( E ) In vitro phosphorylation assay of CST - 1 / 2 to MIG - 2 . Bacterially expressed His - tagged MIG - 2 or His - tagged MIG - 2S139A were incubated with the GST - tagged kinase domain of GST - CST - 1 / 2 ( 1 - 318 aa ) . The kinase may use ATP - γ - S as a phosphodonor to thiophosphorylate its substrate ; thus , the phosphorylation reaction was performed with ATP - γ - S , and anti - thiophosphate ester antibody was used to detect substrate phosphorylation ( Allen et al , 2005 ; Allen et al , 2007 ) . The final reaction was probed via immunoblotting with antibodies as indicated . Thio , thiophosphate ester .

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

(E) In vitro phosphorylation assay of CST-1/2 to MIG-2. Bacterially expressed His-tagged MIG-2 or His-tagged MIG-2S139A were incubated with the GST-tagged kinase domain of GST-CST-1/2 (1-318 aa). The kinase may use ATP-γ-S as a phosphodonor to thiophosphorylate its substrate; thus, the phosphorylation reaction was performed with ATP-γ-S, and anti-thiophosphate ester antibody was used to detect substrate phosphorylation (Allen et al, 2005; Allen et al, 2007). The final reaction was probed via immunoblotting with antibodies as indicated. Thio, thiophosphate ester.


( G H ) Three - dimensional ribbon ( PDB 3TH5 ) representations of the human Rac1 polypeptide which indicate the position of the T135 ( S139 in MIG - 2 ) located between the last two α - helixes ( Krauthammer et al , 2012 ) .

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

(G H) Three-dimensional ribbon (PDB 3TH5) representations of the human Rac1 polypeptide which indicate the position of the T135 (S139 in MIG-2) located between the last two α-helixes (Krauthammer et al, 2012).


( A ) Quantification of AQR position in mig - 2 ( mu28 ) null mutant animals with different rescue transgenes . N = 108 - 114 . * , P < 0 . 05 ; # # , P < 0 . 01 , phenotype enhanced . P values are based on Fisher ' s exact tests .

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

(A) Quantification of AQR position in mig-2(mu28) null mutant animals with different rescue transgenes. N = 108 - 114. *, P < 0.05; ##, P < 0.01, phenotype enhanced. P values are based on Fisher's exact tests.


( B ) Quantification of AQR position in WT and knock - in animals that harbor mig - 2S139 point mutations . N = 100 - 111 . * * * , P < 0 . 001 by Fisher ' s exact test .

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

(B) Quantification of AQR position in WT and knock-in animals that harbor mig-2S139 point mutations. N = 100 - 111. ***, P < 0.001 by Fisher's exact test.


( C - F ) Quantification of the QR . ap migration angle ( C D ) , distance ( E ) and speed ( F ) in mig - 2S139A / E mutant animals . Each line in ( C E ) represents the measurement from one movie of QR . ap migration . Error bars indicate SD . N = 12 - 17 . * * , P 0 . 01 ; * * * , P 0 . 001 by Student ' s t tests .

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

(C - F) Quantification of the QR.ap migration angle (C D), distance (E) and speed (F) in mig-2S139A/E mutant animals. Each line in (C  E) represents the measurement from one movie of QR.ap migration. Error bars indicate SD. N = 12 -17. **, P 0.01; ***, P  0.001 by Student's t tests.


( G ) Inverted fluorescence images ( up ) and schematics ( down ) of QR . ap in WT , cst - 1 ; cst - 2 double or mig - 2 mutant animals . The plasma membrane and histones were labeled with mCherry . Scale bar , 5 μm . ( H ) Area ratio between the cell body and the nucleus in WT and mutant animals . Error bars indicate SD . N = 17 - 22 . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 by Student ' s t tests .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Inverted fluorescence images (up) and schematics (down) of QR.ap in WT, cst-1; cst-2 double or mig-2 mutant animals. The plasma membrane and histones were labeled with mCherry. Scale bar, 5 μm. (H) Area ratio between the cell body and the nucleus in WT and mutant animals. Error bars indicate SD. N = 17 - 22. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student's t tests.


( I - K ) Representative frames ( upper ) and quantification ( lower ) of F - actin enrichment at the QR . ap periphery in WT ( I ) and mig - 2S139A / E mutant animals ( J K ) . The trace starts from the rear of the QR . ap and moves along the cell periphery to the leading edge ( L ) and back to the rear ( R ) . F - actin was labeled with GFP : : Moesin ABD ; the plasma membrane and histone were marked with mCherry fluorescence . Green lines , F - actin fluorescence intensity ; red lines , plasma membrane fluorescence intensity ; dotted lines , cell periphery . Double - headed arrows indicate the F - actin enrichment region , in which the fluorescence intensity is 2 - fold higher than the background . Scale bars , 5 μm . ( L ) Quantification of F - actin enrichment ratio in WT and mig - 2 mutants . Error bars indicate SD . N = 28 - 68 . * * , P 0 . 01 ; * * * , P 0 . 001 by Student ' s t - tests .

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

(I - K) Representative frames (upper) and quantification (lower) of F-actin enrichment at the QR.ap periphery in WT (I) and mig-2S139A/E mutant animals (J  K). The trace starts from the rear of the QR.ap and moves along the cell periphery to the leading edge (L) and back to the rear (R). F-actin was labeled with GFP::Moesin ABD; the plasma membrane and histone were marked with mCherry fluorescence. Green lines, F-actin fluorescence intensity; red lines, plasma membrane fluorescence intensity; dotted lines, cell periphery. Double-headed arrows indicate the F-actin enrichment region, in which the fluorescence intensity is 2-fold higher than the background. Scale bars, 5 μm. (L) Quantification of F-actin enrichment ratio in WT and mig-2 mutants. Error bars indicate SD. N = 28 - 68. **, P  0.01; ***, P  0.001 by Student's t-tests.


( A - C ) Fluorescence images ( upper ) and quantifications ( lower ) of GFP - tagged CST - 1 ( A B ) or GFP - labeled F - actin ( C ) and mCherry tagged plasma membrane and histone in WT and mig - 2 null mutant animals . The membrane protrusions in the white boxes are enlarged 3 times at the bottom . Yellow dashed lines indicate the cell periphery . White lines indicate the line to measure the fluorescence intensity . Green lines denote the CST - 1 fluorescence intensity ; red lines denote the plasma membrane fluorescence intensity ; double - headed arrows denote the distance between the GFP signal and cell periphery . Scale bars , 5 μm . ( D ) Distance between GFP : : CST - 1 or GFP : : Moesin ABD and the plasma membrane at the leading edge in WT and mig - 2 null mutants . Error bars indicate SD . N = 9 - 14 . * * * , P 0 . 001 by Student ' s t test .

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

(A - C) Fluorescence images (upper) and quantifications (lower) of GFP-tagged CST-1 (A  B) or GFP-labeled F-actin (C) and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. The membrane protrusions in the white boxes are enlarged 3 times at the bottom. Yellow dashed lines indicate the cell periphery. White lines indicate the line to measure the fluorescence intensity. Green lines denote the CST-1 fluorescence intensity; red lines denote the plasma membrane fluorescence intensity; double-headed arrows denote the distance between the GFP signal and cell periphery. Scale bars, 5 μm. (D) Distance between GFP::CST-1 or GFP::Moesin ABD and the plasma membrane at the leading edge in WT and mig-2 null mutants. Error bars indicate SD. N = 9 - 14. ***, P  0.001 by Student's t test.


( E - F ) Fluorescence images ( left ) and quantifications ( right ) of GFP - tagged CST - 1 and mCherry tagged plasma membrane and histone in WT and mig - 2 null mutant animals . Blue lines , sites of measurements ; dotted lines , cell periphery ; Green lines ; CST - 1 fluorescence intensity ; red lines , mCherry fluorescence intensity . Scale bars , 5 μm . ( G ) Quantification of the fluorescence intensity ratio of GFP : : CST - 1 and mCherry - membrane between the leading edge and the lagging edge of AQR in WT and mig - 2 null mutant animals . Error bars indicate SD . N = 20 . * * * , P < 0 . 001 by Student ' s t test .

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

(E - F) Fluorescence images (left) and quantifications (right) of GFP-tagged CST-1 and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. Blue lines, sites of measurements; dotted lines, cell periphery; Green lines; CST-1 fluorescence intensity; red lines, mCherry fluorescence intensity. Scale bars, 5 μm. (G) Quantification of the fluorescence intensity ratio of GFP::CST-1 and mCherry-membrane between the leading edge and the lagging edge of AQR in WT and mig-2 null mutant animals. Error bars indicate SD. N = 20. ***, P < 0.001 by Student's t test.


Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton - S control . 24 - hour - sleep curve ( y - axis was average sleep time in every 30 minutes ) , n = 16 . Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118 . 24 - hour - sleep curve , n = 16 .

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

Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton-S control. 24-hour-sleep curve (y-axis was average sleep time in every 30 minutes), n=16. Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118. 24-hour-sleep curve, n=16.


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

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


O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 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 ) .

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

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 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 .

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

(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 ) and nuclei ( blue ) in skin fibroblasts from a representative patient and control . Scale bar :

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

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


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

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

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 analysis with representative blot including NLRP3 , caspase 1 , IL - 1β and actin levels in heart and liver tissues from wild - type and Zmpste24 - / - mice .

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

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(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 ) 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 .

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(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 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 .

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(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 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 .

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(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 the maximum lifespan in WT mice compared with Zmpste24 - / - mice . N = 7 per group .

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


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

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


( 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 .

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

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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 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 ) .

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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 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 .

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

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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 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 ) .

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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 ) 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 ) .

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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 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 ) .

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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 ) 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 .

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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 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 .

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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 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 .

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

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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 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 .

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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 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 )

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 cell maturation as determined by CD71 / Ter119 staining in single dox - treated E10 . 5 control ( left ) and chimeric ( right ) embryos .

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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.


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 .

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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.


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 .

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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 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 .

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

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

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


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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 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 ) .

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

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 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 .

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

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

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

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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 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 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 embryos . Scale bar , 100 µm .

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

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

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 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 ) .

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

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 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 .

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

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 RNA ( at 100 mM NaCl ) or B ) varying NaCl concentrations ( at constant RNA concentration ) . Error bars represent standard deviation of three replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 the MBP tag attached .

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


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

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O 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 the MBP tag .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

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

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

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


( 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 .

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

(H-I) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

(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.


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 .

O O O O O O O O O O O O O O O O O O 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 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


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 .

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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.


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 .

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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 ) 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 )

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(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 ) 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 .

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(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 ) 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 .

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

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


( G - H ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

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


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

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

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


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 .

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

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


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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 . 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

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

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


C . Projection of confocal sections of control inner ear HCs immunolabelled for otoferlin ( magenta ) 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 .

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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 ) 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 .

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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 ( 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 .

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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 ( 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 .

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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 . 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 .

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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 . 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 .

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

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


I - I ' . Acoustic startle reflex measurements of ( I ) live , intact , 5 - dpf control ( n = 20 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . 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 ) .

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

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


E . 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 )

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

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


A . Grand averages of auditory brainstem responses ( ABR ) from Wrbfl / fl : CreA ( red traces ; 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 .

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

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


B . ABRs from Wrbfl / fl : CreA mice showed a progressive threshold increase , Wrb + / + : CreA mice 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 .

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

B. 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.