We calculated ΔΨCr - IBM between neighboring cristae over time as a surrogate measure of cristae individuality functional independence , using live - Airyscan imaging . Stability of cristae ΔΨCr - IBM over time . Representative from time series of HeLa co - stained with MTG TMRE . Top shows merged green ( MTG ) red ( TMRE ) channels . Bottom shows color - coded LUT of TMRE signal of same shown above . arrowheads in image point to specific cristae that were measured over time . Scale bar = 500 nm . N = 5 independent experiments . of A , showing average ΔΨCr - IBM of Cristae " A - D " over time . Note that Cristae C D maintain more than double ΔΨCr - IBM of Cristae A B . Error bars indicate STDEV . of A , showing standard deviation in ΔΨCr - IBM ( mV ) of cristae over time compared to standard deviation in ΔΨCr - IBM ( mV ) among different cristae along a single mitochondrion . N = 5 independent experiments . Note that there is less variability in ΔΨCr - IBM of a single crista over time within same mitochondrion compared to variability of ΔΨCr - IBM among cristae over time , indicating cristae possess unique bioenergetic properties .

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

We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Stability of cristae ΔΨCr-IBM over time. Representative images from time series of HeLa cells co-stained with MTG and TMRE. Top sequence shows merged green (MTG) and red (TMRE) channels. Bottom sequence shows color-coded LUT of TMRE signal of the same sequence shown above. White arrowheads in the image point to specific cristae that were measured over time. Scale bar = 500 nm. N = 5 independent experiments. Quantification of A, showing average ΔΨCr-IBM of Cristae "A-D" over time. Note that Cristae C and D maintain more than double the ΔΨCr-IBM of Cristae A and B. Error bars indicate STDEV. Quantification of A, showing standard deviation in ΔΨCr-IBM (mV) of individual cristae over time compared to the standard deviation in ΔΨCr-IBM (mV) among different cristae along a single mitochondrion. N = 5 independent experiments. Note that there is less variability in ΔΨCr-IBM of a single crista over time within the same mitochondrion compared to the variability of ΔΨCr-IBM among cristae over time, indicating cristae possess unique bioenergetic properties.


We calculated ΔΨCr - IBM between neighboring cristae over time as a surrogate measure of cristae individuality functional independence , using live - Airyscan imaging . Representative live - Airyscan image from time series showing merged green ( MTG ) red ( TMRE ) channels . marked by dashed box is shown in time series in E . Scale bar = 500 nm . Time series of cropped region from D , showing tip of mitochondrion ( arrowhead ) maintaining ΔΨm while neighboring region of same mitochondrion depolarizes . capacity of some cristae to maintain polarity , while adjacent cristae transiently depolarize , indicates cristae can exhibit a significant amount of electrochemical autonomy . N = 3 independent experiments . ( See Movies EV 3 , 4 for time series . ) Zoomed - in LUT of mitochondrion from E ( ~ 2 sec ) , showing some cristae remaining polarized ( red ) while adjacent cristae depolarize ( blue ) . ( See Movie EV 5 for time series . ) of D - F , showing that during a flickering event some cristae maintain their potentials while other cristae depolarize , resulting in large differences in ΔΨCr - IBM along mitochondrion . N = 3 independent experiments .

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

We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Representative live-cell Airyscan image from time series showing merged green (MTG) and red (TMRE) channels. Area marked by dashed box is shown in the time series in E. Scale bar = 500 nm. Time series of cropped region from D, showing tip of mitochondrion (arrowhead) maintaining ΔΨm while neighboring region of same mitochondrion depolarizes. The capacity of some cristae to maintain polarity, while adjacent cristae transiently depolarize, indicates cristae can exhibit a significant amount of electrochemical autonomy. N = 3 independent experiments. (See Movies EV 3, 4 for time series.) Zoomed-in LUT of mitochondrion from E (~2 sec), showing some cristae remaining polarized (red) while adjacent cristae depolarize (blue). (See Movie EV 5 for time series.) Quantification of D-F, showing that during a flickering event some cristae maintain their membrane potentials while other cristae depolarize, resulting in large differences in ΔΨCr-IBM along the individual mitochondrion. N = 3 independent experiments.


Left : representative conventional image of interphase MT network in 1st quadrant of a Human Retinal Pigment Epithelium ( HRPE ) . Grey : α - tubulin ; Yellow : TGN - 46 ; Yellow dashed : Golgi boundary . Right : classified MT subgroups . Red : GaMTs ; Green : non - GaMTs . Scale bar : 5 μm . Box - whisker plot presents proportion of GaMT non - GaMTs . ( data were pooled from 3 independent experiments n = 36 ) . ends of whiskers set as 10 % 90 % of entire population . * * * p < 0 . 001 , two - Mann - Whitney .

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Left: representative conventional image of interphase MT network in the 1st quadrant of a Human Retinal Pigment Epithelium (HRPE) cell. Grey: α-tubulin; Yellow: TGN-46; Yellow dashed line: Golgi boundary. Right: the classified MT subgroups. Red: GaMTs; Green: non-GaMTs. Scale bar: 5 μm. Box-whisker plot presents the proportion of GaMT and non-GaMTs. (data were pooled from 3 independent experiments and n= 36 cells). The ends of whiskers set as the 10% and 90% of the entire population. ***p < 0.001, two-tailed Mann-Whitney.


Six representative of MT networks extracted from MT STORM of HRPE under combinative KDs . were rotated to orient their leading edges locating in 1st quadrant . Upper : control ; CLASPs KD ; GCC185 KD . Lower : CLASPs + GCC185 KD ; CAMSAP2 KD ; AKAP450 KD . Red : GaMT ; Green : non - GaMT . Scale bar : 5 μm . Box - whisker plot presents of GaMT / non - GaMT under combinative KDs . ( 1 representative of 3 independent experiments n = 12 ) . ends of whiskers are set at 10 % 90 % of entire population . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , no significant difference , unpaired t - .

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Six representative images of MT networks extracted from MT STORM images of HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. Upper: control cell; CLASPs KD cell; GCC185 KD cell. Lower: CLASPs + GCC185 KD cell; CAMSAP2 KD cell; AKAP450 KD cell. Red: GaMT; Green: non-GaMT. Scale bar: 5 μm. Box-whisker plot presents the ratio of GaMT/non-GaMT under combinative KDs. (1 representative of 3 independent experiments and n= 12 cells). The ends of the whiskers are set at 10% and 90% of the entire population. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference, unpaired t-test.


Time series show EB1 movement on GaMTs . Red : GaMT ; Green : non - GaMT ; : EB1 tracks . Scale bar : 1 μm . Time series show EB1 movement on non - GaMTs . Red : GaMT ; Green : non - GaMT ; : EB1 tracks . Scale bar : 0 . 5 μm . EB1 velocity distributions of GaMTs non - GaMTs . Red curve green curve represent Gaussian fittings ( data were pooled from 3 independent experiments n = 92 tracks ) . * * * p < 0 . 001 , unpaired t - .

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Time series show EB1 movement on individual GaMTs. Red: GaMT; Green: non-GaMT; White: EB1 tracks. Scale bar: 1 μm. Time series show EB1 movement on individual non-GaMTs. Red line: GaMT; Green line: non-GaMT; White line: EB1 tracks. Scale bar: 0.5 μm. EB1 velocity distributions of GaMTs and non-GaMTs. Red curve and green curve represent the Gaussian fittings (data were pooled from 3 independent experiments and n= 92 tracks). ***p <0.001, unpaired t-test.


Representative trajectories of E - cad cargos tracked over 120 s in an HRPE . Colors represent different average velocity ranges . Red : > 400 nm / s ; Purple : 200 ~ 400 nm / s ; Green : 100 ~ 200 nm / s ; Yellow : 0 ~ 100 nm / s . Scale bar : 20 μm . Total number of cargos within different velocity ranges in 4 quadrants , respectively ( Data were pooled from 3 independent experiments n = 45 ) . Proportions of cargos with different velocity in 4 quadrants . Red : 1st quadrant ; grey : 2nd quadrant ; grey : 3rd quadrant ; : 4th quadrant ( Data were pooled from 3 independent experiments n = 45 ) .

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Representative trajectories of E-cad cargos tracked over 120 s in an HRPE cell. Colors represent different average velocity ranges. Red: > 400 nm/s; Purple: 200~400 nm/s; Green: 100~200 nm/s; Yellow: 0~100 nm/s. Scale bar: 20 μm. Total number of cargos within different velocity ranges in the 4 quadrants, respectively (Data were pooled from 3 independent experiments and n= 45 cells). Proportions of cargos with different velocity in the 4 quadrants. Red: the 1st quadrant; Dark grey: the 2nd quadrant; Light grey: the 3rd quadrant; White: the 4th quadrant (Data were pooled from 3 independent experiments and n= 45 cells).


Representative time series show one fast cargo moving on a GaMT a slow cargo moving on a non - GaMT . Upper , conventional imaging ; lower , STORM imaging . Red : GaMT ; Green : non - GaMT ; circle : cargo position . Scale bar : 2 μm . Marginal distributions show velocity on GaMT ( red dots ) or non - GaMTs ( green dots ) . Red curve green curve , Gaussian fitting curves of velocity distribution on GaMT or non - GaMT , respectively . ( Data were pooled from 3 independent experiments n = 69 MTs ) . Peak of red curve ( 431 . 14 ± 9 . 7 nm / s ) peak of purple curve ( 188 . 96 ± 22 . 03 nm / s ) . * * * p < 0 . 001 , unpaired t - .

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Representative time series show one fast cargo moving on a GaMT and a slow cargo moving on a non-GaMT. Upper, conventional imaging; lower, STORM imaging. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. Scale bar: 2 μm. Marginal distributions show the velocity on GaMT (red dots) or non-GaMTs (green dots). Red curve and green curve, Gaussian fitting curves of velocity distribution on GaMT or non-GaMT, respectively. (Data were pooled from 3 independent experiments and n= 69 MTs). Peak of red curve (431.14 ± 9.7 nm/s) and peak of purple curve (188.96 ± 22.03 nm/s). ***p < 0.001, unpaired t-test.


Upper panel : time series show an example of several fast cargos present on GaMT . Lower panel : time series show one slow cargo on non - GaMT switching onto a GaMT becoming faster . Red : GaMT ; Green : non - GaMT ; circle : cargo position . , cargo trajectory . Scale bar : 2 μm . Box - whisker plot presents averaged acetylation level of fast or slow MT tracks . ( Data were pooled from 2 independent experiments n = 63 MTs ) . ends of whiskers are set at 10 % 90 % of entire population , ns , no significant difference , unpaired t - . Marginal distributions of mean velocity of cargos on segments of Ac - MTs ( orange dots ) non - Ac - MTs ( purple dots ) . Orange curve purple curve represent Gaussian fittings ( Data were pooled from 2 independent experiments n = 63 MTs ) . ns , no significant difference , unpaired t - .

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

Upper panel: time series show an example of several fast cargos present on GaMT. Lower panel: time series show one slow cargo on non-GaMT switching onto a GaMT and becoming faster. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. White line, cargo trajectory. Scale bar: 2 μm. Box-whisker plot presents the averaged acetylation level of fast or slow MT tracks. (Data were pooled from 2 independent experiments and n= 63 MTs). The ends of the whiskers are set at 10% and 90% of the entire population, ns, no significant difference, unpaired t-test. Marginal distributions of the mean velocity of cargos on segments of Ac-MTs (orange dots) and non-Ac-MTs (purple dots). Orange curve and purple curve represent the Gaussian fittings (Data were pooled from 2 independent experiments and n= 63 MTs). ns, no significant difference, unpaired t-test.


Five representative of MT repair sites in HRPE under combinative KDs . were rotated to orient their leading edges locating in 1st quadrant . From left to right , control , CLASPs & GCC185 KD , CAMSAP2 KD , AKAP450 KD Centrinone - B treated . Grey : α - tubulin ; red : GTP - tubulin . Scale bar : 20 μm . Box - whisker plot presents of MT repair sites in HRPE under combinative KDs . ( 1 representative of 3 independent experiments n = 20 ) . * * * p < 0 . 001 , unpaired t - .

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Five representative images of MT repair sites in HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. From left to right, control cell, CLASPs&GCC185 KD cell, CAMSAP2 KD cell, AKAP450 KD cell and Centrinone-B treated cell. Grey: α-tubulin; red: GTP-tubulin. Scale bar: 20 μm. Box-whisker plot presents intensity analysis of MT repair sites in HRPE cells under combinative KDs. (1 representative of 3 independent experiments and n= 20 cells). ***p < 0.001, unpaired t-test.


Conventional image ( left ) STORM image ( right ) of MTs MT repair sites . Grey : α - tubulin ; yellow : GTP - tubulin ; green : Golgi . Scale bar : 5 μm .

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Conventional image (left) and STORM image (right) of MTs and MT repair sites. Grey: α-tubulin; yellow: GTP-tubulin; green: Golgi. Scale bar: 5 μm.


MT repair sites on GaMTs non - GaMTs extracted from C . Red : GaMTs ; Green : non - GaMTs ; yellow : GTP - tubulin . MT repair sites on GaMTs non - GaMTs separately presented in detail . arrows indicates GTP - tubulin labeling sites . Scale bar : 5 μm . Box - whisker plot presents of MT repair sites on GaMTs non - GaMTs separately . ( 1 representative of 3 independent experiments n = 8 ) . * * * p < 0 . 001 , unpaired t - .

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MT repair sites on GaMTs and non-GaMTs extracted from C. Red: GaMTs; Green: non-GaMTs; yellow: GTP-tubulin. MT repair sites on GaMTs and non-GaMTs separately presented in detail. White arrows indicates GTP-tubulin labeling sites. Scale bar: 5 μm. Box-whisker plot presents the ratio of MT repair sites on GaMTs and non-GaMTs separately. (1 representative of 3 independent experiments and n= 8 cells). ***p < 0.001, unpaired t-test.


Representative time series show one fast cargo moving on a GaMT slowing down at MT repaire site . Red : GaMT ; Green : non - GaMT ; Yellow : GTP - tubulin ; circle : cargo position . Scale bar : 2 μm . Representative time series show one cargo moving fast between two MT repair sites slowing down at MTrepair sites . Red : GaMT ; Green : non - GaMT ; Yellow : GTP - tubulin ; circle : cargo position . Scale bar : 2 μm . Pause ( I ) reverse ( J ) events of cargos on MT repair segment non - MTs repair segment , respectively . ( Data were pooled from 2 independent experiments n = 22 ) . * * * p < 0 . 001 , ns , no significant difference , unpaired t - .

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Representative time series show one fast cargo moving on a GaMT and slowing down at the MT repaire site. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Representative time series show one cargo moving fast between two MT repair sites and slowing down at the MTrepair sites. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Pause (I) and reverse (J) events of cargos on MT repair segment and non-MTs repair segment, respectively. (Data were pooled from 2 independent experiments and n= 22 cells). ***p <0.001, ns, no significant difference, unpaired t-test.


A representative image of MT networks extracted from MT STORM image of an HRPE . Red : GaMT ; Green : non - GaMT . Yellow circle : intersection . Blue circle : intersections formed by GaMTs non GaMTs . Scale bar : 5 μm . Rose - plot presents orientation of GaMTs non - GaMTs . Red : GaMTs ; Green : non - GaMTs . Box - whisker plot presents projections of GaMTs non - GaMTs to leading edge direction . ( Data were pooled from 3 independent experiments n = 36 ) . ends of whiskers are set at 10 % 90 % of entire population , * * p < 0 . 01 , unpaired t - . Box - whisker plot presents intersection of GaMTs non - GaMTs . ( data are pooled from 3 independent experiments n = 36 ) . ends of whiskers are set at 10 % 90 % of entire population , * * * p < 0 . 001 , unpaired t - .

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A representative image of MT networks extracted from MT STORM image of an HRPE cell. Red line: GaMT; Green line: non-GaMT. Yellow circle: intersection. Blue circle: intersections formed by GaMTs and non GaMTs. Scale bar: 5 μm. Rose-plot presents the orientation of GaMTs and non-GaMTs. Red: GaMTs; Green: non-GaMTs. Box-whisker plot presents the projections of GaMTs and non-GaMTs to the leading edge direction. (Data were pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, unpaired t-test. Box-whisker plot presents the intersection counts of GaMTs and non-GaMTs. (data are pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, unpaired t-test.


Random migration of HRPE during 24 h . Upper : control ; Middle : CAMSAP2 KD ; Lower : CLASPs & GCC185 KD . Red arrow dashed lines indicate migration direction . Different migration trajectories are distinguished with different colors . Scale bar : 50 μm . Rose - plot presents representative directionality of an HRPE . ( B ) control ; ( C ) CAMSAP2 KD ; ( D ) CLASPs & GCC185 KD . Box - whisker plot presents migration persistence of control , CAMSAP2 KD CLASPs & GCC185 KD . ( 1 representative of 3 independent experiments n = 34 ) . ends of whiskers are set at 10 % 90 % of entire population , * * * p < 0 . 001 , unpaired t - .

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Random migration of HRPE cells during 24 h. Upper: control cell; Middle: CAMSAP2 KD cell; Lower: CLASPs&GCC185 KD cell. Red arrow and white dashed lines indicate the migration direction. Different migration trajectories are distinguished with different colors. Scale bar: 50 μm. Rose-plot presents the representative directionality of an HRPE cell. (B) control cell; (C) CAMSAP2 KD cell; (D) CLASPs&GCC185 KD cell. Box-whisker plot presents the migration persistence of control cell, CAMSAP2 KD cell and CLASPs&GCC185 KD cell. (1 representative of 3 independent experiments and n= 34 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, unpaired t-test.


Controlled migration of 3 representative in PA - Rac1 . spot : 440 nm spot ; Green : original position of ; Red : current position of ; Yellow : overlapped . Scale bar : 10 μm .

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Controlled cell migration of 3 representative cells in PA-Rac1 assay. White spot: 440 nm laser spot; Green area: the original position of the cell; Red area: the current position of the cell; Yellow area: the overlapped area. Scale bar: 10 μm.


Angle change curve of , nucleus Golgi . Red - dashed : angle ; Blue : nucleus angle ; Orange : angle ; Green : Golgi angle . H : control ; I : CAMSAP2 KD ; J : CLASPs & GCC185 KD . Box - whisker plot shows total time required for nucleus , Golgi reaching given angle . Blue : time required for nucleus ; Green : time required for Golgi ; Orange : time required for . ( 1 representative of 3 independent experiments n = 14 ) . ends of whiskers are set at 10 % 90 % of entire population , * * * p < 0 . 001 , ns , no significant difference , unpaired t - .

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Angle change curve of cell, nucleus and Golgi. Red-dashed line: laser angle; Blue line: nucleus angle; Orange line: cell angle; Green line: Golgi angle. H: control cell; I: CAMSAP2 KD cell; J: CLASPs&GCC185 KD cell. Box-whisker plot shows the total time required for the nucleus, Golgi and cell body reaching the given laser angle. Blue: time required for nucleus; Green: time required for Golgi; Orange: time required for cell body. (1 representative of 3 independent experiments and n= 14 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, ns, no significant difference, unpaired t-test.


Projections of 120 frame movies present leading edge delivery of two different types of cargos . Left : Ecad cargo ; Right : Rab5 cargo . Yellow mark boundary . Scale bar : 20 μm .

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Projections of 120 frame movies present leading edge delivery of two different types of cargos. Left: Ecad cargo; Right: Rab5 cargo. Yellow line mark the cell boundary. Scale bar: 20 μm.


Examples of cargo delivery towards leading edge , time series present at 40 s , 80 s 120 s , respectively . B : control ; C : CAMSAP2 KD ; D : CLASPs & GCC185 KD . Red arrow heads indicate cargo current position . of cropped regions : 30 μm x 5 μm .

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Examples of cargo delivery towards the leading edge, time series present at 40 s, 80 s and 120 s, respectively. B: control cell; C: CAMSAP2 KD cell; D: CLASPs&GCC185 KD cell. Red arrow heads indicate cargo current position. Size of cropped regions: 30 μm x 5 μm.


Box - whisker plot presents Ecad cargo number on GaMTs non - GaMTs . ( Data were pooled from 3 independent experiments n = 36 ) . ends of whiskers are set at 10 % 90 % of entire population , * * p < 0 . 01 , unpaired t - .

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Box-whisker plot presents the Ecad cargo number on GaMTs and non-GaMTs. (Data were pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, unpaired t-test.


Box - whisker plot presents number of Ecad cargos finally reached leading edge during 120 s . ( 1 representative of 3 independent experiments n = 24 ) . ends of whiskers are set at 10 % 90 % of entire population , * * p < 0 . 01 , two - Mann - Whitney .

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Box-whisker plot presents the number of Ecad cargos finally reached the leading edge membrane during 120 s. (1 representative of 3 independent experiments and n= 24 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, two-tailed Mann-Whitney.


Box - whisker plot shows number of Rab5 cargos that finally reached leading edge during 120 s . ( 1 representative of 3 independent experiments n = 23 ) . ends of whiskers are set at 10 % 90 % of entire population , ns , no significant difference , two - Mann - Whitney .

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Box-whisker plot shows the number of Rab5 cargos that finally reached the leading edge membrane during 120 s. (1 representative of 3 independent experiments and n= 23 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ns, no significant difference, two-tailed Mann-Whitney.


A ATX gene expression from publicly available datasets in NASH ( n = 9 ) compared to healthy controls ( n = 7 ) ( * P < 0 . 05 ) ; cirrhosis ( n = 40 ) compared to healthy controls ( n = 19 ) ( * * * P < 0 . 001 ) . Bars represent mean ± , statistical was performed by two - student ' s t - . * P < 0 . 05 , * * * P < 0 . 001 denotes significance versus respective healthy controls .

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

A ATX gene expression from publicly available datasets in NASH patients (n=9) compared to healthy controls (n=7) (*P < 0.05); cirrhosis patients (n=40) compared to healthy controls (n=19) (***P < 0.001). Bars represent the mean ± SEM, statistical analysis was performed by two-tailed student's t-test. *P < 0.05, ***P < 0.001 denotes significance versus respective healthy controls.


B Relative ATX gene expression in MCD - diet - fed ( n = 9 ) compared to control mice ( n = 9 ) ( * * * P < 0 . 001 ) ; CCl4 ( n = 8 ) compared to control mice ( n = 5 ) ( * P < 0 . 05 ) . Bars represent mean ± , statistical was performed by two - student ' s t - . * P < 0 . 05 , * * * P < 0 . 001 denotes significance versus respective healthy controls .

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B Relative ATX gene expression in the MCD-diet-fed (n=9) compared to control mice (n=9) (***P < 0.001); CCl4 (n=8) compared to control mice (n=5) (*P < 0.05). Bars represent the mean ± SEM, statistical analysis was performed by two-tailed student's t-test. *P < 0.05, ***P < 0.001 denotes significance versus respective healthy controls.


C Representative ( scale = 50μm ) of ATX stained liver sections from human NASH ( n = 5 ) compared to healthy controls ( n = 4 ) ; human cirrhosis ( n = 5 ) compared to healthy controls ( n = 4 ) .

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C Representative images (scale=50μm) of ATX stained liver sections from human NASH (n=5) compared to healthy controls (n=4); human cirrhosis (n=5) compared to healthy controls (n=4).


D Representative ( scale = 50μm ) of ATX stained liver sections ( using AEC chromogen denoted by red color or DAB chromogen denoted by brown color ; nuclei were stained blue with hematoxylin ) from MCD - diet - fed NASH ( n = 9 ) CCl4 - induced liver fibrosis ( n = 8 ) mouse models compared to respective controls ( n = 9 or n = 5 ) .

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D Representative images (scale=50μm) of ATX stained liver sections (using AEC chromogen denoted by red color staining or DAB chromogen denoted by brown color staining; nuclei were stained blue with hematoxylin) from MCD-diet-fed NASH (n=9) and CCl4-induced liver fibrosis (n=8) mouse models compared to respective controls (n=9 or n=5).


A Structural binding of type I ATX inhibitor PF8380 to ATX ; Potency ( IC50 ) of PF8380 for inhibiting catalysis of LPC to LPA . Bars represent mean + , n = 3 . B Structural binding of type IV ATX inhibitor Cpd17 to ATX ; Potency ( IC50 ) of Cpd17 for inhibiting catalysis of LPC to LPA . Bars represent mean + , n = 3 .

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A Structural binding of type I ATX inhibitor PF8380 to ATX; Potency (IC50) of PF8380 for inhibiting the catalysis of LPC to LPA. Bars represent the mean + SEM, n=3. B Structural binding of type IV ATX inhibitor Cpd17 to ATX; Potency (IC50) of Cpd17 for inhibiting the catalysis of LPC to LPA. Bars represent the mean + SEM, n=3.


A Representative ( scale = 10µm ) of HepG2 stained with oil - red - O after 48 hours of incubation with or without 200μM palmitate with or without 1µM of PF8380 or Cpd17 . Bars represent mean + , n = 3 . Statistical was performed by one - way of variance ( ANOVA ) with Dunnett ' s multiple comparison . # # P < 0 . 01 denotes significance versus control ( non - palmitate ) * P < 0 . 05 denotes significance versus vehicle ( palmitate ) ; ns = non - significant .

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A Representative images (scale=10µm) and quantification of HepG2 cells stained with oil-red-O after 48 hours of incubation with or without 200μM palmitate with or without 1µM of PF8380 or Cpd17. Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. ##P < 0.01 denotes significance versus control (non-palmitate) and *P < 0.05 denotes significance versus vehicle (palmitate); ns=non-significant.


B Representative ( at 0h 24h ) ( scale = 50µm ) , quantitative ( after 24h ) of migration by control ( M0 ) LPS - IFNγ - induced M1 RAW264 . 7 macrophages treated with medium alone , PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent mean + , n = 4 . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # P < 0 . 05 denotes significance versus M0 ( non - stimulated macrophages ) * P < 0 . 05 denotes significance versus vehicle ( LPS - IFNγ - induced M1 macrophages ) ; ns = non - significant .

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B Representative images (at 0h and 24h) (scale=50µm), and quantitative analysis (after 24h) of migration by control (M0) and LPS- and IFNγ-induced M1 RAW264.7 macrophages treated with medium alone, PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus M0 (non-stimulated macrophages) and *P < 0.05 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.


C Relative gene expression ( normalized with GAPDH ) for CCL2 iNOS in control ( M0 ) LPS - IFNγ - induced M1 RAW 264 . 7 macrophages treated with medium alone , PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent mean + , n = 4 . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # # # # P < 0 . 0001 denotes significance versus M0 ( non - stimulated macrophages ) * P < 0 . 05 , * * P < 0 . 01 denotes significance versus vehicle ( LPS - IFNγ - induced M1 macrophages ) ; ns = non - significant .

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C Relative gene expression (normalized with GAPDH) for CCL2 and iNOS in control (M0) and LPS- and IFNγ-induced M1 RAW 264.7 macrophages treated with medium alone, PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus M0 (non-stimulated macrophages) and *P < 0.05, **P < 0.01 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.


D Relative gene expression ( normalized with 18s ) for CCL2 iNOS in control ( M0 ) LPS - induced PMA - differentiated human THP1 macrophages treated with medium alone , PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent mean + , n = 4 . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # # P < 0 . 01 , # # # P < 0 . 001 denotes significance versus M0 ( non - stimulated macrophages ) * * P < 0 . 01 , * * * P < 0 . 001 denotes significance versus vehicle ( LPS - IFNγ - induced M1 macrophages ) ; ns = non - significant .

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

D Relative gene expression (normalized with 18s RNA) for CCL2 and iNOS in control (M0) and LPS-induced PMA-differentiated human THP1 macrophages treated with medium alone, PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ##P < 0.01, ###P < 0.001 denotes significance versus M0 (non-stimulated macrophages) and **P < 0.01, ***P < 0.001 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.


A Collagen I α - SMA stained ( scale = 50µm ) of control TGFβ - activated LX2 with or without PF8380 ( 1μM ) or Cpd17 ( 1μM ) ( n = 3 ) .

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A Collagen I and α-SMA stained images (scale=50µm) of control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM) (n=3).


B Relative Collagen I , α - SMA PDGFβR gene expression ( normalized with GAPDH ) in control TGFβ - activated LX2 with or without PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent mean + , n = 3 . Statistical was performed by two - student ' s t - . # P < 0 . 05 , # # # P < 0 . 001 denotes significance versus control ( non - TGFβ ) * * P < 0 . 01 denotes significance versus vehicle ( TGFβ - activated ) ; ns = non - significant .

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

B Relative Collagen I, α-SMA and PDGFβR gene expression (normalized with GAPDH) in control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by two-tailed student's t-test. #P < 0.05, ###P < 0.001 denotes significance versus control (non-TGFβ cells) and **P < 0.01 denotes significance versus vehicle (TGFβ-activated cells); ns=non-significant.


C , D Representative ( at 0h 24h ) ( scale = 100µm ) ( C ) quantitative ( after 24h ) ( D ) of migration by control TGFβ - activated LX2 with or without PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent mean + , n = 3 . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # # # # P < 0 . 0001 denotes significance versus control ( non - TGFβ ) * * P < 0 . 01 denotes significance versus vehicle ( TGFβ - activated ) ; ns = non - significant .

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C,D Representative images (at 0h and 24h) (scale=100µm) (C) and quantitative analysis (after 24h) (D) of migration by control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (non-TGFβ cells) and **P < 0.01 denotes significance versus vehicle (TGFβ-activated cells); ns=non-significant.


E , F Representative ( E ) quantitative ( F ) showing 3D collagen gel contractility of control TGFβ - activated LX2 with or without PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent mean + , n = 3 . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # # # # P < 0 . 0001 denotes significance versus control ( non - TGFβ ) * * P < 0 . 01 , * * * * P < 0 . 0001 denotes significance versus vehicle ( TGFβ - activated ) .

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E,F Representative images (E) and quantitative analysis (F) showing 3D collagen gel contractility of control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (non-TGFβ cells) and **P < 0.01, ****P < 0.0001 denotes significance versus vehicle (TGFβ-activated cells).


B Top panel shows time - course stimulation to LPA uninhibited or inhibited LPC - treated ATX followed by RhoA activation , measured as a YFP / CFP . Bottom panel depicts quantitation of burst response to stimulants . Bars represent Mean + , n = 3 . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # P < 0 . 05 denotes significance versus control ( non - stimulated ) ; * P < 0 . 05 , * * * P < 0 . 001 denotes significance versus ATX + LPC treated .

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B Top panel shows time-course stimulation to LPA and uninhibited or inhibited LPC-treated ATX followed by RhoA activation, measured as a YFP/CFP fluorescent ratio. Bottom panel depicts quantitation of the burst response to the stimulants. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05, ***P <0.001 denotes significance versus ATX+LPC treated cells.


C Representative showing Western - blot for p - Akt p - ERK normalized with β - actin in control , LPC , LPA , ATX , ATX + LPC , ATX + LPC + PF8380 , ATX + LPC + Cpd17 stimulated LX2 . Bars represent Mean + , n = 3 . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # P < 0 . 05 denotes significance versus control ( non - stimulated ) ; * P < 0 . 05 denotes significance versus ATX + LPC treated .

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C Representative images and quantification showing Western-blot analysis for p-Akt and p-ERK normalized with β-actin in control, LPC, LPA, ATX, ATX+LPC, ATX+LPC+PF8380, and ATX+LPC+Cpd17 stimulated LX2 cells. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05 denotes significance versus ATX+LPC treated cells.


D Representative showing Western - blot for LPAR1 on surface in lysate , normalized with β - actin in lysate , in LPC , ATX + LPC , ATX + LPC + PF8380 , ATX + LPC + Cpd17 stimulated LX2 . Bars represent Mean + , n = 3 . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # P < 0 . 05 denotes significance versus control ( non - stimulated ) ; * P < 0 . 05 denotes significance versus ATX + LPC treated .

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D Representative images and quantification showing Western-blot analysis for LPAR1 on the cell surface and in lysate, normalized with β-actin in lysate, in LPC, ATX+LPC, ATX+LPC+PF8380, and ATX+LPC+Cpd17 stimulated LX2 cells. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05 denotes significance versus ATX+LPC treated cells.


B Liver to total from control ( n = 5 ) , CCl4 ( n = 5 ) CCl4 + Cpd17 ( n = 4 ) mice . Bars represent Mean + . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # # # # P < 0 . 0001 denotes significance versus control ( healthy mice ) ; * * * P < 0 . 001 denotes significance versus CCl4 mice .

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B Liver weight to total body weight ratio from control (n=5), CCl4 (n=5) and CCl4+Cpd17 (n=4) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (healthy mice); ***P < 0.001 denotes significance versus CCl4 mice.


C Serum ALT from control ( n = 5 ) , CCl4 ( n = 5 ) CCl4 + Cpd17 ( n = 4 ) mice . Bars represent Mean + . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # # # P < 0 . 001 denotes significance versus control ( healthy mice ) ; P = 0 . 26 denotes significance versus CCl4 mice .

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C Serum ALT levels from control (n=5), CCl4 (n=5) and CCl4+Cpd17 (n=4) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ###P < 0.001 denotes significance versus control (healthy mice); P = 0.26 denotes significance versus CCl4 mice.


D , E Representative ( scale = 100μm ) ( D ) quantitative ( E ) of liver sections from control ( n = 5 ) , CCl4 ( n = 5 ) CCl4 + Cpd17 ( n = 4 ) mice stained with H & E , collagen - I F4 / 80 . Bars represent Mean + . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # P < 0 . 05 , # # # P < 0 . 001 denotes significance versus control ( healthy mice ) ; P = 0 . 098 or P = 0 . 08 denotes significance versus CCl4 mice .

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D,E Representative images (scale=100μm) (D) and quantitative analysis (E) of liver sections from control (n=5), CCl4 (n=5) and CCl4+Cpd17 (n=4) mice and stained with H&E, collagen-I and F4/80. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05, ###P < 0.001 denotes significance versus control (healthy mice); P = 0.098 or P = 0.08 denotes significance versus CCl4 mice.


D , Representative ( scale = 100μm ) ( D ) of liver sections from control ( n = 5 ) , MCD ( n = 6 ) MCD + Cpd17 ( n = 6 ) mice stained with H & E , oil - red - O Collagen I .

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D, Representative images (scale=100μm) (D) of liver sections from control (n=5), MCD (n=6) and MCD+Cpd17 (n=6) mice and stained with H&E, oil-red-O and Collagen I.


quantitative ( E ) of liver sections from control ( n = 5 ) , MCD ( n = 6 ) MCD + Cpd17 ( n = 6 ) mice stained with oil - red - O Collagen I . Bars represent Mean + . Statistical was performed by one - way of variance ( ANOVA ) with Bonferroni post - hoc . # P < 0 . 05 , # # # P < 0 . 001 denotes significance versus control ( healthy mice ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 denotes significance versus MCD - diet fed mice .

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quantitative analysis (E) of liver sections from control (n=5), MCD (n=6) and MCD+Cpd17 (n=6) mice stained with oil-red-O and Collagen I. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05, ###P < 0.001 denotes significance versus control (healthy mice); *P < 0.05, **P < 0.01, ***P < 0.001 denotes significance versus MCD-diet fed mice.


B . β - catenin - mediated reporter activity in HEK293T expressing indicated RNF43 cancer mutants . were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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B. β-catenin-mediated reporter activity in HEK293T cells expressing the indicated RNF43 cancer mutants. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


C . Western blot showing effect of RNF43 cancer mutants on V5 - FZD5 expression in HEK293T . Open closed arrows indicate mature ( post - Golgi ) immature ( ER - associated ) FZD5 , respectively .

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C. Western blot analysis showing the effect of RNF43 cancer mutants on V5-FZD5 expression in HEK293T cells. Open and closed arrows indicate mature (post-Golgi) and immature (ER-associated) FZD5, respectively.


D . Confocal microscopy of surface labeled SNAP - FZD5 in HEK293T upon expression of RNF43 WT or R519X . were chased for 30 min . Scale bars represent 10 μm .

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D. Confocal microscopy of surface labeled SNAP-FZD5 in HEK293T cells upon expression of RNF43 WT or R519X. Cells were chased for 30 min. Scale bars represent 10 μm.


E . Confocal microscopy of β - catenin localization in HEK293T expressing indicated RNF43 cancer mutants . Scale bars represent 10 μm .

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E. Confocal microscopy of β-catenin localization in HEK293T cells expressing the indicated RNF43 cancer mutants. Scale bars represent 10 μm.


F . Western blot of RNF43 WT R519X for β - catenin .

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F. Western blot analysis of RNF43 WT and R519X for cytoplasmic β-catenin levels.


G . β - catenin - mediated reporter activity in HEK293T expressing Wnt3a WT RNF43 , oncogenic RNF43 ( R519X ) or a LOF RNF43 variant ( I48T ) after o / n treatment with DMSO or PORCN inhibitor C59 ( 1 μM ) .

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G. β-catenin-mediated reporter activity in HEK293T cells expressing Wnt3a and WT RNF43, oncogenic RNF43 (R519X) or a LOF RNF43 variant (I48T) after o/n treatment with DMSO or the PORCN inhibitor C59 (1 μM).


A . β - catenin - mediated reporter activity in HEK293T expressing WT RNF43 oncogenic RNF43 ( R519X ) harboring mutations C290S / H292S in RING domain ( M1 ) . were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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A. β-catenin-mediated reporter activity in HEK293T cells expressing WT RNF43 and oncogenic RNF43 (R519X) harboring mutations C290S/H292S in the RING domain (M1). Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


C . β - catenin - mediated reporter activity in HEK293T expressing indicated RNF43 constructs . were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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C. β-catenin-mediated reporter activity in HEK293T cells expressing the indicated RNF43 constructs. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


D . β - catenin - mediated reporter activity in HEK293T co - expressing oncogenic RNF43 ( R519X ) Dishevelled DEP - C tail . were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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D. β-catenin-mediated reporter activity in HEK293T cells co-expressing oncogenic RNF43 (R519X) and the Dishevelled DEP-C tail. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


E . β - catenin - mediated reporter activity in HEK293T co - expressing oncogenic RNF43 ( R519X ) dominant negative ∆ N - TCF4 . were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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E. β-catenin-mediated reporter activity in HEK293T cells co-expressing oncogenic RNF43 (R519X) and dominant negative ∆N-TCF4. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


A . Volcano plot showing enriched after streptavidin pull - down of biotin - treated HEK293 stably expressing dox - inducible RNF43 - BirA * . Striped demarcates empirical 0 . 01 False discovery rate ( FDR ) cut off . Significantly enriched Wnt / β - catenin pathway components are highlighted in red RNF43 in orange .

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A. Volcano plot showing proteins enriched after streptavidin pull-down of biotin-treated HEK293 cells stably expressing dox-inducible RNF43-BirA*. Striped line demarcates the empirical 0.01 False discovery rate (FDR) cut off. Significantly enriched Wnt/β-catenin pathway components are highlighted in red and RNF43 in orange.


B . Western blot of endogenous destruction components that co - precipitated with indicated RNF43 cancer variants expressed in HEK293T . Oncogenic RNF43 truncations are indicated in red . Data information : IP ; immunoprecipitation , IB ;

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B. Western blot analysis of endogenous destruction complex components that co-precipitated with the indicated RNF43 cancer variants expressed in HEK293T cells. Oncogenic RNF43 truncations are indicated in red. Data information: IP; immunoprecipitation, IB; immunoblot


Confocal microscopy of subcellular localization of Axin1 - GFP upon expression of WT or oncogenic RNF43 ( R519X ) . Scale bars represent 10 μm . RNF43 is visualised by Flag - . Asterisks indicate RNF43 - expressing .

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Confocal microscopy analysis of the subcellular localization of Axin1-GFP upon expression of WT or oncogenic RNF43 (R519X). Scale bars represent 10 μm. RNF43 is visualised by Flag-staining. Asterisks indicate RNF43-expressing cells.


Confocal microscopy of subcellular localization of endogenous CK1α upon expression of WT or oncogenic RNF43 ( R519X ) . Scale bars represent 10 μm . RNF43 is visualised by Flag - . Asterisks indicate RNF43 - expressing .

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Confocal microscopy analysis of the subcellular localization of endogenous CK1α upon expression of WT or oncogenic RNF43 (R519X). Scale bars represent 10 μm. RNF43 is visualised by Flag-staining. Asterisks indicate RNF43-expressing cells.


E . β - catenin - mediated reporter activity in HEK293T expressing non - mutated RNF43 R519X ( SLSS ) or ALAA , DLDD ∆ 486 - 89 mutants . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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E. β-catenin-mediated reporter activity in HEK293T cells expressing non-mutated RNF43 R519X (SLSS) or the ALAA, DLDD and ∆486-89 mutants. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


A . Bright - field microscopy of WT , TP53KO onco - RNF43 / TP53KO human colon organoid lines grown in medium with high Wnt / Rspo ( 20 % conditioned medium ( CM ) ) or without Wnt / Rspo ( 20 , 2 or 0 . 2 % CM ) . were taken 6 days after splitting . Scale bars represent 1000 μm . Non - cystic , non - proliferative organoids are indicated with red asterisks .

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A. Bright-field microscopy images of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines grown in medium with high Wnt/Rspo (20% conditioned medium (CM)) or without Wnt/Rspo (20, 2 or 0.2% CM). Images were taken 6 days after splitting. Scale bars represent 1000 μm. Non-cystic, non-proliferative organoids are indicated with red asterisks.


C . Bright - field microscopy microscopy pictures of WT , TP53KO onco - RNF43 / TP53KO human colon organoid lines grown in two different media transduced with TOP - GFP reporter . were taken 6 days after splitting . Scale bars represent 1000 μm .

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C. Bright-field microscopy and fluorescence microscopy pictures of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines grown in two different media and transduced with the TOP-GFP reporter. Images were taken 6 days after splitting. Scale bars represent 1000 μm.


D . Gene Set Enrichment of onco - RNF43 / P53KO compared to TP53KO organoids in medium without Wnt / low Rspo ( 0 . 2 % ) . Significantly changed intestinal - type gene sets from Haber et al . , 2017 ( ) are shown ( FDR < 0 . 05 ) .

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D. Gene Set Enrichment Analysis of onco-RNF43/P53KO compared to TP53KO organoids in medium without Wnt/low Rspo (0.2%). Significantly changed intestinal cell-type gene sets from Haber et al., 2017() are shown (FDR < 0.05).


A . Top : Schematic experimental set up of clonogenic . Bottom : Bright - field microscopy of WT , TP53KO onco - RNF43 / TP53KO human colon organoid lines at day 2 14 . Scale bars represent 1000 μm .

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A. Top: Schematic experimental set up of the clonogenic assay. Bottom: Bright-field microscopy images of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines at day 2 and 14. Scale bars represent 1000 μm.


B . Relative outgrowth of WT , TP53KO onco - RNF43 / TP53KO organoid lines treated with PORCN inhibitor C59 ( 1 μM ) for 7 days . Graph shows number of organoids at 5 days after splitting ( day 14 ) relative to number of organoids at day 2 . Error bars represent ± s . d . of mean of n = 3 experiments .

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B. Relative outgrowth of WT, TP53KO and onco-RNF43/TP53KO organoid lines treated with the PORCN inhibitor C59 (1 μM) for 7 days. Graph shows the number of organoids at 5 days after splitting (day 14) relative to the number of organoids at day 2. Error bars represent ±s.d. of the mean of n = 3 experiments.


( B ) Western blot analyses of whole - lysates of wild - type ( CC - 124 ) , bld10 , bld10 : : B10 - N - HA , bld10 : : B10 - M - HA , bld10 : : B10 - C - HA using antibodies against C - terminal region of Bld10p ( Anti - Bld10 - C antibody , upper blot ) HA antigen ( Anti - HA antibody , lower blot ) . apparent molecular weights of tagged Bld10ps are slightly larger than that of native Bld10p due to their HA tags .

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(B) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld10, bld10::B10-N-HA, bld10::B10-M-HA, and bld10::B10-C-HA cells using antibodies against the C-terminal region of Bld10p (Anti-Bld10-C antibody, upper blot) and the HA antigen (Anti-HA antibody, lower blot). The apparent molecular weights of tagged Bld10ps are slightly larger than that of native Bld10p due to their HA tags.


( C ) Percentages of flagellated in wild - type ( CC - 124 ) , bld10 , bld10 : : B10 - N - HA , bld10 : : B10 - M - HA , bld10 : : B10 - C - HA , bld12 , bld10bld12 , bld10bld12 : : ∆ N3 , bld10bld12 : : ∆ C2 , bld10 : : ∆ N3 , bld10 : : ∆ C2 . with two ( red ) , one ( green ) , zero ( blue ) flagella were counted . n , number of counted .

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(C) Percentages of flagellated cells in wild-type (CC-124), bld10, bld10::B10-N-HA, bld10::B10-M-HA, bld10::B10-C-HA, bld12, bld10bld12, bld10bld12::∆N3, bld10bld12::∆C2, bld10::∆N3, and bld10::∆C2 cells. Cells with two (red), one (green), and zero (blue) flagella were counted. n, number of cells counted.


( D ) Immunoelectron of wild type centrioles treated with anti - Bld10p ( B10 - C ) after embedding in resin sectioning . Localization of 16 gold particles in 13 centriole are plotted on triangular map ( right panel ) . Data information : Scale bar , 100 nm .

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(D) Immunoelectron micrographs of wild type centrioles treated with anti-Bld10p (B10-C) after embedding in the resin and sectioning. Localization of 16 gold particles in 13 centriole images are plotted on the triangular map (the right panel). Data information: Scale bar, 100 nm.


( C ) A schematic diagram showing positions of observed filamentous structures ( L1 , L2 , L3 ) . between each filamentous structure inner side of A - tubule is indicated in parentheses ( mean ± ) . numbers of observed L1 , L2 , L3 are 25 , 19 , 13 , respectively . ( D ) Slices from reconstructed cryo - electron tomograms of probasal bodies . Lower panels show regions with L1 , L2 L3 , boxed from upper panels . Red dots denote connection points between spokes filamentous structures . Data information : Scale bar , 100 nm .

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(C) A schematic diagram showing the positions of the observed filamentous structures (L1, L2, and L3). The distance between each filamentous structure and the inner side of the A-tubule is indicated in parentheses (mean ± SEM). The numbers of observed L1, L2, and L3 are 25, 19, and 13, respectively. (D) Slices from the reconstructed cryo-electron tomograms of probasal bodies. Lower panels show the regions with L1, L2 and L3, boxed from the upper panels. Red dots denote the connection points between the spokes and filamentous structures. Data information: Scale bar, 100 nm.


( A ) Western blot analyses of whole - lysates of wild - type ( CC - 124 ) , bld12 , bld10 , bld10bld12 , bld10bld12 : : ∆ N3 using an antibody against C - terminal one - third of Bld10p ( Anti - Bld10 - C antibody ) . band corresponding to truncated Bld10p ( Bld10p ∆ N3 ) overlaps with a non - specific band at 75 kDa . ( B ) Western blot analyses of whole - lysates of wild - type ( CC - 124 ) , bld12 , bld10 , bld10bld12 , bld10bld12 : : ∆ C2 using an antibody raised against N - terminal 120 - acid of Bld10p ( Anti - Bld10 - N antibody ) .

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(A) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld12, bld10, bld10bld12, and bld10bld12::∆N3 cells using an antibody against the C-terminal one-third of Bld10p (Anti-Bld10-C antibody). The band corresponding to truncated Bld10p (Bld10p ∆N3) overlaps with a non-specific band at 75 kDa. (B) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld12, bld10, bld10bld12, and bld10bld12::∆C2 cells using an antibody raised against the N-terminal 120 amino-acid sequence of Bld10p (Anti-Bld10-N antibody).


( C ) Cross - sectional electron of centrioles in bld12 , bld10 : : ∆ N3 , bld10bld12 : : ∆ N3 , bld10 : : ∆ C2 , bld10bld12 : : ∆ C2 . Scale bar , 100 nm . ( D ) Distribution of number of triplets in centrioles of bld12 , bld10 : : ∆ N3 , bld10bld12 : : ∆ N3 , bld10 : : ∆ C2 , bld10bld12 : : ∆ C2 . Red lines denote normal number of microtubules in centriole ( nine ) . n , number of centrioles observed .

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(C) Cross-sectional electron micrographs of centrioles in bld12, bld10::∆N3, bld10bld12::∆N3, bld10::∆C2, and bld10bld12::∆C2 cells. Scale bar, 100 nm. (D) Distribution of the number of triplets in centrioles of bld12, bld10::∆N3, bld10bld12::∆N3, bld10::∆C2, and bld10bld12::∆C2 cells. Red lines denote the normal number of microtubules in the centriole (nine). n, number of centrioles observed.


( A B ) Cross - sectional electron of proximal ( A ) distal ( B ) regions of centrioles containing eight or nine triplets in bld12 , bld10bld12 : : ∆ N3 , bld10bld12 : : ∆ C2 . arrangement of triplets in of axonemes were circularized using CentrioleJ program colored circles represent circles inscribed to innermost positions of A - tubules in centriole . Scale bar , 100 nm . ( C D ) Distances between adjacent A - tubules at proximal ( C ) distal ( D ) regions of centrioles containing seven , eight , nine , ten triplets in wild - type ( cw92 ) , bld12 , bld10bld12 : : ∆ N3 , bld10bld12 : : ∆ C2 . distances between contact points of A - tubules with inscribed circle were measured . These distances were ~ 10 % shorter in expressing truncated Bld10ps than in wild - type bld12 . * * * , P < 0 . 0001 . n , number of centrioles measured . Data information : Data were analyzed by unpaired t - . graphs show mean ± .

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(A and B) Cross-sectional electron micrographs of the proximal (A) and distal (B) regions of centrioles containing eight or nine triplets in bld12, bld10bld12::∆N3, and bld10bld12::∆C2 cells. The arrangement of triplets in images of axonemes were circularized using CentrioleJ program The colored circles represent the circles inscribed to the innermost positions of the A-tubules in the centriole. Scale bar, 100 nm. (C and D) Distances between adjacent A-tubules at the proximal (C) and distal (D) regions of centrioles containing seven, eight, nine, and ten triplets in wild-type (cw92), bld12, bld10bld12::∆N3, and bld10bld12::∆C2 cells. The distances between the contact points of A-tubules with the inscribed circle were measured. These distances were ~10% shorter in cells expressing truncated Bld10ps than in wild-type and bld12 cells. ***, P<0.0001. n, number of centrioles measured. Data information: Data were analyzed by unpaired t-test. The graphs show mean ± SEM.


( E ) Lengths of A - C linkers in wild - type ( cw92 ) , bld12 , bld10bld12 : : ∆ N3 , bld10bld12 : : ∆ C2 centrioles . Centrioles containing nine triplets eight triplets were separately analyzed . n , number of A - Clinkers measured . ( F ) Angles between adjacent triplets in centrioles analyzed in panel € . angle ( θ ) measured was defined as indicated in schematic diagram ; angle between connecting centers of B - tubules of two adjacent triplets ( black ) connecting centers of A - C - tubules of triplet of two ( brown ) . * , P < 0 . 01 . n , number of triplet angles measured . Data information : Data were analyzed by unpaired t - . graphs show mean ± .

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 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

(E) Lengths of A-C linkers in wild-type (cw92), bld12, bld10bld12::∆N3, and bld10bld12::∆C2 centrioles. Centrioles containing nine triplets and eight triplets were separately analyzed. n, number of A-Clinkers measured. (F) Angles between adjacent triplets in centrioles analyzed in panel €. The angle (θ) measured was defined as indicated in the schematic diagram; the angle between the line connecting the centers of B-tubules of the two adjacent triplets (black line) and the line connecting the centers of A- and C-tubules of the triplet of the two (brown line). *, P<0.01. n, number of triplet angles measured. Data information: Data were analyzed by unpaired t-test. The graphs show mean ± SEM.


( A , B ) Flies form well - organized compound eyes when they carry GMR - GAL4 without a target gene ( A ) but develop rough eyes when GMR - GAL4 drives expression of UAS - Foxn1 ( B ) . Panel B shows typical severity of rough - eye phenotype . Data information : Representative are shown in panels . Scale bars , 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 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

(A, B) Flies form well-organized compound eyes when they carry GMR-GAL4 without a target gene (A) but develop rough eyes when GMR-GAL4 drives expression of UAS-Foxn1 (B). Panel B shows the typical severity of the rough-eye phenotype. Data information: Representative phenotypes are shown in all panels. Scale bars, 100 μm.


( C , D ) Bristles are pigmented properly shaped when flies carry hs - GAL4 without a target gene ( C ) but are often , bent , shortened when hs - GAL4 drives expression of UAS - Foxn1 ( D ) . Flies in panels C D were raised at 25 ° C . Arrows denote examples of bristles . Data information : Representative are shown in panels . Scale bars , 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 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

(C, D) Bristles are pigmented and properly shaped when flies carry hs-GAL4 without a target gene (C) but are often white, bent, and shortened when hs-GAL4 drives expression of UAS-Foxn1 (D). Flies in panels C and D were raised at 25°C. Arrows denote examples of white bristles. Data information: Representative phenotypes are shown in all panels. Scale bars, 100 μm.


AFF4 ( green ) FOXN1 ( red ) are shown by immunofluorescence in following wild - type tissue samples : ( B ) murine hair follicles , P9 ; is stained by Hoechst dye 33258 ( blue ) . Co - localization of AFF4 FOXN1 generates yellow color , visible in nuclei of samples . Arrowheads mark CH , cortex of hair ; Scale bars , 20 µm .

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 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

AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (B) murine hair follicles, P9; DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. Arrowheads mark the CH, cortex of the hair; Scale bars, 20 µm.


AFF4 ( green ) FOXN1 ( red ) are shown by immunofluorescence in following wild - type tissue samples : ( C ) developing murine epidermis , E16 . 5 ; ( D ) human epidermis , adult ; ( E ) murine thymus , P0 , outer region . is stained by Hoechst dye 33258 ( blue ) . Co - localization of AFF4 FOXN1 generates yellow color , visible in nuclei of samples . In panel C , signal in periderm ( P ) is nonspecific , as it is present when antibodies are omitted from . Inner regions of murine thymus ( not shown ) yielded same extensive overlap of FOXN1 AFF4 as that shown in panel E . Arrowheads mark dermal / epidermal border ( C , D ) . CT , cortex of thymus . Scale bars , 20 µm .

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

AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (C) developing murine epidermis, E16.5; (D) human epidermis, adult; (E) murine thymus, P0, outer region. DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. In panel C, the fluorescent signal in the periderm (P) is nonspecific, as it is present when primary antibodies are omitted from the staining. Inner regions of the murine thymus (not shown) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E. Arrowheads mark the dermal/epidermal border (C, D). CT, cortex of the thymus. Scale bars, 20 µm.


Representative skin of wild - type ( WT ) mice shown macroscopically compared are as follows : hair coats , 4 - month - old

O 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 skin phenotypes of wild-type (WT) mice shown macroscopically The phenotypes compared are as follows: hair coats, 4-month-old adults


Representative skin of Foxn1nu / nu ( Nude ) , or KRT14 - cre ; Aff4flox / flox ( Aff4 cko ) mice are shown macroscopically or histologically after hematoxylin eosin ( H - N ) . compared are as follows : hair coats , 4 - month - old adults ; ( E - G ) vibrissae , P7 ; ( H ) hair follicles with growing hairs , juveniles ; ( I - K ) regressing hair follicles , juveniles ; ( L - N ) epidermis , P9 . In panel H , from left to right are WT , Nude , Aff4 cko . In panels I - K , HF marks examples of hair follicles . In panels L - M , arrowheads indicate dermal / epidermal border ; arrows mark examples of bent hair shafts . Scale bars : H , L - N , 20 µm ; I - K , 40 µm .

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

Representative skin phenotypes of Foxn1nu/nu (Nude), or KRT14-cre; Aff4flox/flox (Aff4 cko) mice are shown macroscopically or histologically after hematoxylin and eosin staining (H-N). The phenotypes compared are as follows: hair coats, 4-month-old adults; (E-G) vibrissae, P7; (H) hair follicles with growing hairs, juveniles; (I-K) regressing hair follicles, juveniles; (L-N) epidermis, P9. In panel H, micrographs from left to right are WT, Nude, and Aff4 cko. In panels I-K, HF marks examples of hair follicles. In panels L-M, arrowheads indicate the dermal/epidermal border; arrows mark examples of bent hair shafts. Scale bars: H, L-N, 20 µm; I-K, 40 µm.


( A - C ) Thymi from E18 . 5 are shown : ( A ) intact with genotypes indicated , or ( B , C ) after sectioning , immunofluorescent for DLL4 ( red ) , counterstaining of ( blue ) . Sectioned samples are wild - type ( B ) or Aff4ko / ko ; Foxn1 + / - ( C ) . Scale bars : A , 1 mm ; B - C , 50 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O 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

(A-C) Thymi from E18.5 embryos are shown: (A) intact with genotypes indicated, or (B, C) after sectioning, immunofluorescent staining for DLL4 (red), and counterstaining of DNA (blue). Sectioned samples are wild-type (B) or Aff4ko/ko; Foxn1+/- (C). Scale bars: A, 1 mm; B-C, 50 µm.


( C - E ) KRT86 ( red ) is shown by immunofluorescence in skin sections from wild - type ( WT ) , Foxn1 null , or Aff4 cko mice at P9 . is stained blue . HS marks examples of hair shafts . Scale bar , 40 µm .

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

(C-E) KRT86 (red) is shown by immunofluorescence in skin sections from wild-type (WT), Foxn1 null, or Aff4 cko mice at P9. DNA is stained blue. HS marks examples of hair shafts. Scale bar, 40 µm.


Graphs present pre - mRNA ( B , C ) assays of Krt86 using skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P6 ( B , C ) values are derived from 3 independent experiments presented as mean ± S . D . Pre - mRNA measurements were analyzed statistically using two - way ANOVA with post - hoc Sidak ' s multiple comparison at a 95 % confidence interval .

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

Graphs present pre-mRNA (B, C) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P6 (B, C) All values are derived from 3 independent experiments and presented as mean ± S.D. Pre-mRNA measurements were analyzed statistically using two-way ANOVA with post-hoc Sidak's multiple comparison test at a 95% confidence interval.


Graphs present ChIP ( D - G ) assays of Krt86 using skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P7 ( D - G ) . ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated . values are derived from 3 independent experiments presented as mean ± S . D . ChIP assays were analyzed statistically using one - way ANOVA with post - hoc Tukey ' s multiple comparison at a 95 % confidence interval . * * * * , p ≤ 0 . 0001 .

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

Graphs present ChIP (D-G) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7 (D-G). ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated. All values are derived from 3 independent experiments and presented as mean ± S.D. ChIP assays were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.


( B - G ) Graphs present ChIP assays of Krt86 using antibodies to total ( B , C ) , P - Ser5 ( D , E ) , or P - Ser2 ( F , G ) Pol II skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P7 . values are derived from at least 3 independent experiments presented as mean ± S . D . results were analyzed statistically using one - way ANOVA with post - hoc Tukey ' s multiple comparison at a 95 % confidence interval . * * * * , p ≤ 0 . 0001 .

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

(B-G) Graphs present ChIP assays of Krt86 using antibodies to total (B, C), P-Ser5 (D, E), or P-Ser2 (F, G) Pol II and skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7. All values are derived from at least 3 independent experiments and presented as mean ± S.D. Assay results were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.


( B ) Immunoblotting of whole extracts obtained from parental RAP80 - / - clones in RPE - 1 hTERT p53 - / - Cas9 , RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 , U2OS , U2OS - 2 - 6 - 3 U2OS Flp - In / TREx lines with RAP80 antibodies . Tubulin is used as loading control . Representative of at least two independent immunoblots . See Figure EV1 for further details on gene editing strategy used to knockout RAP80 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O 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) Immunoblotting of whole cell extracts obtained from parental and RAP80-/- clones in the RPE-1 hTERT p53-/- Cas9, RPE-1 hTERT p53-/- BRCA1-/- Cas9, U2OS, U2OS-2-6-3 and U2OS Flp-In/TREx cell lines with RAP80 antibodies. Tubulin is used as loading control. Representative of at least two independent immunoblots. See Figure EV1 for further details on the gene editing strategy used to knockout RAP80.


( C , D ) indicated parental RAP80 - / - lines were processed for immunofluorescence 1 h post - irradiation ( 10 Gy ) stained with antibodies against ABRAXAS1 γH2AX . Shown in ( C ) is of with > 5 ABRAXAS1 foci that colocalize with γH2AX . A minimum of 100 per replicate were analyzed bars represent mean ± S . D . ( n = 3 biological replicates ) . Representative of RPE1 - hTERT p53 - / - Cas9 wild type ( WT ) RAP90 - / - are shown in ( D ) .

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

(C, D) The indicated parental and RAP80-/- cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against ABRAXAS1 and γH2AX. Shown in (C) is the percentage of cells with > 5 ABRAXAS1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs of RPE1-hTERT p53-/- Cas9 wild type (WT) and RAP90-/- cells are shown in (D).


( E , indicated lines were processed for immunofluorescence 1 h post - irradiation ( 10 Gy ) stained with antibodies against BRCA1 γH2AX . Shown in ( E ) is of with > 5 BRCA1 foci that colocalize with γH2AX . A minimum of 100 per replicate were analyzed bars represent mean ± S . D . ( n = 3 biological replicates for conditions except n = 5 for U2OS WT , n = 2 for U2OS 2 - 6 - 3 , n = 7 , 5 , 4 for U2OS Flp - In / T - REx ) .

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(E, The indicated cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against BRCA1 and γH2AX. Shown in (E) is the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates for all conditions except n=5 for U2OS WT, n=2 for U2OS 2-6-3 cells, n=7, 5, 4 for U2OS Flp-In/T-REx cells).


( G ) RPE - 1 hTERT p53 - / - Cas9 parental ( WT ) RAP80 - / - were untreated ( t = 0 h ) or irradiated with a 10 Gy dose . Samples were collected at indicated time points , processed for immunofluorescence stained with antibodies against BRCA1 γH2AX . Shown is of with > 5 BRCA1 foci . A minimum of 100 per replicate were analyzed datapoints represent mean ± S . D . ( n = 3 biological replicates ) . scale bars are 5 μm .

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 B-MEASURED_VAR I-MEASURED_VAR 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

(G) RPE-1 hTERT p53-/- Cas9 parental (WT) and RAP80-/- cells were untreated (t=0 h) or irradiated with a 10 Gy dose. Samples were collected at the indicated time points, processed for immunofluorescence and stained with antibodies against BRCA1 and γH2AX. Shown is the percentage of cells with > 5 BRCA1 foci. A minimum of 100 cells per replicate were analyzed and the datapoints represent the mean ± S.D. (n=3 biological replicates). All scale bars are 5 μm.


( A , B ) U2OS Flp - In / T - Rex parental ( WT ) RAP80 - / - were transfected with siRNA pools targeting BRCA1 , RNF8 , RNF168 or BARD1 or with a non - targeting control siRNA ( CTRL ) . 48 h post - transfection were irradiated ( 2 Gy ) processed for immunofluorescence 1 h post - IR treatment using antibodies against BRCA1 γH2AX . Quantitation of of with > 5 BRCA1 foci that colocalize with γH2AX is shown in ( A ) . A minimum of 100 per replicate were analyzed bars represent mean ± S . D . ( n = 3 biological replicates ) . Representative are shown in ( B ) .

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

(A, B) U2OS Flp-In/T-Rex parental (WT) and RAP80-/- cells were transfected with siRNA pools targeting BRCA1, RNF8, RNF168 or BARD1 or with a non-targeting control siRNA (CTRL). 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX is shown in (A). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (B).


( C , D ) Parental ( WT ) RPE1 - hTERT p53 - / - Cas9 , RNF8 - / - , RNF168 - / - were treated with either a non - targeting siRNA pool ( CTRL ) or a pool targeting RAP80 . 48 h post - transfection were irradiated ( 2 Gy ) processed for immunofluorescence 1 h post - IR treatment using antibodies against BRCA1 γH2AX . Quantitation of of with > 10 BRCA1 foci that colocalize with γH2AX is shown in ( C ) . A minimum of 100 per replicate were analyzed bars represent mean ± S . D . ( n = 3 biological replicates ) . Representative are shown in ( D ) . scale bars are 5 μm .

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

(C, D) Parental (WT) RPE1-hTERT p53-/- Cas9, RNF8-/-, and RNF168-/- cells were treated with either a non-targeting siRNA pool (CTRL) or a pool targeting RAP80. 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 10 BRCA1 foci that colocalize with γH2AX is shown in (C). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.


( A , B ) U2OS Flp - In / T - REx with integrated transgenes encoding GFP - BRCA1BRCT or - BRCA1BRCT S1655A were transfected with non - targeting siRNA ( CTRL ) or siRNA targeting RAP80 or BRCA1 . Following doxycycline treatment to induce transgene expression ( 5 μg / mL , 24 h ) , were irradiated ( 10 Gy ) processed for immunofluorescence 1 h post - IR for GFP antibodies against RAP80 γH2AX . Shown in ( A ) is quantitation of a minimum of 100 per replicate where bars represent mean ± S . D ( n = 4 ) . Representative are shown in ( B ) .

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

(A, B) U2OS Flp-In/T-REx cells with integrated transgenes encoding GFP-BRCA1BRCT or -BRCA1BRCT S1655A were transfected with non-targeting siRNA (CTRL) or siRNA targeting RAP80 or BRCA1. Following doxycycline treatment to induce transgene expression (5 μg/mL, 24 h), cells were irradiated (10 Gy) and processed for immunofluorescence 1 h post-IR for GFP and antibodies against RAP80 and γH2AX. Shown in (A) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=4). Representative micrographs are shown in (B).


( C , D ) U2OS Flp - In / T - REx stably integrated with indicated transgenes were treated with doxycycline ( 5 μg / mL , 36 h ) to induce expression transfected with an siRNA targeting BRCA1 also either non - targeting siRNA ( CTRL ) or siRNAs targeting RAP80 . 1 h post - irradiation ( 10 Gy ) were processed for immunofluorescence using GFP ( to detect BRCA1 fusions ) antibodies against γH2AX . Shown in ( C ) is quantitation of a minimum of 100 per replicate where bars represent mean ± S . D ( n = 3 biological replicates ) . Representative are shown in ( D ) . scale bars are 5 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 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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) U2OS Flp-In/T-REx cells stably integrated with the indicated transgenes were treated with doxycycline (5 μg/mL, 36 h) to induce protein expression and transfected with an siRNA targeting BRCA1 and also either non-targeting siRNA (CTRL) or siRNAs targeting RAP80. 1 h post-irradiation (10 Gy) cells were processed for immunofluorescence using GFP (to detect the BRCA1 fusions) and antibodies against γH2AX. Shown in (C) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.


( A , B ) U2OS 2 - 6 - 3 parental ( WT ) or RAP80 - / - lines were transfected with siRNAs targeting BRCA1 followed by nucleofection of indicated GFP fusion . 48 h post - nucleofection , mCherry - LacR - FokI expression was induced for 5 h prior to being processed for microscopy for GFP mCherry . Shown in ( A ) is quantitation of GFP at mCherry focus where bars represent mean ± S . D ( n = 50 , 50 , 40 , 40 , 40 , 40 , 20 , 20 , 30 , 30 analyzed from 3 biological replicates ) . Representative for BRCA1 BRCA1 - I26A conditions are shown in ( B ) . Additional for other conditions are in Figure EV2C .

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

(A, B) U2OS 2-6-3 parental (WT) or RAP80-/- cell lines were transfected with siRNAs targeting BRCA1 followed by nucleofection of the indicated GFP fusion proteins. 48 h post-nucleofection, mCherry-LacR-FokI expression was induced for 5 h prior to being processed for fluorescence microscopy for GFP and mCherry. Shown in (A) is the quantitation of GFP fluorescence at the mCherry focus where the bars represent the mean ± S.D (n= 50, 50, 40, 40, 40, 40, 20, 20, 30, 30 cells analyzed from 3 biological replicates). Representative micrographs for the BRCA1 and BRCA1-I26A conditions are shown in (B). Additional micrographs for the other conditions are in Figure EV2C.