Laser - induced mitochondrial membrane depolarization in L6 myoblasts shows gradual depolarization of elements along the IMM , visualized using live - cell Airyscan microscopy . Representative images of laser - induced depolarization time series from L6 myoblast , stained with ΔΨm - dependent dye , Rho123 . Images show LUT color - coded for Rho123 FI . White and blue pixels represent most and least intense ΔΨm , respectively . See legend of LUT colors on the right . Note that at the first time point ( ~ 0 . 15 sec ) white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2 - photon laser ( ≤ 5 msec ) , inducing depolarization . Ensuing frames show wavelike depolarization away from site of perturbation ( arrowheads ) . Scale bar = 500 nm . N = 3 independent experiments . ( See Movie EV 2 for time series . ) Profile plot showing Rho123 pixel intensity as function of distance along mitochondrion in A . Note that zero value in the X axis corresponds to top area of the mitochondrion shown in A , near site of 2 - photon laser exposure ( box ) . Red line corresponds to frame immediately before depolarization ( ~ 0 . 15 sec ) ; green , blue , purple , and dark purple lines correspond to ~ 0 . 30 , ~ 0 . 60 , ~ 1 . 05 , and ~ 2 . 10 sec , respectively , after initial depolarization , showing wavelike dissipation of ΔΨm over time .

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

Laser-induced mitochondrial membrane depolarization in L6 myoblasts shows gradual depolarization of elements along the IMM, visualized using live-cell Airyscan microscopy. Representative images of laser-induced depolarization time series from L6 myoblast, stained with ΔΨm-dependent dye, Rho123. Images show LUT color-coded for Rho123 FI. White and blue pixels represent most and least intense ΔΨm, respectively. See legend of LUT colors on the right. Note that at the first time point (~0.15 sec) white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Ensuing frames show wavelike depolarization away from site of perturbation (arrowheads). Scale bar = 500 nm. N = 3 independent experiments. (See Movie EV 2 for time series.) Profile plot showing Rho123 pixel intensity as function of distance along mitochondrion in A. Note that zero value in the X axis corresponds to top area of the mitochondrion shown in A, near site of 2-photon laser exposure (box). Red line corresponds to frame immediately before depolarization (~0.15 sec); green, blue, purple, and dark purple lines correspond to ~0.30, ~0.60, ~1.05, and ~2.10 sec, respectively, after initial depolarization, showing wavelike dissipation of ΔΨm over time.


Laser - induced mitochondrial membrane depolarization in living human skin fibroblasts co - stained with TMRE and MTG and imaged with Airyscan , showing gradual depolarization of elements along the IMM . Time - lapse images of merged green ( MTG ) and red ( TMRE ) channels following laser - induced mitochondrial membrane depolarization . Note that , at the first time point ( 0 . 3 sec ) , white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2 - photon laser ( ≤ 5 msec ) , inducing depolarization . Depolarization is marked by the dissipation of the ΔΨm - dependent dye ( TMRE ) while the green ( MTG ) signal persists . Scale bar = 1 µm . N = 4 independent experiments . TMRE channel from C , showing gradual of the loss of TMRE along the IMM . Note that although depolarization has largely completed at the area near the phototoxic stimulus already at 0 . 9 sec , some specific regions at the very top maintain ΔΨm while others depolarize . Quantification of ΔΨm using TMRE FI from C - D . Measurement of TMRE pixel intensities immediately after laser - induced depolarization at site distal ( ≥ 10 µm ) vs . proximal ( ≤ 1 µm ) to box . N = 4 independent experiments ; see specific p values in panel . Quantification of C - D . Percentage of mitochondria that depolarize in a wavelike ( i . e . , gradual ) vs . instantaneous manner after laser - induced depolarization . Note : imaging at high temporal resolution ( ~ 100 - 500 msec / frame ) reveals wavelike depolarizations predominate , suggesting the ΔΨm is composed of multiple , disparate electrochemical domains along the IMM . The time scale of propagation of depolarization is slower than the propagation of electrical phenomena . N = 4 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Laser-induced mitochondrial membrane depolarization in living human skin fibroblasts co-stained with TMRE and MTG and imaged with Airyscan, showing gradual depolarization of elements along the IMM. Time-lapse images of merged green (MTG) and red (TMRE) channels following laser-induced mitochondrial membrane depolarization. Note that, at the first time point (0.3 sec), white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Depolarization is marked by the dissipation of the ΔΨm-dependent dye (TMRE) while the green (MTG) signal persists. Scale bar = 1 µm. N = 4 independent experiments. TMRE channel from C, showing gradual of the loss of TMRE along the IMM. Note that although depolarization has largely completed at the area near the phototoxic stimulus already at 0.9 sec, some specific regions at the very top maintain ΔΨm while others depolarize. Quantification of ΔΨm using TMRE FI from C-D. Measurement of TMRE pixel intensities immediately after laser-induced depolarization at site distal (≥ 10 µm) vs. proximal (≤ 1 µm) to box. N = 4 independent experiments; see specific p values in panel. Quantification of C-D. Percentage of mitochondria that depolarize in a wavelike (i.e., gradual) vs. instantaneous manner after laser-induced depolarization. Note: imaging at high temporal resolution (~100-500 msec/frame) reveals wavelike depolarizations predominate, suggesting the ΔΨm is composed of multiple, disparate electrochemical domains along the IMM. The time scale of propagation of depolarization is slower than the propagation of electrical phenomena. N = 4 independent experiments.


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 .

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

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

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

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 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 .

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

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 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 .

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

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 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 ) .

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

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

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

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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 ) and STORM image ( right ) of MTs and 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 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 .

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

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 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 .

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

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

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

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 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 .

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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 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 .

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

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 line mark the cell 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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 B-CONTROLLED_VAR 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

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 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 .

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

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 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 .

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

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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells upon expression of RNF43 WT or R519X . Cells 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 cells expressing the 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 analysis of RNF43 WT and R519X for cytoplasmic β - catenin levels .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ;

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 - 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 - 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 ) .

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

(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 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 .

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

(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 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 .

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

(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 , 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 .

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 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

(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 the typical severity of the rough - eye phenotype . Data information : Representative phenotypes are shown in all 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 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 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 .

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

(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 ) 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 .

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 B-MEASURED_VAR 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

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 ) 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 .

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 O B-MEASURED_VAR 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 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

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 phenotypes of wild - type ( WT ) mice shown macroscopically The phenotypes 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 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 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 .

O 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 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

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 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 .

O O O O O O O O O O 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 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 . DNA 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 B-MEASURED_VAR 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 ) 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 .

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 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 . 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 .

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 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 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 .

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 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

(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.