O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Rescue experiment of stx mutant. The stx mutant phenotype can be rescued by stx over expression driven by stx-Gal4 (in female flies, P) or EB1-Gal4 (Q). For P, from left to right females mean±SEM: 226.0±11.76, n=49; 336.6±14.10, n=86; 382.7±16.43, n=50; 357.8±16.55, n=72; 296.2±16.04, n=68; 484.9±12.97, n=49; 507.6±13.83, n=86; 539.5±12.99, n=50. 577.0±11.34, n=72; 520.7±14.96, n=68; For Q, from left to right mean±SEM; 398.3±9.535, n=63; 378.4±23.78, n=32; 324.4±12.33, n=45; 495.8±16.43, n=28; 478.5±14.65, n=53; 328.7±16.99, n=34; 540.7±7.690, n=63; 489.9±21.39, n=32; 490.9±13.65, n=45; 494.7±11.99, n=28; 537.8±12.07, n=53; 450.3±15.47, n=34.


Statistical of Pc stx double mutant stxd77 ; ; pcXT109 sleep profile compared with controls . Quantitative RT - PCR of Octβ2R in adult head of stxd77 w1118 control . ( Data represent mean ± : w1118 , 0 . 8853 ± 0 . 1177 ; N = 3 ; stxd77 / Y , 0 . 3263 ± 0 . 1575 ； N = 3 ) .

O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. Quantitative RT-PCR of Octβ2R in adult head of stxd77 and w1118 control. (Data represent mean±SEM: w1118, 0.8853±0.1177; N=3; stxd77/Y, 0.3263±0.1575；N=3).


O O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Rescue of stxd77 by over expressing Octβ2R in ellipsoid body. From left to right mean±SEM: 399.6±11.49, n=50; 400.3±10.48, n=60; 349.9±14.77, n=48; 495.8±16.43, n=28; 470.1±13.04, n=44; 423.6±14.32, n=47; 548.6±8.707, n=50; 499.0±8.225, n=60; 490.7±16.26, n=48; 494.7±11.99, n=28; 480.2±14.64, n=44; 437.4±14.85, n=47.


O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O

Rescue of stxd77 by Pc RNAi in ellipsoid body. From left to right mean±SEM: 399.9±11.88, n=48; 409.8±11.50, n=49; 348.7±20.19, n=32; 495.8±16.43, n=28; 459.3±14.78, n=61; 407.5±15.66, n=40; 550.7±8.881, n=48; 516.0±11.76, n=49; 502.3±15.08, n=32; 494.7±11.99, n=28; 546.3±11.35, n=61; 514.2±18.94, n=40. Note that the same data is used for genotype stxd77;; EB1-Gal4 in panel F and G.


B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Sleep phenotype of OctβR double and triple mutants. (From left to right mean±SEM: 392.0±12.91, n=60; 395.1±8.56, n=79; 433.7±13.03, n=58; 422.8±10.34; n=62; 474.4±8.36, n=73; 517.4±6.30, n=119; 513.2±7.10, n=79; 536.6±9.05 n=78; 499.1±11.95, n=60; 546.0±8.51, n=79; 519.6±10.31, n=58; 518.9±13.13; n=62; 584.3±8.59, n=73; 579.2±8.35, n=119; 560.9±6.80, n=79; 606.4±6.38, n=78).


B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR of stx after OA treatment in w1118 control and Octβ1R, Octβ2R mutants, Octβ3R mutants. (From left to right mean±SEM: 1.000±0.0288, 0.781±0.0176, 1.477±0.043，1.403±0.0578, 1.157±0.0617, 0.881±0.0689, 1.149±0.0398, 0.953±0.0543, 1.017±0.0600, 1.090±0.0378, N=3).


Ellipsoid knock down of Octβ2R results in sleep rebound increase . In response to OA , ellipsoid knock down of Octβ2R rescues control phenotype . Data represent mean ± : 13 . 80 ± 2 . 439 , n = 31 ; 5 . 465 ± 2 . 036 , n = 25 ; 19 . 55 ± 2 . 275 , n = 27 ; 12 . 47 ± 2 . 684 , n = 24 ; 30 . 03 ± 3 . 428 , n = 25 ; 43 . 67 ± 4 . 616 , n = 25 .

O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ellipsoid body knock down of Octβ2R results in sleep rebound increase. In response to OA, ellipsoid knock down of Octβ2R rescues control phenotype. Data represent mean±SEM: 13.80±2.439, n=31; 5.465±2.036, n=25; 19.55±2.275, n=27; 12.47±2.684, n=24; 30.03±3.428, n=25; 43.67±4.616, n=25.


Sleep profile of PcXT109 mutant with or without OA treatment . In both control PcXT109 mutant flies , treatment of OA results in sleep decrease , n = 16 ( G ) . Quantifications of decreasing amount showed that PcXT109 mutant fly loss significantly more sleep that control flies ( From left to right mean ± : 13 . 90 ± 1 . 397 ; 21 . 25 ± 1 . 493 , N = 4 ) ( H ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Sleep profile of PcXT109 mutant with or without OA treatment. In both control and PcXT109 mutant flies, the treatment of OA results in sleep decrease, n=16 (G). Quantifications of the decreasing amount showed that PcXT109 mutant fly loss significantly more sleep that control flies (From left to right mean±SEM: 13.90±1.397; 21.25±1.493, N=4) (H).


Total sleep for Tβh mutant control flies in video - method ( From left to right mean ± : 688 . 2 ± 32 . 71 , n = 48 ; 123 . 2 ± 11 . 68 , n = 46 ) .

O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Total sleep for Tβh mutant and control flies in video-based method (From left to right mean±SEM: 688.2±32.71, n=48; 123.2±11.68, n=46).


( A ) Western blot with representative blot including lamin A / C , NLRP3 , caspase 1 , actin in skin fibroblats from with HGPS , n = 2 controls 2 . Positive control correspond to THP1 stimulated with LPS + ATP .

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

(A) Western blot analysis with representative blot including lamin A/C, NLRP3, caspase 1, and actin levels in skin fibroblats from patients with HGPS, n = 2 controls and 2 patients. Positive control correspond to THP1 cells stimulated with LPS+ATP.


( B ) Immunofluorescence ( IF ) visualization of NLRP3 ( green ) nuclei ( blue ) in skin fibroblasts from a representative control . Scale bar :

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

(B) Immunofluorescence (IF) visualization of NLRP3 (green) and nuclei (blue) in skin fibroblasts from a representative patient and control. Scale bar: 30µm


( C ) expression of NLRP3 caspase 1 in lymphoblasts from control one after stimulation with uric acid cholesterol crystal .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O

(C) Protein expression of NLRP3 and caspase 1 in lymphoblasts from control and one patient after stimulation with uric acid and cholesterol crystal.


( D ) IL - 1β IL - 18 medium release from lymphoblasts which were assessed after a 24 hr incubation with uric acid cholesterol . * * * P < 0 . 001 , * * P < 0 . 005 , * treatment vs no treatment ; aaaP < 0 . 001 ; aaP < 0 . 01 control vs .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) IL-1β and IL-18 medium release from lymphoblasts which were assessed after a 24 hr incubation with uric acid and cholesterol. ***P < 0.001, **P < 0.005, * treatment vs no treatment; aaaP < 0.001; aaP < 0.01 control cells vs patient cells.


( E ) Western blot with representative blot including NLRP3 , caspase 1 , IL - 1β actin in heart liver tissues from wild - type Zmpste24 - / - mice .

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

(E) Western blot analysis with representative blot including NLRP3, caspase 1, IL-1β and actin levels in heart and liver tissues from wild-type and Zmpste24-/- mice.


( F ) NLRP3 Caspase 1 transcript expression were determined in heart liver tissues by real - time quantitative RT - PCR . n = 5 for Zmpste24 + / + n = 5 for Zmpste24 - / - groups respectively . * * * P < 0 . 001 , * * P < 0 . 005 , wild - type vs Zmpste24 - / - mice . Data are showed means ± SD , n = 4 mice per group .

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

(F) NLRP3 and Caspase 1 transcript expression levels were determined in heart and liver tissues by real-time quantitative RT-PCR. n=5 for Zmpste24+/+ and n=5 for Zmpste24-/- groups respectively. ***P < 0.001, **P < 0.005, wild-type vs Zmpste24-/- mice. Data are showed means ± SD, n = 4 mice per group.


( A ) growthn ( lower subpanel left 1 right 2 ) morphological aspect ( upper subpanel ) with MCC950 determined in healthy representative HGPS fibroblasts . * * P < 0 . 01 , * P < 0 . 05 no treatment vs treatment . Results are presented as mean ± SD of three independent experiments .

O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-DISEASE B-CELL_TYPE O O O O O O O O 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) Cell growthn (lower subpanel left patient 1 and right patient 2) and morphological aspect (upper subpanel) with MCC950 determined in healthy and representative HGPS fibroblasts. **P < 0.01, *P < 0.05 patient cells no treatment vs treatment. Results are presented as the mean ± SD of three independent experiments.


( B ) Western blot with representative blots including lamin A / C , NLRP3 , IL - 1β actin in skin fibroblats from control HGPS after 48 hr of vehicle MCC950 treatment .

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

(B) Western blot analysis with representative blots including lamin A/C, NLRP3, IL-1β and actin levels in skin fibroblats from control and HGPS patient after 48 hr of vehicle and MCC950 treatment.


( C - E ) Representative of HGPS control fibroblasts to evaluate effect of MCC950 in ( D ) NLRP3 expression ( E ) . Scale bar : 30µm . Results are presented as mean ± SD of three independent experiments . * * * P < 0 . 01 vs control , aaaP < 0 . 01 no treatment vs treatment . One - way ANOVA was used for statistical .

O O O O O O O B-DISEASE O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-E) Representative fluorescence images of HGPS and control fibroblasts to evaluate the effect of the MCC950 in the nuclear morphology (D) and NLRP3 expression (E). Scale bar: 30µm. Results are presented as the mean ± SD of three independent experiments. ***P < 0.01 patient vs control cells, aaaP < 0.01 patient cells no treatment vs treatment. One-way ANOVA test was used for statistical analysis.


( F ) Kaplan - Meier graph showing a significant increase in maximum lifespan in WT mice compared with Zmpste24 - / - mice . N = 7 per group .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O B-ORGANISM O O O O O O O

(F) Kaplan-Meier graph showing a significant increase in the maximum lifespan in WT mice compared with Zmpste24-/- mice. N=7 per group.


( G ) weights evolution of groups over time . Data are showed means ± SD , n = 6 mice per group .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O O O

(G) Body weights evolution of the groups over time. Data are showed means ± SD, n = 6 mice per group.


( H ) of serum concentrations of IL - 1β measured by ELISA . N = 6 per group . Data are shown as means ± SD . * * * P < 0 . 001 , wild - type vs Zmpste24 - / - mice ; aaaP < 0 . 001 ; vehicle vs MCC950 .

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

(H) Analysis of serum concentrations of IL-1β measured by ELISA. N=6 per group. Data are shown as means ± SD. ***P < 0.001, wild-type vs Zmpste24-/- mice; aaaP < 0.001; vehicle vs MCC950.


A . Representative immunoblots quantifications ( below ) of indicated in lysates of ARPE - 19 treated with 1 mM DFP for indicated time ( h , hours ) .

O O O B-EXP_ASSAY O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O

A. Representative immunoblots and quantifications (below) of the indicated proteins in lysates of ARPE-19 cells treated with 1 mM DFP for the indicated time (h, hours).


B - C . Representative confocal of ARPE - 19 stably expressing mito - QC reporter ( B ) , or pexo - QC reporter ( C ) treated with 1 mM DFP for indicated time ( h , hours ) . Enlarged of outlined in are shown in lower corners . ' red - only ' mask is generated during highlights red - only ( autolysosomes ) on mCherry / GFP thresholding criteria . Nuclei were stained in blue ( Hoechst ) . Scale bar : 10μm . At right , of total red - only punctate per ( mitolysosomes or pexolysosomes ) .

O O O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O 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-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O

B-C. Representative confocal images of ARPE-19 cells stably expressing the mito-QC reporter (B), or the pexo-QC reporter (C) treated with 1 mM DFP for the indicated time (h, hours). Enlarged images of the area outlined in white are shown in the lower corners. The 'red-only' mask is generated during images analysis and highlights red-only puncta (autolysosomes) based on the mCherry/GFP ratio and thresholding criteria. Nuclei were stained in blue (Hoechst). Scale bar: 10μm. At right, quantification of total red-only punctate per cell (mitolysosomes or pexolysosomes).


D . ARPE - 19 were transfected with 50 pmol of non - targeting siRNA ( siNT ) or 50 pmol of siRNA targeting ATG13 or ULK1 . 48 hours post knockdown , were treated with 1 mM DFP for an additional 48 hours . lysates were subject to immunoblotting with indicated antibodies . is shown on right .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

D. ARPE-19 cells were transfected with 50 pmol of non-targeting siRNA (siNT) or 50 pmol of siRNA targeting ATG13 or ULK1. 48 hours post knockdown, cells were treated with 1 mM DFP for an additional 48 hours. Cell lysates were subject to immunoblotting with the indicated antibodies. Quantification is shown on the right.


A . Representative immunoblots of indicated in lysates of WT or HIF1α KO U2OS , treated with 1mM DFP for 48 hours .

O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O

A. Representative immunoblots of the indicated proteins in lysates of WT or HIF1α KO U2OS cells, treated with 1mM DFP for 48 hours.


B . Representative confocal of WT or HIF1α KO U2OS stably expressing pexo - QC reporter treated with 1 mM DFP for 48 hours ( left panel ) flow cytometry of mCherry / GFP ( right panel ) .

O O O B-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O

B. Representative confocal images of WT or HIF1α KO U2OS cells stably expressing the pexo-QC reporter and treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).


C . Representative immunoblots ( left panel ) ( right panel ) of indicated in lysates of WT or two NIX KO ARPE - 19 clones ( Cl ) , treated with 1 mM DFP for indicated time ( h , hours ) .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

C. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins in lysates of WT or two NIX KO ARPE-19 clones (Cl), treated with 1 mM DFP for the indicated time (h, hours).


D - E . Representative confocal of WT or NIX KO ARPE - 19 stably expressing mito - QC reporter ( D ) or pexo - QC reporter ( E ) treated with 1 mM DFP for 48 hours ( left panel ) flow cytometry of mCherry / GFP ( right panel ) .

O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O

D-E. Representative confocal images of WT or NIX KO ARPE-19 cells stably expressing the mito-QC reporter (D) or the pexo-QC reporter (E) treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).


F . Representative immunoblots ( left panel ) ( right panel ) of indicated . NIX KO ARPE - 19 ( Cl 31 ) stably expressing a pBabe empty vector ( ∅ ) or a pBabe NIX vector , were treated with 1 mM DFP for 48 hours prior lysis . Two biological replicates are shown in immunoblot .

O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O

F. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins. NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe empty vector (∅) or a pBabe NIX vector, were treated with 1 mM DFP for 48 hours prior lysis. Two biological replicates are shown in the immunoblot.


A - B . Representative confocal of untreated ( CTRL ) DFP - treated ARPE - 19 ( 1mM , 48 hours ) , stably expressing GFP - WT NIX ( green ) stained with anti - catalase antibody ( A , red ) or anti - PMP70 antibody ( B , red ) . At right , of Pearson correlation coefficients between NIX peroxisomal markers catalase PMP70 , with or without DFP treatment for 48 hours . Peroxisomes enriched for GFP - NIX or having minimal signal for GFP - NIX are depicted with arrowheads or arrows , respectively .

O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

A-B. Representative confocal images of untreated (CTRL) and DFP-treated ARPE-19 cells (1mM, 48 hours), stably expressing GFP-WT NIX (green) and stained with anti-catalase antibody (A, red) or anti-PMP70 antibody (B, red). At right, quantification of Pearson correlation coefficients between NIX and the peroxisomal markers catalase and PMP70, with or without DFP treatment for 48 hours. Peroxisomes enriched for GFP-NIX or having minimal signal for GFP-NIX are depicted with white arrowheads or white arrows, respectively.


C . Representative confocal of ARPE - 19 stably expressing GFP - WT NIX , GFP - NIXΔTM or GFP - NIX TM only ( green ) , treated with DFP ( 1mM ) for 48 hours immunostained with anti - catalase antibody ( red ) . At right , of Pearson correlation coefficients between green red signal .

O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O

C. Representative confocal images of ARPE-19 cells stably expressing GFP-WT NIX, GFP-NIXΔTM or GFP-NIX TM only (green), treated with DFP (1mM) for 48 hours and immunostained with anti-catalase antibody (red). At right, quantification of Pearson correlation coefficients between the green and the red signal.


B . Representative immunoblots of indicated in lysates of WT or HA - Parkin ( PRKN ) - ARPE - 19 treated as in A .

O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

B. Representative immunoblots of the indicated proteins in lysates of WT or HA-Parkin(PRKN)- ARPE-19 cells treated as in A.


B . Representative immunoblots of indicated in lysates of NIX KO ARPE - 19 ( Cl 31 ) stably expressing a pBabe Flag vector ( ∅ ) , a pBabe Flag - NIX vector ( NIX ) , a pBabe Flag - NIX W36A \ L39A vector ( W36A \ L39A ) or a pBabe Flag - NIX S34E \ S35E vector ( S34E \ S35E ) , treated with 1 mM DFP for 48 hours prior lysis .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

B. Representative immunoblots of the indicated proteins in lysates of NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe Flag vector (∅), a pBabe Flag-NIX vector (NIX), a pBabe Flag-NIX W36A\L39A vector (W36A\L39A) or a pBabe Flag-NIX S34E\S35E vector (S34E\S35E), and treated with 1 mM DFP for 48 hours prior lysis.


C - D . Representative confocal of as in B , stably expressing mito - QC reporter ( C ) or pexo - QC reporter ( D ) treated with 1 mM DFP for 24 hours ( C ) or 48 hours ( D ) ( left panel ) flow cytometry of mCherry / GFP ( right panel ) . Enlarged of outlined in are shown in lower corners . Nuclei were stained in blue ( Hoechst )

O O O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-SMALL_MOLECULE O

C-D. Representative confocal images of cells as in B, stably expressing the mito-QC reporter (C) or the pexo-QC reporter (D) and treated with 1 mM DFP for 24 hours (C) or 48 hours (D) (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel). Enlarged images of the area outlined in white are shown in the lower corners. Nuclei were stained in blue (Hoechst)


A . Representative confocal of optical section from WT NIX KO mouse retina immunostained with peroxisomal markers ( PMP70 catalase ) or a lysosomal marker LAMP1 . Nuclei are coloured in ( DAPI ) . RPE , retinal pigment epithelium ; ONL , outer layer ; OPL , outer plexiform layer ; INL ; inner layer ; IPL , inner plexiform layer ; RGC , retinal ganglion . B . of signal in ONL INL from as A . Each data point represents a single mouse ( n = 3 - 4 per group ) .

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

A. Representative confocal images of optical section from WT and NIX KO mouse retina immunostained with peroxisomal markers (PMP70 and catalase) or a lysosomal marker LAMP1. Nuclei are coloured in white (DAPI). RPE, retinal pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL; inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cells. B. Quantification of signal area in the ONL and INL from images as A. Each data point represents a single mouse (n=3-4 per group).


Dox - induced prolongation of Nanog expression in Nanogtg up to E9 . 5 results in lack of blood ( left ) downregulation of erythropoietic gene expression . center right panels show whole - mount in situ hybridization for Hbb - bh1 ( in with intact yolk sacs ) for long non - coding Redrum . Asterisks mark aorta - gonad - mesonephros region ( AGM ) arrows tailbud . of same genotype but not treated with dox were used as controls ( - dox ) . Scale bars , 500 µm .

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

Dox-induced prolongation of Nanog expression in Nanogtg embryos up to E9.5 results in lack of blood (left) and downregulation of erythropoietic gene expression. The center and right panels show whole-mount in situ hybridization for Hbb-bh1 (in embryos with intact yolk sacs) and for the long non-coding RNA Redrum. Asterisks mark the aorta-gonad-mesonephros region (AGM) and arrows the tailbud. Embryos of the same genotype but not treated with dox were used as controls (-dox). Scale bars, 500 µm.


Endomucin of vessels in control ( - dox ) or treated ( + dox ) E9 . 5 Nanogtg . On right , higher magnifications of boxed areas . Scale bar , 500 µm .

B-GENEPROD O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O

Endomucin staining of vessels in control (-dox) or treated (+dox) E9.5 Nanogtg embryos. On the right, higher magnifications of the boxed areas. Scale bar, 500 µm.


Representative FACS plot of distribution of CD71 Ter119 populations in dissected yolk sacs from untreated dox - treated E9 . 5 Nanogtg . of CD71 + Ter119 + population in controls ( - dox , black dots ; n = 8 ) Nanog expressing ( + dox , red dots ; n = 7 ) E9 . 5 yolk sacs . Each replicate contained a pool of 5 ( - dox ) or 8 ( + dox ) E9 . 5 Nanogtg . * * * P . < 0 . 0005 ; Student ' s t - . Horizontal represents mean values error bars standard deviation ( SD ) .

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

Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox-treated E9.5 Nanogtg embryos. Quantification of the CD71+ Ter119+ population in controls (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 yolk sacs. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. ***P. < 0.0005; Student's t-test. Horizontal line represents mean values and error bars standard deviation (SD).


Representative FACS plots showing distribution of cKit CD41 populations in yolk sacs from untreated controls ( - dox ) Nanog expressing ( + dox ) E9 . 5 Nanogtg . of different progenitor populations in yolk sacs from control ( - dox , black dots ; n = 8 ) Nanog expressing ( + dox , red dots ; n = 7 ) E9 . 5 . Each replicate contained a pool of 5 ( - dox ) or 8 ( + dox ) E9 . 5 Nanogtg . Horizontal represents mean values error bars SD .

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

Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls (-dox) and Nanog expressing (+dox) E9.5 Nanogtg embryos. Quantification of different progenitor populations in yolk sacs from control (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 embryos. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. Horizontal line represents mean values and error bars SD.


Differences in expression of Nanog selected hematopoietic genes in CD71 + Ter119 + population of control ( - dox ; n = 7 ) Nanog - expressing ( + dox ; n = 4 ) E9 . 5 . * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - . Horizontal represents mean values error bars SD .

O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71+ Ter119+ population of control (-dox; n=7) and Nanog-expressing (+dox; n=4) E9.5 embryos. **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Freshly dissected dox - treated Nanogtg E10 . 5 without ( control ) with ( chimera ) contribution of wt - ESGFP ( left , brightfield ; right , GFP ) . Arrows mark presence of blood in chimeric that is absent from controls . Scale bar , 500 µm .

O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O O O

Freshly dissected dox-treated Nanogtg E10.5 embryos without (control) and with (chimera) contribution of wt-ESGFP cells (left, brightfield; right, GFP). Arrows mark the presence of blood in chimeric embryos that is absent from controls. Scale bar, 500 µm.


Representative FACS plots showing of red blood maturation as determined by CD71 / Ter119 in single dox - treated E10 . 5 control ( left ) chimeric ( right ) .

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

Representative FACS plots showing of red blood cell maturation as determined by CD71/Ter119 staining in single dox-treated E10.5 control (left) and chimeric (right) embryos.


of CD71 + Ter119 + population in single dox - treated E10 . 5 ( D ; control , n = 18 ; chimera , n = 10 ) , untreated ( E ; control , n = 7 ; chimera , n = 12 ) , in GFP - ( not derived from wild type ES ) from dox - treated ( F ; control , n = 18 ; chimera , n = 10 ) . * * * P < 0 . 0005 ; Student ' s t - . Horizontal represents mean values error bars SD .

O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the CD71+ Ter119+ population in single dox-treated E10.5 embryos (D; control, n=18; chimera, n=10), untreated embryos (E; control, n=7; chimera, n=12), and in GFP- cells (not derived from wild type ES cells) from dox-treated embryos (F; control, n=18; chimera, n=10). ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


of colony - forming units generated by wild type ( wt ) knockout ( Nanog - / - ) ES after culture of EBs for 5 ( D5 ) , 6 ( D6 ) , or 7 ( D7 ) days plating disaggregated in different hemogenic - promoting conditions ) . Panels on left show representative of mouse hematopoietic colonies obtained after 12 days of culture in specific media . CFU - GEMM , progenitors giving rise to granulocytes , erythrocytes , monocytes , megakaryocytes ; BFU - E , burst forming units - erythroid ; Ery - P , colony forming primitive erythroid ; CFU - GM , granulocyte - monocyte precursors ; CFU - M , monocyte precursors ; CFU - G , granulocyte precursors . No CFU - GEMM are detected at D5 no BFU - E at D7 . For both wt knockout , n = 3 each with 3 technical replicates . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 00005 ; Student ' s t - . Horizontal represents mean values error bars SD .

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

Quantification of colony-forming units generated by wild type (wt) and knockout (Nanog-/-) ES cells after culture of EBs for 5 (D5), 6 (D6), or 7 (D7) days and plating disaggregated cells in different hemogenic-promoting conditions). Panels on the left show representative images of mouse hematopoietic colonies obtained after 12 days of culture in specific media. CFU-GEMM, progenitors giving rise to granulocytes, erythrocytes, monocytes, and megakaryocytes; BFU-E, burst forming units-erythroid; Ery-P, colony forming primitive erythroid; CFU-GM, granulocyte-monocyte precursors; CFU-M, monocyte precursors; CFU-G, granulocyte precursors. No CFU-GEMM are detected at D5 and no BFU-E at D7. For both wt and knockout cells, n=3 each with 3 technical replicates. *P < 0.05, **P < 0.005, ***P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.


RT - qPCR determination of relative expression of Brachyury selected hematopoietic genes in control ( wt , right ) knockout ( Nanog - / - , left ) ES ( n = 3 ) during 10 days of EB differentiation in hematopoietic - cytokine - enriched medium . Black arrowheads indicate peak of Brachyury expression arrowheads time of maximum hematopoietic - gene expression .

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

RT-qPCR determination of the relative expression of Brachyury and selected hematopoietic genes in control (wt, right) and knockout (Nanog-/-, left) ES cells (n=3) during 10 days of EB differentiation in hematopoietic-cytokine-enriched medium. Black arrowheads indicate the peak of Brachyury expression and white arrowheads the time of maximum hematopoietic-gene expression.


Representative FACS plots showing distribution of different populations distinguished by CD71 / Ter119 in whole bone marrow from untreated ( - dox ) or treated ( + dox ) adult mice . S0 ( double negative ) , S1 ( proerythroblast ) , S2 ( basophylic erythroblast ) , S3 ( polychromatic erythroblast ) , S4 ( orthochromatic erythroblast ) are different stages of blood maturation . of S1 - S4 erythroid populations ( - dox , n = 4 ; + dox , n = 5 ) . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - . Horizontal represents mean values error bars SD .

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

Representative FACS plots showing the distribution of different populations distinguished by CD71/Ter119 staining in whole bone marrow from untreated (-dox) or treated (+dox) adult mice. S0 (double negative cell), S1 (proerythroblast), S2 (basophylic erythroblast), S3 (polychromatic erythroblast), and S4 (orthochromatic erythroblast) are different stages of blood maturation. Quantification of the S1-S4 erythroid populations (-dox, n=4; +dox, n=5). *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Representative FACS plots showing distribution of CD16 / 32 CD34 hematopoietic precursors sorted from cKit + Sca1 - LIN - bone marrow of untreated ( - dox ) or treated ( + dox ) adult Nanogtg mice .

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

Representative FACS plots showing the distribution of CD16/32 and CD34 hematopoietic precursors sorted from the cKit+Sca1-LIN- bone marrow of untreated (-dox) or treated (+dox) adult Nanogtg mice.


of precursor populations on CD16 / 32 CD34 sorting , as total number of per femur ( - dox , n = 5 ; + dox , n = 6 ) . * P < 0 . 05 , * * P < 0 . 005 ; Student ' s t - . Horizontal represents mean values error bars SD .

O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of precursor populations based on CD16/32 and CD34 sorting, as total number of cells per individual femur (-dox, n=5; +dox, n=6). *P < 0.05, **P < 0.005; Student's t-test. Horizontal line represents mean values and error bars SD.


RT - qPCR of relative expression of hematopoietic genes in megakaryocyte - erythroid progenitors ( MEP ) ( - dox , n = 8 ; + dox , n = 5 ) . * P < 0 . 05 , * * * * P < 0 . 00005 ; Student ' s t - . Horizontal represents mean values error bars SD .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCR quantification of the relative expression of hematopoietic genes in megakaryocyte-erythroid progenitors (MEP) (-dox, n=8; +dox, n=5). *P < 0.05, ****P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.


Contribution of Nanogtg transplanted bone marrow to peripheral blood before ( left ) after ( right ) dox treatment . of host derived ( CD45 . 1 + ) are shown in black , of donor derived ( CD45 . 1 / CD45 . 2 double + ) in red . mice are indicated on x - axis ( n = 7 ) .

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

Contribution of Nanogtg transplanted bone marrow cells to peripheral blood before (left) and after (right) dox treatment. Percentage of host derived cells (CD45.1+) are shown in black, and of donor derived cells (CD45.1/CD45.2 double +) in red. Individual mice are indicated on the x-axis (n=7).


Contribution of Nanogtg transplanted to LSK , CMP , GMP MEP populations purified from bone marrow . of host derived ( CD45 . 1 + ) are show in black , of donor derived ( CD45 . 1 / CD45 . 2 double + ) in blue . mice are indicated on x - axis ( n = 7 ) .

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

Contribution of Nanogtg transplanted cells to LSK, CMP, GMP and MEP populations purified from bone marrow. Percentage of host derived cells (CD45.1+) are show in black, and of donor derived cells (CD45.1/CD45.2 double +) in blue. Individual mice are indicated on the x-axis (n=7).


Expected observed number of mesodermal ( Flk1 + ) of E7 . 0 mouse expressing Nanog selected mesodermal or hematopoietic gene expression , on single - seq data ( Scialdone et al . , 2016 ) . Statistical significance was calculated with a hypergeometric .

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

Expected and observed number of mesodermal (Flk1+) cells of the E7.0 mouse embryo expressing Nanog and selected mesodermal or hematopoietic gene expression, based on single cell RNA-seq data (Scialdone et al., 2016). Statistical significance was calculated with a hypergeometric test.


PCA showing distribution of Flk1 + E7 . 0 mesoderm expressing Nanog ( green ) or Tal1 ( red ) . single expressing both genes is shown in yellow indicated by an arrow .

O O O O B-GENEPROD O O O O B-CELL_TYPE B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O

PCA showing the distribution of Flk1+ E7.0 mesoderm cells expressing Nanog (green) or Tal1 (red). The single cell expressing both genes is shown in yellow and indicated by an arrow.


E6 . 5 Nanogtg after 8 hours ex - utero culture in presence ( + dox ) or absence ( - dox ) of doxycycline . Scale bar , 100 µm .

O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O

E6.5 Nanogtg embryos after 8 hours ex-utero culture in the presence (+dox) or absence (-dox) of doxycycline. Scale bar, 100 µm.


RT - qPCR of relative expression of Nanog , Tal1 , Klf1 in untreated ( - dox ) or treated ( + dox ) ( n = 5 ) . * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - . Horizontal represents mean values error bars SD .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCR quantification of the relative expression of Nanog, Tal1, and Klf1 in individual untreated embryos (-dox) or treated embryos (+dox) (n=5). **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Whole mount in situ hybridization of Tal1 in E7 . 5 untreated ( - dox ) or in utero treated ( + dox ) Nanogtg . Scale bar , 100 µm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O

Whole mount in situ hybridization of Tal1 in E7.5 untreated (-dox) or in utero treated (+dox) Nanogtg embryos. Scale bar, 100 µm.


RT - qPCR determination of relative expression in wild type CRISPR - deleted ( n = 5 ) of Tal1 ( wt , n = 19 ; deleted , n = 13 ) , Klf1 ( wt , n = 3 ; deleted , n = 6 ) , Gfi1b ( wt , n = 10 ; deleted , n = 8 ) , Runx1 ( wt , n = 13 ; deleted , n = 5 ) Stil ( wt , n = 19 ; deleted , n = 13 ) . * * P < 0 . 005 , Student ' s t - . Horizontal represents mean values error bars SD .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCR determination of relative expression in wild type and CRISPR-deleted embryos (n=5) of Tal1 (wt, n=19; deleted, n=13), Klf1 (wt, n=3; deleted, n=6), Gfi1b (wt, n=10; deleted, n=8), Runx1 (wt, n=13; deleted, n=5) and Stil (wt, n=19; deleted, n=13). **P < 0.005, Student's t-test. Horizontal line represents mean values and error bars SD.


Experimental design for ES to EpiL differentiation of Nanogtg two independent clones ( Nanogtg ; dTal1del # 1 Nanogtg ; dTal1del # 2 ) where binding site for NANOG distal to Tal1 has been deleted ( left ) . On right , relative expression of Tal1 determined by RT - qPCR for each ES ( ESC ; n = 9 for three lines ) EpiL without ( EpiLC ; Nanogtg Nanogtg ; dTal1del # 1 , n = 8 ; Nanogtg ; dTal1del # 2 , n = 6 ) or with dox treatment ( EpiLC + dox ; n = 9 for three lines ) . genotype of lines is indicated below . Values were normalized to Nanogtg ESC . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ; ANOVA with Fisher post - . Horizontal represents mean values error bars standard error of mean ( ) .

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

Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones (Nanogtg;dTal1del#1 and Nanogtg;dTal1del#2) where the binding site for NANOG distal to Tal1 has been deleted (left). On the right, relative expression of Tal1 determined by RT-qPCR for each ES cell line (ESC; n=9 for all three lines) and EpiL cells without (EpiLC; Nanogtg and Nanogtg;dTal1del#1, n=8; Nanogtg;dTal1del#2, n=6) or with dox treatment (EpiLC +dox; n=9 for all three lines). The genotype of the cell lines is indicated below. Values were normalized to Nanogtg ESC. *P < 0.05, **P < 0.01, ns = not significant; ANOVA with Fisher post-test. Horizontal line represents mean values and error bars standard error of the mean (SEM).


A ) Phase separation over time as monitored by turbidity of 50 μM MBP - N after addition of TEV protease in presence of salt ( 183 mM NaCl ) 0 . 3 mg / ml at pH 7 . 4 ( 50 mM Tris ) , pH 6 . 1 , pH 5 . 5 , or pH 4 . 9 ( pH 6 . 1 below in 20 mM MES ) . Error bars represent standard deviation of three replicates . For samples at B ) pH 7 . 4 or C ) pH 5 . 5 , DIC of 50 μM MBP - N in presence of salt ( 183 mM NaCl ) without or with TEV protease ( to cleave MBP from N ) without or with 0 . 3 mg / mL desalted total torula yeast . Scale bars represent 50 μm .

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

A) Phase separation over time as monitored by turbidity of 50 μM MBP-N after addition of TEV protease in the presence of salt (183 mM NaCl) and 0.3 mg/ml RNA at pH 7.4 (50 mM Tris), pH 6.1, pH 5.5, or pH 4.9 (pH 6.1 and below in 20 mM MES). Error bars represent standard deviation of three replicates. For samples at B) pH 7.4 or C) pH 5.5, DIC micrographs of 50 μM MBP-N in the presence of salt (183 mM NaCl) without or with TEV protease (to cleave MBP from N) and without or with 0.3 mg/mL desalted total torula yeast RNA. Scale bars represent 50 μm.


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

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

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


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

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

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


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

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

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


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

O O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O O

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


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

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O O

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


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

O O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O O

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


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

O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O B-SUBCELLULAR O

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


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

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

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


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

O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE 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-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

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


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

O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE 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-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

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


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

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

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


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

O O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

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


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

O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

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


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

O O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

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


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

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

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


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

O O O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

O O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

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


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

O O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

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


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

B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O

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


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

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


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

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


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

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


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

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O

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


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

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O O O B-GENEPROD O O O O B-GENEPROD B-ORGANISM B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O 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-GENEPROD O O O O O 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-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O

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


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

O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O 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-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O

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


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

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


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

O O O O O O O B-GENEPROD O O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

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


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

O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

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


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

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O

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


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

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O

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


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

O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O

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