickness of interventricular (IVS) and lateral left ventricular wall (LLVH). Scale bar: 1mm. (F) Scatter graph shows increased ventricular wall thickness in FlnAΔECS mice hearts. Six male mice (6months old) analyzed for each genotype. P value <0.05 was considered significant. (Student's t test)


( G ) Heart cross - sections were stained with FITC - WGA ( Wheat germ agglutinin ) to visualize cell membranes of cardiomyocytes . Scale bar : 20 μm . ( H ) Respective cell areas measured by Image J software and represented by box plots for each animal ( six per genotype ) . Boxes represent the 25th and the 75th percentile with median represented by the black line in the box . The whiskers depict the minimum and the maximum value . P value < 0 . 05 ( mixed model approach ) was considered significant .

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(G) Heart cross-sections were stained with FITC-WGA (Wheat germ agglutinin) to visualize cell membranes of cardiomyocytes. Scale bar: 20 μm. (H) Respective cell areas measured by Image J software and represented by box plots for each animal (six per genotype). Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value. P value <0.05 (mixed model approach) was considered significant.


( A ) Graph showing the individual blood velocity profile determined by MRI over time in 4 wt mice ( shades of grey ) and 5 FLNAΔECS mice ( shades of red ) . The mouse shown in dotted purple line is clearly an outlier within the FLNAΔECS cohort . Aortic velocity waveform represents velocity during different stages of cardiac cycle . A significant reduction of velocity was observed at 40ms in the systolic cycle .

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(A) Graph showing the individual blood velocity profile determined by MRI over time in 4 wt mice (shades of grey) and 5 FLNAΔECS mice (shades of red). The mouse shown in dotted purple line is clearly an outlier within the FLNAΔECS cohort. Aortic velocity waveform represents velocity during different stages of cardiac cycle. A significant reduction of velocity was observed at 40ms in the systolic cycle.


( B ) Representative wt ( top ) and FLNAΔECS ( below ) mouse showing the blood flow just above the aortic valve . Note : Reduced blood flow in FLNAΔECS mouse as depicted by pseudocoloring . The red circle represents the time window at which the images are shown .

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(B) Representative wt (top) and FLNAΔECS (below) mouse showing the blood flow just above the aortic valve. Note: Reduced blood flow in FLNAΔECS mouse as depicted by pseudocoloring. The red circle represents the time window at which the images are shown.


( A - C ) Bone marrow derived mouse macrophages ( BMDMs ) from wild type C57BL / 6 mice were treated with 4µ8c ( 100 µM ) or vehicle ( dimethyl sulfoxide ; DMSO ) for 24 hours prior to sample processing for lipidomics analysis : ( A ) PI ( 3 , 4 , 5 ) P3 to PIP2 ; ( B ) PI ( 3 , 4 , 5 ) P3 to PI ; ( C ) PI ( 3 , 4 , 5 ) P3 to PIP ratio are shown . Data represent mean values ± standard error of mean ( SEM ) of peak areas normalized to internal standards ( n = 3 biological replicates ) .

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(A-C) Bone marrow derived mouse macrophages (BMDMs) from wild type C57BL/6 mice were treated with 4µ8c (100 µM) or vehicle (dimethyl sulfoxide; DMSO) for 24 hours prior to sample processing for lipidomics analysis: (A) PI(3,4,5)P3 to PIP2; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP ratio are shown. Data represent mean values ± standard error of mean (SEM) of peak areas normalized to internal standards (n=3 biological replicates).


( D ) BMDMs were treated with 4µ8c ( 100 µM ) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 and β - actin . Western blot quantifications are shown next to the figure ( n = 3 biological replicates ; a representative blot is shown ) .

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(D) BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates; a representative blot is shown).


( E ) Wild type C57BL / 6 mice were injected with tunicamycin ( 1 mg / kg ) and 4µ8c ( 10 µg / kg ) for 8 hours prior to sacrifice and collection of thiogylcolate - elicited peritoneal macrophages . Protein lysates were analyzed by western blotting using specific antibodies against , pIRE1S742 , IRE1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 and β - actin ( n = 3 biological replicates ; a representative blot is shown ) . Western blot quantifications are shown next to the figure .

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(E) Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 hours prior to sacrifice and collection of thiogylcolate-elicited peritoneal macrophages. Protein lysates were analyzed by western blotting using specific antibodies against, pIRE1S742, IRE1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin (n=3 biological replicates; a representative blot is shown). Western blot quantifications are shown next to the figure.


( F ) IRE1 + / + and IRE1 - / - BMDMs were treated with PA ( 500 µM ) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742 , IRE1 , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 and β - actin . Western blot quantifications are shown next to the figure ( n = 3 biological replicates , a representative blot is shown ) .

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

(F) IRE1+/+ and IRE1-/- BMDMs were treated with PA (500 µM) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742, IRE1, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates, a representative blot is shown).


IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( B ) RNA lysates from the same experiment were analyzed by qRT - PCR for miR - 2137 expression .

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

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (B) RNA lysates from the same experiment were analyzed by qRT-PCR for miR-2137 expression.


IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( C ) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742 , IRE1 and β - actin ( n = 3 biological replicates ) .

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

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (C) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742, IRE1 and β-actin (n=3 biological replicates).


IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( D ) RNA lysates were analyzed by qRT - PCR for sXBP1 expression ( n = 3 biological replicates ) .

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

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (D) RNA lysates were analyzed by qRT-PCR for sXBP1 expression (n=3 biological replicates).


IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( E - F ) RNA lysates from the same experiment were analyzed by qRT - PCR for ( E ) BLOC1S1 and ( F ) SCARA3 expression ( n = 3 biological replicates ) .

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

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (E-F) RNA lysates from the same experiment were analyzed by qRT-PCR for (E) BLOC1S1 and (F) SCARA3 expression (n=3 biological replicates).


( A - B ) BMDMs were treated with PA , steric acid ( SA ) , palmitoleic acid ( PAO ) or oleic acid ( OA ) ( 500 µM ) for 16 hours . RNA lysates were analyzed by qRT - PCR for the expression of ( A ) miR - 2137 and ( B ) sXBP1 ( n = 3 biological replicates ) .

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

(A-B) BMDMs were treated with PA, steric acid (SA), palmitoleic acid (PAO) or oleic acid (OA) (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for the expression of (A) miR-2137 and (B) sXBP1 (n=3 biological replicates).


( C , D ) ( C ) C57BL / 6 ( WT ) and ( D ) Apoe - / - mice were fed with chow or WD for 16 weeks followed by peritoneal macrophage isolation . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( WT ; n = 4 - 4 mice per group , Apoe - / - ; n = 5 - 5 mice per group ) .

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 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 O O O O O O O O

(C, D) (C) C57BL/6 (WT) and (D) Apoe-/- mice were fed with chow or WD for 16 weeks followed by peritoneal macrophage isolation. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (WT; n=4-4 mice per group, Apoe-/-; n=5-5 mice per group).


( E - F ) Apoe - / - mice were fed with chow or WD for 12 weeks and injected with 4µ8c ( 10 mg / kg / day ) or vehicle ( DMSO ) in the final 6 weeks of the diet ( n = 5 - 7 mice per group ) : ( E ) RNA lysates from bone marrow were analyzed by qRT - PCR for miR - 2137 expression ( n = 5 - 7 mice per group ) . ( F ) Total RNA isolated from the aortic root plaques was analyzed by qRT - PCR for miR - 2137 expression ( n = 5 - 7 mice per group ) .

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

(E-F) Apoe-/- mice were fed with chow or WD for 12 weeks and injected with 4µ8c (10 mg/kg/day) or vehicle (DMSO) in the final 6 weeks of the diet (n=5-7 mice per group): (E) RNA lysates from bone marrow were analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group). (F) Total RNA isolated from the aortic root plaques was analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group).


( A ) BMDMs were transfected with scrambled ( Scr ) or IRE1 - specific siRNA ( 40 nM ) and treated with palmitate ( PA , 500 µM ) or vehicle for 6 and 9 hours ( hr ) . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

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

(A) BMDMs were transfected with scrambled (Scr) or IRE1-specific siRNA (40 nM) and treated with palmitate (PA, 500 µM) or vehicle for 6 and 9 hours (hr). RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( B ) BMDMs were treated with PA ( 500 µM ) and 4µ8c ( 100 µM ) or vehicle for 9 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) . ( C ) BMDM cells were treated with tunicamycin ( Tun ; 5 µg / ml ) and 4µ8c ( 100 µM ) or vehicle ( DMSO ) for 9 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

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

(B) BMDMs were treated with PA (500 µM) and 4µ8c (100 µM) or vehicle for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates). (C) BMDM cells were treated with tunicamycin (Tun; 5 µg/ml) and 4µ8c (100 µM) or vehicle (DMSO) for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( D ) IRE1 - / - MEFs were transfected with vector ( control ) , WT - IRE1 or K907A - IRE1 ( RNase dead mutant ) plasmids for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

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

(D) IRE1-/- MEFs were transfected with vector (control), WT-IRE1 or K907A-IRE1 (RNase dead mutant) plasmids for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( E ) BMDMs were transfected with scrambled or XBP - 1 specific siRNA ( 70 nM ) for 24 hours , followed by treatment with PA ( 500 µM ) for 16 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

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 B-CONTROLLED_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

(E) BMDMs were transfected with scrambled or XBP-1 specific siRNA (70 nM) for 24 hours, followed by treatment with PA (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( F ) XBP1 - / - MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . RNA lysates were analyzed for miR - 2137 expression by qRT - PCR ( n = 3 biological replicates ) .

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-CONTROLLED_VAR 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

(F) XBP1-/- MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed for miR-2137 expression by qRT-PCR (n=3 biological replicates).


( G ) IRE1 cleavage assay performed using synthetic pre - miR - 2137 ( 50 nM ) as substrate with active , recombinant IRE1 ( 100 ng ) at 37 ℃ for 2 hours , followed by sample separation in Urea - PAGE and detection with SYBR gold staining . M indicates micro RNA marker .

O O O B-CONTROLLED_VAR 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 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

(G) IRE1 cleavage assay performed using synthetic pre-miR-2137 (50 nM) as substrate with active, recombinant IRE1 (100 ng) at 37℃ for 2 hours, followed by sample separation in Urea-PAGE and detection with SYBR gold staining. M indicates micro RNA marker.


( H - I ) BMDMs isolated from WT ( C57BL6 ) and DICER - / - mice were treated with PA ( 500 µM ) for 12 hours . RNA lysates were analyzed by qRT - PCR for ( H ) miR - 2137 and ( I ) sXBP1 expression ( n = 3 biological replicates ) .

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

(H-I) BMDMs isolated from WT (C57BL6) and DICER-/- mice were treated with PA (500 µM) for 12 hours. RNA lysates were analyzed by qRT-PCR for (H) miR-2137 and (I) sXBP1 expression (n=3 biological replicates).


( B ) HEK293 cells were co - transfected with an INPPL1 3 ` UTR - luciferase plasmid and scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) or miR - 205 mimic ( 40 nM ) for 24 hours . Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase ( SEAP ) reporter activity ( n = 3 biological replicates ) .

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

(B) HEK293 cells were co-transfected with an INPPL1 3` UTR-luciferase plasmid and scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours. Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase (SEAP) reporter activity (n=3 biological replicates).


( C ) BMDMs were transfected with scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) and miR - 205 mimic ( 40 nM ) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β - actin . Protein quantifications are displayed in graph next to the blots ( n = 3 biological replicates ) .

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 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 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

(C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) and miR-205 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications are displayed in graph next to the blots (n=3 biological replicates).


( D ) BMDMs were transfected with negative control antagomir ( 200 nM ) or miR - 2137 antagomir ( 200 nM ) for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β - actin . Protein quantifications shown in the graph next to blots ( n = 3 biological replicates ) .

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

(D) BMDMs were transfected with negative control antagomir (200 nM) or miR-2137 antagomir (200 nM) for 24 hours, followed by PA (500 µM) treatment for 9 hours. Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications shown in the graph next to blots (n=3 biological replicates).


( A - C ) BMDMs were transfected with scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) or miR - 205 mimic ( 40 nM ) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section . The ratio of lipids from this analysis is shown for ( A ) PI ( 3 , 4 , 5 ) P3 to PIP2 . ; ( B ) PI ( 3 , 4 , 5 ) P3 to PI ; ( C ) PI ( 3 , 4 , 5 ) P3 to PIP . Data from LC / MS / MS using a Waters Xevo TQ - S MS / MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods . ( n = 3 biological replicates )

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

(A-C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section. The ratio of lipids from this analysis is shown for (A) PI(3,4,5)P3 to PIP2.; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP. Data from LC/MS/MS using a Waters Xevo TQ-S MS/MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods. (n=3 biological replicates)


BMDMs were transfected with ( D ) scrambled miRNA mimic ( 40 nM ) or miR - 2137 mimic ( 40 nM ) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 , pmTORS2448 , mTOR and β - actin . Protein quantifications are shown next to the western blots ( n = 3 biological replicates ) .

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

BMDMs were transfected with (D) scrambled miRNA mimic (40 nM) or miR-2137 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).


BMDMs were transfected ( E ) with control antagomir ( 200 nM ) or miR - 2137 antagomir for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 , pmTORS2448 , mTOR and β - actin . Protein quantifications are shown next to the western blots ( n = 3 biological replicates ) .

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

BMDMs were transfected (E) with control antagomir (200 nM) or miR-2137 antagomir for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).


( A ) In situ hybridizations for Elp3 transcripts in the developing inner ear . Transcripts are present at E12 . 5 in a ventro - medial region of the developing cochlear duct ( CD ) and in the cochleo - vestibular ganglion ( CVG ) . At E16 . 5 , transcripts are present in the spiral ganglion neurons ( SGNs ) and in the newly formed hair cells ( HCs ) at the base of the cochlea , where they remain strongly expressed at P1 . Scale bar = 50µm ; inset = 25µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 , OC : Organ of Corti .

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

(A) In situ hybridizations for Elp3 transcripts in the developing inner ear. Transcripts are present at E12.5 in a ventro-medial region of the developing cochlear duct (CD) and in the cochleo-vestibular ganglion (CVG). At E16.5, transcripts are present in the spiral ganglion neurons (SGNs) and in the newly formed hair cells (HCs) at the base of the cochlea, where they remain strongly expressed at P1. Scale bar=50µm; inset=25µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: Organ of Corti.


( B ) Elp3 specific immunostaining confirms the presence of the protein in HCs and SGNs at birth . Scale bar = 10µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 , OC : Organ of Corti .

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(B) Elp3 specific immunostaining confirms the presence of the protein in HCs and SGNs at birth. Scale bar=10µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: Organ of Corti.


Validation of Elp3 depletion from the cochlea in Elp3cKO animals . ( C ) At P1 , Elp3 mRNA is absent from Elp3cKO . Scale bars = 50µm . In situ hybridizations were repeated 4 times .

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Validation of Elp3 depletion from the cochlea in Elp3cKO animals. (C) At P1, Elp3 mRNA is absent from Elp3cKO. Scale bars=50µm. In situ hybridizations were repeated 4 times.


Validation of Elp3 depletion from the cochlea in Elp3cKO animals . ( D ) Western Blots from P1 WT and Elp3cKO cochlear extracts confirm the loss of Elp3 at the protein level ( cochleae from 3 different animals are pooled per sample ) .

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Validation of Elp3 depletion from the cochlea in Elp3cKO animals. (D) Western Blots from P1 WT and Elp3cKO cochlear extracts confirm the loss of Elp3 at the protein level (cochleae from 3 different animals are pooled per sample).


( E ) Representative ABR recording from P19 WT and Elp3cKO animals reveal an absence of sound - evoked potentials in Elp3cKO animals , indicating that they suffer from severe hearing loss . ( F ) Elp3cKO animals present increased ABR thresholds ( WT / Elp3cKO n = 5 / 3 . Unpaired two - tailed t - test , * * p = 0 . 0017 , t = 5 . 369 , DF = 6 , mean ± SD ) .

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

(E) Representative ABR recording from P19 WT and Elp3cKO animals reveal an absence of sound-evoked potentials in Elp3cKO animals, indicating that they suffer from severe hearing loss. (F) Elp3cKO animals present increased ABR thresholds (WT/Elp3cKO n=5/3. Unpaired two-tailed t-test, **p=0.0017, t=5.369, DF=6, mean±SD).


( A ) Toluidine blue stainings of semi - thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss . Scale bar = 100µm , OC : Organ of Corti .

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

(A) Toluidine blue stainings of semi-thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss. Scale bar=100µm, OC: Organ of Corti.


( B ) Specific labellings of the pre - synaptic marker Ctbp2 ( in IHCs ) and the post - synaptic marker GluR2 / 3 ( in SGN terminals ) indicate a decrease in the number of double - labelled dots in P15 Elp3cKO whole - mounted cochlea compared to WT . The remaining synapses still show coupling of the pre - and post - synaptic markers , suggesting they may be functional . Scale bar = 20µm . ( C ) The mean number of Ctbp2 - GluR2 / 3 double - positive dots in IHCs from the basal region of P15 Elp3cKO cochleae is reduced by 65 % compared to control ( n = 6 animals per genotype ; Unpaired two - tailed t - test , * * * p < 0 . 001 , t = 13 . 48 , DF = 10 , mean ± SD ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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) Specific labellings of the pre-synaptic marker Ctbp2 (in IHCs) and the post-synaptic marker GluR2/3 (in SGN terminals) indicate a decrease in the number of double-labelled dots in P15 Elp3cKO whole-mounted cochlea compared to WT. The remaining synapses still show coupling of the pre- and post-synaptic markers, suggesting they may be functional. Scale bar=20µm. (C) The mean number of Ctbp2-GluR2/3 double-positive dots in IHCs from the basal region of P15 Elp3cKO cochleae is reduced by 65% compared to control (n=6 animals per genotype; Unpaired two-tailed t-test, ***p<0.001, t=13.48, DF=10, mean±SD).


( D ) NF - 1 and Mafb stainings on P0 cochlear sections indicate that SGN loss occurs before birth in Elp3cKO cochleae . Scale bar = 100µm .

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

(D) NF-1 and Mafb stainings on P0 cochlear sections indicate that SGN loss occurs before birth in Elp3cKO cochleae. Scale bar=100µm.


( E ) SGN quantifications on cochlear sections from different embryonic stages reveal a progressive loss of SGNs from E14 . 5 onwards ( n = 3 - 6 animals ; one - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 ; * p < 0 . 05 ; F = 20 . 43 ; DF = 7 ; mean ± SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SGN quantifications on cochlear sections from different embryonic stages reveal a progressive loss of SGNs from E14.5 onwards (n=3-6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; *p<0.05; F=20.43; DF=7; mean±SD).


( F ) Detection of apoptotic SGNs from E13 . 5 cochlear sections with cleaved caspase - 3 and B - tubIII stainings . Scale bar = 100µm .

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

(F) Detection of apoptotic SGNs from E13.5 cochlear sections with cleaved caspase-3 and B-tubIII stainings. Scale bar=100µm.


( G ) The number of apoptotic SGN per section is significantly increased in E13 . 5 and E14 . 5 Elp3cKO compared to WT cochleae , suggesting SGNs are properly specified but die during their differentiation process ( n = 3 - 4 animals ; one - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 ; * * p < 0 . 01 ; F = 23 . 74 ; DF = 7 ; mean ± SD ) .

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 O O O O O O

(G) The number of apoptotic SGN per section is significantly increased in E13.5 and E14.5 Elp3cKO compared to WT cochleae, suggesting SGNs are properly specified but die during their differentiation process (n=3-4 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; **p<0.01; F=23.74; DF=7; mean±SD).


( A ) Proteostat staining of WT and Elp3cKO E13 . 5 cochlear sections reveals the presence of numerous aggresome - like structures in Elp3 - deficient SGNs . Scale bar = 100µm .

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

(A) Proteostat staining of WT and Elp3cKO E13.5 cochlear sections reveals the presence of numerous aggresome-like structures in Elp3-deficient SGNs. Scale bar =100µm.


( B ) Transmission electron micrographs from E13 . 5 Elp3cKO SGNs confirm the presence of apoptotic neurons ( marked by a red arrowhead ) and a large electron dense juxtanuclear structures typical of aggresome ( dashed circle ) . Scale bars = 10µm and 2µm ( last panel ) .

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

(B) Transmission electron micrographs from E13.5 Elp3cKO SGNs confirm the presence of apoptotic neurons (marked by a red arrowhead) and a large electron dense juxtanuclear structures typical of aggresome (dashed circle). Scale bars =10µm and 2µm (last panel).


( C ) Chop and Chac1 transcripts are upregulated in E14 Elp3cKO compared to WT cochleae ( n = 8 / 6 for WT / KO ; unpaired t - test two - tailed ; p = 0 . 0001 / 0 . 0015 / 0 . 0010 ; t = 9 . 535 / 4 . 090 / 4 . 307 ; DF = 12 / 12 / 12 for Elp3 , Chop and Chac1 respectively ; mean ± SEM presented ) indicating that the pro - apoptotic arm of the unfolded protein response ( UPR ) is activated in Elp3 - deficient SGNs .

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

(C) Chop and Chac1 transcripts are upregulated in E14 Elp3cKO compared to WT cochleae (n=8/6 for WT/KO; unpaired t-test two-tailed; p=0.0001/0.0015/0.0010; t=9.535/4.090/4.307; DF=12/12/12 for Elp3, Chop and Chac1 respectively; mean±SEM presented) indicating that the pro-apoptotic arm of the unfolded protein response (UPR) is activated in Elp3-deficient SGNs.


( D - E ) Proteostat staining of whole - mounted P0 organs of Corti reveal the presence of aggregates in HCs upon loss of Elp3 . Orthogonal slices ( Orthog ) reveal aggregated proteins localize at the apical surface of Elp3cKO HCs . Scale bars = 5µm . # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 .

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

(D-E) Proteostat staining of whole-mounted P0 organs of Corti reveal the presence of aggregates in HCs upon loss of Elp3. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of Elp3cKO HCs. Scale bars =5µm. #IHC; *OHC1; **OHC2; ***OHC3.


( F ) Protein extracts from P1 cochleae show increased levels of polyubiquitinated conjugates upon loss of Elp3 , confirming proteostasis disruption .

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

(F) Protein extracts from P1 cochleae show increased levels of polyubiquitinated conjugates upon loss of Elp3, confirming proteostasis disruption.


( G ) The levels of Chop and Chac1 transcripts are unaffected by Elp3 depletion in P0 organs of Corti ( n = 3 animals per genotype ; unpaired t - test two - tailed ; p = 0 . 0003 / 0 . 4296 / 0 . 056 ; t = 11 . 62 / 0 . 8778 / 2 . 662 ; DF = 4 / 4 / 4 for Elp3 , Chop and Chac1 respectively ; mean ± SEM presented ) suggesting that proteostasis disruption in Elp3cKO HCs is not sufficient to induce UPR - mediated apoptosis .

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

(G) The levels of Chop and Chac1 transcripts are unaffected by Elp3 depletion in P0 organs of Corti (n=3 animals per genotype; unpaired t-test two-tailed; p=0.0003/0.4296/0.056; t=11.62/0.8778/2.662; DF=4/4/4 for Elp3, Chop and Chac1 respectively; mean±SEM presented) suggesting that proteostasis disruption in Elp3cKO HCs is not sufficient to induce UPR-mediated apoptosis.


( A ) Scanning electron micrographs of cochlear tissue from P19 animals indicate no HC loss in the absence of Elp3 but illustrate some defects of polarity , as misaligned HCs and dysmorphic stereociliary bundles are visible ( red arrows and white arrowheads , respectively ) . Scale bars =

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

(A) Scanning electron micrographs of cochlear tissue from P19 animals indicate no HC loss in the absence of Elp3 but illustrate some defects of polarity, as misaligned HCs and dysmorphic stereociliary bundles are visible (red arrows and white arrowheads, respectively). Scale bars=5µ


( B ) Surface view of P1 actin - stained organ of Corti confirms defective establishment of polarity in Elp3cKO as numerous stereociliary bundles are misaligned along the medio - lateral axis ( M - L ) or misshaped . Scale bars = 5µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

(B) Surface view of P1 actin-stained organ of Corti confirms defective establishment of polarity in Elp3cKO as numerous stereociliary bundles are misaligned along the medio-lateral axis (M-L) or misshaped. Scale bars=5µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3


( C ) Elp3cKOs exhibit a broader distribution of hair cell orientation . The angles of deviation between the stereociliary bundle and the medio - lateral axis are plotted in rose diagrams , for each OHC row . Whereas the majority of WT bundles are pointing to the most lateral region of the tissue ( within a range of 10 ° ) , many Elp3cKO bundles of the two last rows of OHCs present increased angles of deviation ( WT / Elp3cKO OHC1 : n = 99 / 99 , p > 0 . 1 , DF = 99 ; OHC2 : n = 99 / 100 , p < 0 . 001 , DF = 99 ; OHC3 : n = 99 / 98 , p < 0 . 001 , DF = 98 ; pooled data from 6 animals per genotype ; Watson U2 test ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

(C) Elp3cKOs exhibit a broader distribution of hair cell orientation. The angles of deviation between the stereociliary bundle and the medio-lateral axis are plotted in rose diagrams, for each OHC row. Whereas the majority of WT bundles are pointing to the most lateral region of the tissue (within a range of 10°), many Elp3cKO bundles of the two last rows of OHCs present increased angles of deviation (WT/Elp3cKO OHC1: n=99/99, p>0.1, DF=99; OHC2: n=99/100, p<0.001, DF=99; OHC3: n=99/98, p<0.001, DF=98; pooled data from 6 animals per genotype; Watson U2 test). Data information: #IHC; *OHC1; **OHC2; ***OHC3


Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( D ) Scanning electron micrographs of HCs from P1 WT and Elp3cKO cochleae ( scale bar = 2µm ) illustrating the misshaped stereociliary bundles and the shorter kinocilium ( false coloured in pink ) of Elp3cKO HCs . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (D) Scanning electron micrographs of HCs from P1 WT and Elp3cKO cochleae (scale bar=2µm) illustrating the misshaped stereociliary bundles and the shorter kinocilium (false coloured in pink) of Elp3cKO HCs. Data information: #IHC; *OHC1; **OHC2; ***OHC3


Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( E ) Graph presents the mean angle of HC bundle ( n = 6 animals ; one - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 , F = 13 . 69 , DF = 7 ; mean ± SD ) .

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

Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (E) Graph presents the mean angle of HC bundle (n=6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001, F=13.69, DF=7; mean±SD).


Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( F ) Elp3cKO HCs possess a significantly larger bare zone ( WT / KO IHC : n = 23 / 24 cells ; OHC1 : n = 27 / 27 ; OHC2 : n = 27 / 29 ; OHC3 : n = 30 / 29 ; cumulative data collected from 3 animals per genotype ; One - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 , * p < 0 . 05 F = 16 . 72 , DF = 7 ; mean ± SD ) .

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

Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (F) Elp3cKO HCs possess a significantly larger bare zone (WT/KO IHC: n= 23/24 cells; OHC1: n=27/27; OHC2: n=27/29; OHC3: n=30/29; cumulative data collected from 3 animals per genotype; One-way ANOVA, Tukey's multiple comparisons test; ***p<0.001, *p<0.05 F=16.72, DF=7; mean±SD).


Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( G ) Elp3cKO HCs possess a significantly smaller medial zone ( WT / KO IHC : n = 23 / 24 cells ; OHC1 : n = 27 / 27 ; OHC2 : n = 27 / 29 ; OHC3 : n = 27 / 29 ; cumulative data collected from 3 animals per genotype ; One - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 , * p < 0 . 05 , F = 63 . 3 , DF = 7 ; mean ± SD ) .

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

Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (G) Elp3cKO HCs possess a significantly smaller medial zone (WT/KO IHC: n= 23/24 cells; OHC1: n=27/27; OHC2: n=27/29; OHC3: n=27/29; cumulative data collected from 3 animals per genotype; One-way ANOVA, Tukey's multiple comparisons test; ***p<0.001, *p<0.05, F=63.3, DF=7; mean±SD).


( H ) Kinocilia are shorter in Elp3cKO HCs ( WT / KO IHC : n = 15 / 15 ; OHC1 : n = 30 / 32 ; OHC3 : n = 31 / 30 ; OHC3 : n = 27 / 30 ; data collected from 3 animals per genotype ; One - way ANOVA , Tukey ' s multiple comparisons test , * * * p < 0 . 001 , * * p < 0 . 01F = 19 . 6 , DF = 7 ; mean ± SD ) .

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

(H) Kinocilia are shorter in Elp3cKO HCs (WT/KO IHC: n=15/15; OHC1: n=30/32; OHC3: n=31/30; OHC3: n=27/30; data collected from 3 animals per genotype; One-way ANOVA, Tukey's multiple comparisons test, ***p<0.001, **p<0.01F=19.6, DF=7; mean±SD).


( I ) High magnification of representative HCs from P1 WT and Elp3cKO cochlea stained for F - actin and gamma - tubulin ( white ) . The basal body of kinocilia is shifted centrally at the apical surface of Elp3cKO HCs . Scale bars = 2µm . Polar plotting of kinocilium basal body position for each row of HCs of WT and Elp3cKO ( WT / KO IHC : n = 42 / 32 ; OHC1 : n = 47 / 48 ; OHC2 : n = 47 / 45 ; OHC3 : n = 44 / 44 ; cumulative data collected from 3 animals per genotype ) . Cellular orientations were tested using Watson U2 test ( p < 0 . 001 between WT / Elp3cKO , for all HC rows ) and the mean vector lengths , determined from the cell centre to the basal body , were compared using t - tests ( p < 0 . 001 between WT / Elp3cKO , for all HC rows ) .

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

(I) High magnification of representative HCs from P1 WT and Elp3cKO cochlea stained for F-actin and gamma-tubulin (white). The basal body of kinocilia is shifted centrally at the apical surface of Elp3cKO HCs. Scale bars=2µm. Polar plotting of kinocilium basal body position for each row of HCs of WT and Elp3cKO (WT/KO IHC: n= 42/32; OHC1: n= 47/48; OHC2: n= 47/45; OHC3: n= 44/44; cumulative data collected from 3 animals per genotype). Cellular orientations were tested using Watson U2 test (p<0.001 between WT/Elp3cKO, for all HC rows) and the mean vector lengths, determined from the cell centre to the basal body, were compared using t-tests (p<0.001 between WT/Elp3cKO, for all HC rows).


The localization of apical polarity proteins is affected by Elp3 loss . Surface views of mid - basal turn of P1 cochleae from WT or Elp3cKO mice stained with LGN ( J ) and F - actin . The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs . Scale bars = 5µm ( upper panels ) and 2µm ( lower panels ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

The localization of apical polarity proteins is affected by Elp3 loss. Surface views of mid-basal turn of P1 cochleae from WT or Elp3cKO mice stained with LGN (J) and F-actin. The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs. Scale bars=5µm (upper panels) and 2µm (lower panels). Data information: #IHC; *OHC1; **OHC2; ***OHC3


The localization of apical polarity proteins is affected by Elp3 loss . Surface views of mid - basal turn of P1 cochleae from WT or Elp3cKO mice stained with Gαi3 ( K ) and F - actin . The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs . Scale bars = 5µm ( upper panels ) and 2µm ( lower panels ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

The localization of apical polarity proteins is affected by Elp3 loss. Surface views of mid-basal turn of P1 cochleae from WT or Elp3cKO mice stained with Gαi3 (K) and F-actin. The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs. Scale bars=5µm (upper panels) and 2µm (lower panels). Data information: #IHC; *OHC1; **OHC2; ***OHC3


The localization of apical polarity proteins is affected by Elp3 loss . Surface views of mid - basal turn of P1 cochleae from WT or Elp3cKO mice stained with aPKC ( L ) and F - actin . The enrichment of these apical polarity regulators at the medial ( L ) edges of HCs is disrupted in Elp3cKO HCs . Scale bars = 5µm ( upper panels ) and 2µm ( lower panels ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

The localization of apical polarity proteins is affected by Elp3 loss. Surface views of mid-basal turn of P1 cochleae from WT or Elp3cKO mice stained with aPKC (L) and F-actin. The enrichment of these apical polarity regulators at the medial (L) edges of HCs is disrupted in Elp3cKO HCs. Scale bars=5µm (upper panels) and 2µm (lower panels). Data information: #IHC; *OHC1; **OHC2; ***OHC3


( A - B ) Polarized enrichment of LGN at the cortices of dividing HEK293T cells is disrupted upon Elp3 or Alkbh8 knockdown . ( A ) HEK293T cells transfected with siRNAs for Elp3 , Alkbh8 or control and immunolabelled with LGN , acetylated alpha - tubulin and DAPI . Scale bar = 5µm . ( B ) The mean fluorescence intensity profile of LGN across the radii perpendicular to division in HEK293T cells was quantified ( siRNA Control / Elp3 / Alkbh8 : n = 27 / 30 / 27 ; pooled from 3 independent experiments ; mean ± SEM ) .

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

(A-B) Polarized enrichment of LGN at the cortices of dividing HEK293T cells is disrupted upon Elp3 or Alkbh8 knockdown. (A) HEK293T cells transfected with siRNAs for Elp3, Alkbh8 or control and immunolabelled with LGN, acetylated alpha-tubulin and DAPI. Scale bar=5µm. (B) The mean fluorescence intensity profile of LGN across the radii perpendicular to division in HEK293T cells was quantified (siRNA Control/Elp3/Alkbh8: n=27/30/27; pooled from 3 independent experiments; mean±SEM).


( C - D ) The length of the primary cilium is reduced in non - dividing HEK293T cells upon Elp3 or Albh8 knockdown . ( C ) Control , Elp3 or Alkbh8 siRNA - treated HEK293T cells were immunolabelled for basal body marker ALMS1 , axoneme marker Arl13b and DAPI . Scale bar = 5µm . ( D ) Primary cilium length of non - dividing HEK293T was measured and pooled data were plotted ( siRNA Control / Elp3 / Alkbh8 : n = 94 / 81 / 90 ; data collected from 3 independent experiments ; One - way ANOVA , Tukey ' s multiple comparisons test , * * * p < 0 . 001 , F = 21 . 46 , DF = 4 ; minimum , first quartile , median , third quartile and maximum presented ) .

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

(C-D) The length of the primary cilium is reduced in non-dividing HEK293T cells upon Elp3 or Albh8 knockdown. (C) Control, Elp3 or Alkbh8 siRNA-treated HEK293T cells were immunolabelled for basal body marker ALMS1, axoneme marker Arl13b and DAPI. Scale bar=5µm. (D) Primary cilium length of non-dividing HEK293T was measured and pooled data were plotted (siRNA Control/Elp3/Alkbh8: n=94/81/90; data collected from 3 independent experiments; One-way ANOVA, Tukey's multiple comparisons test, ***p<0.001, F=21.46, DF=4; minimum, first quartile, median, third quartile and maximum presented).


( E - F ) Elp3 knockdown increases the amount of polyubiquitinated conjugates in HEK293T cells ( n = 3 ; One - way ANOVA , Tukey ' s multiple comparisons test , * * p < 0 . 01 , * p < 0 . 05 , F = 16 . 51 , DF = 2 ; mean ± SD ) .

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

(E-F) Elp3 knockdown increases the amount of polyubiquitinated conjugates in HEK293T cells (n=3; One-way ANOVA, Tukey's multiple comparisons test, **p<0.01, *p<0.05, F=16.51, DF=2; mean±SD).


( G ) Increased formation of aggresome - like structures in Elp3 - or Alkbh8 - depleted HEK293T . Aggresomes ( red ) ; DAPI ( blue ) . Enlarged images depict examples of non - dividing ( left ) and dividing ( right ) cells . Scale bar = 10µm .

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

(G) Increased formation of aggresome-like structures in Elp3- or Alkbh8-depleted HEK293T. Aggresomes (red); DAPI (blue). Enlarged images depict examples of non-dividing (left) and dividing (right) cells. Scale bar=10µm.


Inducing aggresomes with MG - 132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells . ( H ) HEK293T cells treated with DMSO vehicle or MG - 132 and stained for aggresomes ( left panels ) or LGN , F - actin and DAPI ( green , red and blue , respectively in right panels ) . Scale bar = 10µm .

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

Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (H) HEK293T cells treated with DMSO vehicle or MG-132 and stained for aggresomes (left panels) or LGN, F-actin and DAPI (green, red and blue, respectively in right panels). Scale bar=10µm.


Inducing aggresomes with MG - 132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells . ( I ) Fluorescence intensity profile of LGN across the radii perpendicular to division in dividing HEK293T cells . Mean fluorescence values per percentage of cell length with SEM are plotted . Fluorescence intensity profiles collected from 3 independent experiments ( DMSO / MG - 132 : n = 24 / 24 ) .

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

Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (I) Fluorescence intensity profile of LGN across the radii perpendicular to division in dividing HEK293T cells. Mean fluorescence values per percentage of cell length with SEM are plotted. Fluorescence intensity profiles collected from 3 independent experiments (DMSO/MG-132: n= 24/24).


( J ) Proteostat staining of MG132 - treated whole - mounted P0 organs of Corti reveals the presence of aggresome - like structures in HCs . Orthogonal slices ( Orthog ) reveal aggregated proteins localize at the apical surface of MG132 - treated HCs . Scale bars = 5µm . # IHC ; * OHC1 ; * * OHC2 ; * * *

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 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

(J) Proteostat staining of MG132-treated whole-mounted P0 organs of Corti reveals the presence of aggresome-like structures in HCs. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of MG132-treated HCs. Scale bars=5µm. #IHC; *OHC1; **OHC2; ***OHC3


( K ) Localization of LGN at the lateral apical surface of E16 . 5 WT HCs is disrupted by MG - 132 . LGN ( white / green ) and F - actin ( red ) . Scale bars = 5µm ; magnified views = 2µm .

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(K) Localization of LGN at the lateral apical surface of E16.5 WT HCs is disrupted by MG-132. LGN (white/green) and F-actin (red). Scale bars=5µm; magnified views=2µm.


LGN and Gαi3 protein levels are not affected by Elp3 loss . ( A ) Western blots with total lysates from control and Elp3 siRNA - treated HEK293T cells and corresponding quantifications of normalized LGN , Gαi3 and Elp3 levels ( n = 6 from 2 independent cultures ; Unpaired two - tailed t - test , LGN : p = 0 . 749 , t = 0 . 328 , DF = 10 , Gαi3 : p = 0 . 275 , t = 1 . 155 , DF = 10 , Elp3 : * * * p = 0 . 0003 , t = 5 . 438 , DF = 10 , mean ± SEM ) .

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

LGN and Gαi3 protein levels are not affected by Elp3 loss. (A) Western blots with total lysates from control and Elp3 siRNA-treated HEK293T cells and corresponding quantifications of normalized LGN, Gαi3 and Elp3 levels (n=6 from 2 independent cultures; Unpaired two-tailed t-test, LGN: p=0.749, t=0.328, DF=10, Gαi3: p=0.275, t=1.155, DF=10, Elp3: ***p=0.0003, t=5.438, DF=10, mean±SEM).


LGN and Gαi3 protein levels are not affected by Elp3 loss . ( B ) Western blots on P0 WT and Elp3cKO cochlear extracts and corresponding quantifications of normalized LGN and Gαi3 levels ( n = 3 , each lysate obtained from cochleae pooled from 2 animals ; Unpaired two - tailed t - test , LGN : p = 0 . 586 , t = 0 . 591 , DF = 4 , Gαi3 : p = 0 . 963 , t = 0 . 0491 , DF = 4 , mean ± SEM ) .

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

LGN and Gαi3 protein levels are not affected by Elp3 loss. (B) Western blots on P0 WT and Elp3cKO cochlear extracts and corresponding quantifications of normalized LGN and Gαi3 levels (n=3, each lysate obtained from cochleae pooled from 2 animals; Unpaired two-tailed t-test, LGN: p=0.586, t=0.591, DF=4, Gαi3: p=0.963, t=0.0491, DF=4, mean±SEM).


( C ) Mislocalized LGN and Gαi3 are not found in protein aggregates formed upon Elp3 depletion . HEK293T transfected with Elp3 siRNA were co - stained for aggresomes , Dapi and LGN or Gαi3 . Scale bar = 5µm .

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

(C) Mislocalized LGN and Gαi3 are not found in protein aggregates formed upon Elp3 depletion. HEK293T transfected with Elp3 siRNA were co-stained for aggresomes, Dapi and LGN or Gαi3. Scale bar=5µm.


Microtubular transport speed is reduced by the presence of aggresome - like structures in HEK293T cells and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HEK293T cells labelled with LysoTracker ® . ( D ) : DMSO / MG - 132 / MG - 132 + 4PBA n = 555 / 337 / 453 , pooled from 3 experiments , * * * p < 0 . 001 , * * p < 0 . 01 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum .

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

Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (D): DMSO/MG-132/MG-132+4PBA n=555/337/453, pooled from 3 experiments, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.


Microtubular transport speed is reduced by the presence of aggresome - like structures in HEK293T cells and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HEK293T cells labelled with LysoTracker ® . ( E ) : siCtl / + 4PBA n = 622 / 677 ; siElp3 / + 4PBA n = 674 / 577 ; siAlkbh8 / + 4PBA n = 460 / 484 , pooled from 4 experiments , * * * p < 0 . 001 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum .

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

Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (E): siCtl/+4PBA n=622/677; siElp3/+4PBA n=674/577; siAlkbh8/+4PBA n=460/484, pooled from 4 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.


Microtubular transport speed is reduced by the presence of aggresome - like structures in cultured HCs , and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HCs , at the level of their apical surface labelled with LysoTracker ® . ( F ) : DMSO / MG - 132 / MG - 132 + 4PBA n = 210 / 225 / 266 , pooled from 3 experiments , * * * p < 0 . 001 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum .

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

Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (F): DMSO/MG-132/MG-132+4PBA n=210/225/266, pooled from 3 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.


Microtubular transport speed is reduced by the presence of aggresome - like structures in cultured HCs , and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HCs , at the level of their apical surface labelled with LysoTracker ® . ( G ) : WT / WT + 4PBA / Elp3cKO / Elp3cKO + 4PBA n = 732 / 763 / 730 / 738 , pooled from 4 OCs per condition , * * * p < 0 . 001 , * * p < 0 . 01 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (G): WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA n=732/763/730/738, pooled from 4 OCs per condition, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.


Intrinsic polarity is disrupted upon MG - 132 treatment of cultured HCs and restored by the chemical chaperone 4 - PBA . ( A ) LGN and F - actin immunostainings of E16 . 5 + 1DIV OCs reveal that MG - 132 perturbs LGN distribution at the apical surface of HCs , while co - treatment with 4 - PBA ameliorates its enrichment at the lateral edge . Scale bars = 5µm ; magnified views = 2µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (A) LGN and F-actin immunostainings of E16.5+1DIV OCs reveal that MG-132 perturbs LGN distribution at the apical surface of HCs, while co-treatment with 4-PBA ameliorates its enrichment at the lateral edge. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3


Intrinsic polarity is disrupted upon MG - 132 treatment of cultured HCs and restored by the chemical chaperone 4 - PBA . ( B ) Acetylated alpha - tubulin immunostaining reveals that the kinocilium is centrally shifted by MG - 132 and partially restored by 4 - PBA in E16 . 5 + 1DIV HCs ( yellow arrowheads = kinocilium base ; scale bar = 2µm ) .

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

Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (B) Acetylated alpha-tubulin immunostaining reveals that the kinocilium is centrally shifted by MG-132 and partially restored by 4-PBA in E16.5+1DIV HCs (yellow arrowheads = kinocilium base; scale bar=2µm).


Intrinsic polarity is disrupted upon MG - 132 treatment of cultured HCs and restored by the chemical chaperone 4 - PBA . ( C ) Kinocilium position plotted as a fraction of HC diameter for each HC row ( DMSO / MG - 132 / MG - 132 + 4PBA IHC : n = 57 / 67 / 53 , * * * p < 0 . 001 , F = 34 . 52 , DF = 2 ; OHC1 : n = 61 / 66 / 53 , * * * p < 0 . 001 , F = 57 . 88 , DF = 2 ; OHC2 : n = 55 / 73 / 58 , * * * p < 0 . 001 , F = 124 . 7 , DF = 2 ; OHC3 : n = 48 / 66 / 43 , * * * p < 0 . 001 , F = 67 . 16 , DF = 2 ; pooled from 4 explants per condition ; mean ± SD ; One - way ANOVA with Tukey ' s multiple comparisons test ) .

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

Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (C) Kinocilium position plotted as a fraction of HC diameter for each HC row (DMSO/MG-132/MG-132+4PBA IHC: n=57/67/53, ***p<0.001, F=34.52, DF=2; OHC1: n=61/66/53, ***p<0.001, F=57.88, DF=2; OHC2: n=55/73/58, ***p<0.001, F=124.7, DF=2; OHC3: n=48/66/43, ***p<0.001, F=67.16, DF=2; pooled from 4 explants per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).


In vivo administration of 4 - PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs . Pregnant mice were treated daily with saline or 4 - PBA from E13 . 5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17 . 5 . ( D ) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration . Scale bars = 5µm ; magnified views = 2µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (D) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3


In vivo administration of 4 - PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs . Pregnant mice were treated daily with saline or 4 - PBA from E13 . 5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17 . 5 . ( E ) Acetylated alpha - tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4 - PBA ( yellow arrowheads = base of kinocilium ; scale bar = 2µm ) .

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

In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (E) Acetylated alpha-tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4-PBA (yellow arrowheads = base of kinocilium; scale bar=2µm).


In vivo administration of 4 - PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs . Pregnant mice were treated daily with saline or 4 - PBA from E13 . 5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17 . 5 . ( F ) Kinocilium position plotted as a fraction of HC diameter for each HC row ( WT / WT + 4PBA / Elp3cKO / Elp3cKO + 4PBA IHC : n = 35 / 31 / 35 / 36 , p = 0 . 6812 , F = 4 . 510 , DF = 3 ; OHC1 : n = 36 / 32 / 37 / 40 , * * * p < 0 . 001 , * * p < 0 . 01 , F = 27 . 60 , DF = 3 ; OHC2 : n = 35 / 33 / 38 / 42 , * * * p < 0 . 001 , F = 76 . 62 , DF = 3 ; OHC3 : n = 36 / 32 / 38 / 39 , * * * p < 0 . 001 , * p < 0 . 05 , F = 27 . 86 , DF = 3 ; pooled from 3 animals per condition ; mean ± SD ; One - way ANOVA with Tukey ' s multiple comparisons test ) .

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

In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (F) Kinocilium position plotted as a fraction of HC diameter for each HC row (WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA IHC: n=35/31/35/36, p=0.6812, F=4.510, DF=3; OHC1: n=36/32/37/40, ***p<0.001, **p<0.01, F=27.60, DF=3; OHC2: n=35/33/38/42, ***p<0.001, F=76.62, DF=3; OHC3: n=36/32/38/39, ***p<0.001, *p<0.05, F=27.86, DF=3; pooled from 3 animals per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).


Non - endosomal ubiquitin - positive structures accumulate in ESCRT depleted cells . HeLa cells were left untransfected ( A ) or transfected with control ( B ) , Hrs ( C ) , Tsg101 ( D ) , Vps22 ( E ) , or Vps24 ( F ) siRNA for 5 d and processed for immunofluorescence microscopy . Cells were labeled with antibodies against EEA1 ( blue ) , ubiquitin ( mono and poly - Ub , red ) , and Lamp2 ( green ) . Colocalization between EEA1 and Ub is indicated in purple and between Ub and Lamp2 in yellow . Bar , 10 μm . Single channel images of the insets are shown in Fig . S1 ( available at http : / / www . jcb . org / cgi / content / full / jcb . 200702115 / DC1 ) .

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

Non-endosomal ubiquitin-positive structures accumulate in ESCRT depleted cells. HeLa cells were left untransfected (A) or transfected with control (B), Hrs (C), Tsg101 (D), Vps22 (E), or Vps24 (F) siRNA for 5 d and processed for immunofluorescence microscopy. Cells were labeled with antibodies against EEA1 (blue), ubiquitin (mono and poly-Ub, red), and Lamp2 (green). Colocalization between EEA1 and Ub is indicated in purple and between Ub and Lamp2 in yellow. Bar, 10 μm. Single channel images of the insets are shown in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200702115/DC1).


( G ) Knock - down efficiencies were analyzed by Western blotting . Equal loading was verified by anti - α - tubulin immunoblotting .

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

(G) Knock-down efficiencies were analyzed by Western blotting. Equal loading was verified by anti-α-tubulin immunoblotting.


p62 - and Alfy - positive structures accumulate in cells depleted of Tsg101 or Vps24 . HeLa cells transfected with siRNA against Tsg101 or Vps24 were fixed , permeabilized , and stained with antibodies against ubiquitin ( red ) and p62 ( green ) ( A ) or against ubiquitin ( green ) and Alfy ( red ) ( B ) . Colocalization is indicated in yellow . Bar , 10 μm . ( C ) The number and size ( area ) of p62 - positive particles were quantified using ImageJ software . Approximately 100 cells from three different experiments ( n = 334 ( control ) , 309 ( Tsg101 ) , and 246 ( Vps24 ) ) were used for the quantification and the data is presented as average number per 100 cells . Error bars = SEM .

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

p62- and Alfy-positive structures accumulate in cells depleted of Tsg101 or Vps24. HeLa cells transfected with siRNA against Tsg101 or Vps24 were fixed, permeabilized, and stained with antibodies against ubiquitin (red) and p62 (green) (A) or against ubiquitin (green) and Alfy (red) (B). Colocalization is indicated in yellow. Bar, 10 μm. (C) The number and size (area) of p62-positive particles were quantified using ImageJ software. Approximately 100 cells from three different experiments (n = 334 (control), 309 (Tsg101), and 246 (Vps24)) were used for the quantification and the data is presented as average number per 100 cells. Error bars = SEM.