Figure 6 ( A ) Effect of flg22 on the NbFLS2 - GmLMM1 interaction . The indicated constructs were transiently expressed in N . benthamiana by Agrobacterium - mediated transient expression for 2 days , and the leaves were treated with flg22 ( 1 μM ) or water for 10 min before being subjected to Co - IP assay . Data information : The experiments were performed three times , as biological replicates , with similar results . ( B ) Regulation of NbBAK1 - GmLMM1 interaction by flg22 . Data information : The experiments were performed three times as biological replicates , with similar results . ( C ) Suppression of flg22 - induced NbFLS2 - NbBAK1 interaction by GmLMM1 . NbFLS2 and NbBAK1 were co - expressed in N . benthamiana along with GmLMM1 or GmLMM1L407H , treated with H2O or flg22 and subjected to co - IP assays . Data information : The experiments were performed three times as biological replicates , with similar results . ( D Suppression effect of GmLMM1 on the flg22 - induced interaction of GmFLS2 with GmBAK1a ( D ) Data information : The experiments were performed two times ( D as biological replicates , with similar results . E ) Suppression effect of GmLMM1 on the flg22 - induced interaction of GmFLS2 with GmBAK1b ( E ) . Data information : The experiments were performed two times E ) , as biological replicates , with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Effect of flg22 on the NbFLS2-GmLMM1 interaction. The indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transient expression for 2 days, and the leaves were treated with flg22 (1 μM) or water for 10 min before being subjected to Co-IP assay. Data information: The experiments were performed three times , as biological replicates, with similar results.(B) Regulation of NbBAK1-GmLMM1 interaction by flg22. Data information: The experiments were performed three times as biological replicates, with similar results.(C) Suppression of flg22-induced NbFLS2-NbBAK1 interaction by GmLMM1. NbFLS2 and NbBAK1 were co-expressed in N. benthamiana along with GmLMM1 or GmLMM1L407H, treated with H2O or flg22 and subjected to co-IP assays. Data information: The experiments were performed three times as biological replicates, with similar results.(D Suppression effect of GmLMM1 on the flg22-induced interaction of GmFLS2 with GmBAK1a (D) Data information: The experiments were performed two times (D as biological replicates, with similar results.E) Suppression effect of GmLMM1 on the flg22-induced interaction of GmFLS2 with GmBAK1b (E). Data information: The experiments were performed two times E), as biological replicates, with similar results.


Fig . 1 ( B ) Growth kinetics of PiCoVacc ( CN2 ) P5 stock in Vero cells . ( D ) Protein composition and purity evaluation of PiCoVacc by NuPAGE 4 - 12 % Bis - Tris Gel . ( E ) Representative electron micrograph of PiCoVacc .

O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O

Fig. 1(B) Growth kinetics of PiCoVacc (CN2) P5 stock in Vero cells.(D) Protein composition and purity evaluation of PiCoVacc by NuPAGE 4-12% Bis-Tris Gel.(E) Representative electron micrograph of PiCoVacc.


Fig . 2isolates . BALB / c mice and Wistar rats were immunized with various doses of PiCoVacc or control ( adjuvant only as the sham group ) ( n = 10 ) . Serums from recovered COVID19 patients ( RECOV ) and non - infected people ( NI ) act as positive and negative controls , respectively . The antibody responses were analyzed in mice ( A ) , humans ( B ) and rats ( C ) . Top : SARS - CoV - 2 - specific IgG response measured by ELISAThe spectrum of neutralizing activities elicited by PiCoVacc was investigated in mice ( D ) and rats ( E ) . Neutralization assays against the other 9 isolated SARS - CoV - 2 strains were performed using mouse and rat serums collected 3 weeks post vaccination . Points represent individual animals and humans ; dotted lines indicate the limit of detection ; horizontal lines indicate the geometric mean titer ( GMT ) of EC50 for each group .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig. 2isolates.BALB/c mice and Wistar rats were immunized with various doses of PiCoVacc or control (adjuvant only as the sham group) (n=10). Serums from recovered COVID19 patients (RECOV) and non-infected people (NI) act as positive and negative controls, respectively. The antibody responses were analyzed in mice (A), humans (B) and rats (C). Top: SARS-CoV-2-specific IgG response measured by ELISAThe spectrum of neutralizing activities elicited by PiCoVacc was investigated in mice (D) and rats (E). Neutralization assays against the other 9 isolated SARS-CoV-2 strains were performed using mouse and rat serums collected 3 weeks post vaccination. Points represent individual animals and humans; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC50 for each group.


Fig . 3primates . Macaques were immunized three times through the intramuscular route with various doses of PiCoVacc or adjuvant only ( sham ) or placebo ( n = 4 ) . SARS - CoV - 2 - specific IgG response ( A ) and neutralizing antibody titer ( B ) were measured . Points represent individual macaques ; dotted lines indicate the limit of detection ; horizontal lines indicate the geometric mean titer ( GMT ) of EC50 for each group . Protective efficacy of PiCoVacc against SARS - CoV - 2 challenge at week 3 after immunization was evaluated in macaques ( C - F ) . Viral loads of throat ( C ) and anal ( D ) swab specimens collected from the inoculated macaques at day 3 , 5 and 7 pi were monitored . Viral loads in various lobe of lung tissue from all the inoculated macaques at day 7 post infection were measured ( E ) . RNA was extracted and viral load was determined by qRT - PCR . All data are presented as mean ± SEM . Histopathological examinations ( F ) in lungs from all the inoculated macaques at day 7 post infection . Lung tissue was collected and stained with hematoxylin and eosin .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig. 3primates.Macaques were immunized three times through the intramuscular route with various doses of PiCoVacc or adjuvant only (sham) or placebo (n=4). SARS-CoV-2-specific IgG response (A) and neutralizing antibody titer (B) were measured. Points represent individual macaques; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC50 for each group. Protective efficacy of PiCoVacc against SARS-CoV-2 challenge at week 3 after immunization was evaluated in macaques(C-F). Viral loads of throat (C) and anal (D) swab specimens collected from the inoculated macaques at day 3, 5 and 7 pi were monitored. Viral loads in various lobe of lung tissue from all the inoculated macaques at day 7 post infection were measured (E). RNA was extracted and viral load was determined by qRT-PCR. All data are presented as mean ± SEM.Histopathological examinations (F) in lungs from all the inoculated macaques at day 7 post infection. Lung tissue was collected and stained with hematoxylin and eosin.


Fig . 4primates . Macaques were immunized three times at day 0 , 7 and 14 through the intramuscular route with low dose ( 1 . 5 μg per dose ) or high dose ( 6 μg per dose ) of PiCoVacc or adjuvant only ( sham ) or placebo . ( A and B ) Hematological analysis in all four groups of macaques ( n = 4 ) . Lymphocyte subset percents ( A ) , including CD3 + , CD4 + and CD8 + were monitored at day − 1 ( 1 day before vaccination ) , 18 ( 3 day after the second vaccination ) and 29 ( 7 day after the third vaccination ) . Key cytokines ( B ) , containing TNF - α , IFN - γ and IL - 2 were examined at day − 1 , 1 ( the day for the first vaccination ) , 4 , 18 and 29 after vaccination . Values are mean ± s . d . ( C ) Histopathological evaluations in lungs from four groups of macaques at day 29 . Lung tissue was collected and stained with hematoxylin and eosin .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig. 4primates.Macaques were immunized three times at day 0, 7 and 14 through the intramuscular route with low dose (1.5 μg per dose) or high dose (6 μg per dose) of PiCoVacc or adjuvant only (sham) or placebo. (A and B) Hematological analysis in all four groups of macaques (n=4). Lymphocyte subset percents (A), including CD3+, CD4+ and CD8+ were monitored at day −1 (1 day before vaccination), 18 (3 day after the second vaccination) and 29 (7 day after the third vaccination). Key cytokines (B), containing TNF-α, IFN-γ and IL-2 were examined at day −1, 1 (the day for the first vaccination), 4, 18 and 29 after vaccination. Values are mean ± s.d.(C) Histopathological evaluations in lungs from four groups of macaques at day 29. Lung tissue was collected and stained with hematoxylin and eosin.


Figure 1Left : 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 . 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 .

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Figure 1Left: 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.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.


Figure 2Representative 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative 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.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.


Figure 3Five 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Five 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.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.


Figure 4A 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 .

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Figure 4A 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.


Figure 5Random 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 . 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 .

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Figure 5Random 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.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.


Figure 6Projections 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 . 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 . 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 .

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Figure 6Projections 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.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.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.


Figure 1Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Body weight curve . Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Cumulative body weight . Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Fasting and fed blood glucose . Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Rectal temperature . Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Locomotor tolerance . Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . 24 - h oxygen consumption . Respiratory exchange ratio ( RER : VCO2 / VO2 ) . Energy expenditure .

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Figure 1Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Body weight curve.Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Cumulative body weight.Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Fasting and fed blood glucose.Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Rectal temperature.Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Locomotor tolerance.Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. 24-h oxygen consumption. Respiratory exchange ratio (RER: VCO2/VO2). Energy expenditure.


Figure 2Eight - week - old male WT and DOCK5 - / - mice were fed a SD or HFD for 3 months . Blood glucose levels and area under curve ( AUC ) during the glucose tolerance test . Blood glucose levels and AUC during the insulin tolerance test . Eight - week - old male WT and DOCK5 - / - mice were fed a SD or HFD for 3 months . Glucose infusion rates ( GIR ) . Glucose disposal rate ( GDR ) . Hepatic glucose production ( HGP ) . Eight - week - old male WT and DOCK5 - / - mice were fed a SD or HFD for 3 months . Percentage of suppression of hepatic glucose production ( HGP ) . Eight - week - old male WT and DOCK5 - / - mice Glucose infusion rates ( GIR ) during lipid infusion .

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Figure 2Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Blood glucose levels and area under curve (AUC) during the glucose tolerance test. Blood glucose levels and AUC during the insulin tolerance test.Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP).Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Percentage of suppression of hepatic glucose production (HGP).Eight-week-old male WT and DOCK5-/- mice Glucose infusion rates (GIR) during lipid infusion.


Figure 3Eight - week - old male WT and DOCK5 - / - mice were fed a SD or a HFD for 3 months . Mice were treated with insulin ( 1 U / kg ) or control ( PBS ) by intraperitoneal injection for 10 minutes ; animals were killed and livers were collected . The expression of expression of phosphoenolpyruvate carboxykinase ( PEPCK ) and glucose - 6 - phosphatase ( G6Pase ) mRNA and protein . Eight - week - old male WT and DOCK5 - / - mice were fed a SD or a HFD for 3 months . Mice were treated with insulin ( 1 U / kg ) or control ( PBS ) by intraperitoneal injection for 10 minutes ; animals were killed and livers were collected . Total and phosphorylated insulin receptor ( InsR ) , IRS - 1ser1101 and Akt . Eight - week - old male WT and DOCK5 - / - mice were fed a SD or a HFD for 3 months . Mice were treated with insulin ( 1 U / kg ) or control ( PBS ) by intraperitoneal injection for 10 minutes ; animals were killed and livers were collected . Total and phosphorylated mTOR , S6K1 , and the levels of Raptor and Rictor protein .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. The expression of expression of phosphoenolpyruvate carboxykinase(PEPCK) and glucose-6- phosphatase(G6Pase) mRNA and protein.Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. Total and phosphorylated insulin receptor (InsR), IRS-1ser1101 and Akt.Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. Total and phosphorylated mTOR, S6K1, and the levels of Raptor and Rictor protein.


Figure 4Hep1 - 6 cells were infected with pCDNA3 . 1 - DOCK5 ( DOCK5 + ) or pCDNA3 . 1 ( DOCK5 - ) for 48 h . mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . The mRNA and protein expression of PEPCK and G6Pase in Hep1 - 6 cells . Hep1 - 6 cells were infected with pCDNA3 . 1 - DOCK5 ( DOCK5 + ) or pCDNA3 . 1 ( DOCK5 - ) for 48 h . - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt in Hep1 - 6 cells . Hep1 - 6 cells were infected with pCDNA3 . 1 - DOCK5 ( DOCK5 + ) or pCDNA3 . 1 ( DOCK5 - ) for 48 h . mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated mTOR and S6K1 , and the levels of Raptor and Rictor protein in Hep1 - 6 cells . MPHs isolated from WT and DOCK5 - / - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . The mRNA and protein expression of PEPCK and G6Pase in MPHs . MPHs isolated from WT and DOCK5 - / - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt in MPHs . MPHs isolated from WT and DOCK5 - / - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated mTOR and S6K1 , and the levels of Raptor and Rictor protein in MPHs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. The mRNA and protein expression of PEPCK and G6Pase in Hep1-6 cells.Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. - mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in Hep1-6 cells.Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in Hep1-6 cells.MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. The mRNA and protein expression of PEPCK and G6Pase in MPHs.MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in MPHs.MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in MPHs.


Figure 5Eight - week - old male Raptor flox / flox mice ( Raptorflox / flox Cre + ) were fed a HFD for 12 weeks and injected with AAV8 - shDOCK5 ± AAV8 - Cre or AAV8 - GFP ( at a dose of 3 × 1011 vg / 200 μL / mouse ) via the tail - vein 14 days prior to the in vivo study . Fasting and fed blood glucose 14 days post - infection . Eight - week - old male Raptor flox / flox mice ( Raptorflox / flox Cre + ) were fed a HFD for 12 weeks and injected with AAV8 - shDOCK5 ± AAV8 - Cre or AAV8 - GFP ( at a dose of 3 × 1011 vg / 200 μL / mouse ) via the tail - vein 14 days prior to the in vivo study . Blood glucose levels and AUC during glucose tolerance tests . Blood glucose levels and AUC during insulin tolerance tests . Glucose infusion rates ( GIR ) . Glucose disposal rate ( GDR ) . Hepatic glucose production ( HGP ) . Percentage of suppression of hepatic glucose production ( HGP ) .

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Figure 5Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Fasting and fed blood glucose 14 days post-infection.Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Blood glucose levels and AUC during glucose tolerance tests. Blood glucose levels and AUC during insulin tolerance tests. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP). Percentage of suppression of hepatic glucose production (HGP).


Figure 6Male Raptor flox / flox mice ( 8 weeks ) were fed a HFD for 12 weeks and injected with AAV8 - GFP or AAV8 - shDOCK5 or AAV8 - shDOCK5 + AAV8 - Cre via the tail vein . The mRNA and protein expression of PEPCK and G6Pase in the liver . Male Raptor flox / flox mice ( 8 weeks ) were fed a HFD for 12 weeks and injected with AAV8 - GFP or AAV8 - shDOCK5 or AAV8 - shDOCK5 + AAV8 - Cre via the tail vein . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt in the liver . Male Raptor flox / flox mice ( 8 weeks ) were fed a HFD for 12 weeks and injected with AAV8 - GFP or AAV8 - shDOCK5 or AAV8 - shDOCK5 + AAV8 - Cre via the tail vein . The levels of DOCK5 and Raptor protein , and total and phosphorylated mTOR and S6K1 in the liver . MPHs from Raptor flox / flox mice were infected with Ad - GFP or Ad - shDOCK5 or Ad - shDOCK5 + Ad - Cre . The lysates were immunoblotted with indicated antibodies or β - actin . The expression of PEPCK and G6Pase mRNA and protein . MPHs from Raptor flox / flox mice were infected with Ad - GFP or Ad - shDOCK5 or Ad - shDOCK5 + Ad - Cre . The lysates were immunoblotted with indicated antibodies or β - actin . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt . MPHs from Raptor flox / flox mice were infected with Ad - GFP or Ad - shDOCK5 or Ad - shDOCK5 + Ad - Cre . The lysates were immunoblotted with indicated antibodies or β - actin . The levels of DOCK5 and Raptor protein , and total and phosphorylated mTOR and S6K1 .

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Figure 6Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. The mRNA and protein expression of PEPCK and G6Pase in the liver.Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in the liver.Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1 in the liver.MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. The expression of PEPCK and G6Pase mRNA and protein.MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. Total and phosphorylated InsR, IRS-1Ser1101 and Akt.MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1.


Figure 7Rac1 GEF activation in MPHs from WT and DOCK5 - / - mice . SEW2871 treatment promotes Rac1 GEF activation and eliminates the influence of a DOCK5 deficiency on Raptor / S6K1 signaling in the mouse primary hepatocytes ( MPHs ) of DOCK5 - / - mice . Total and phosphorylated mTOR and S6K1 and the level of Raptor protein expression in plasmids expressing a WT DOCK5 or mutant DOCK5 treated - Hep1 - 6 cells . Coimmunoprecipitation ( Co - IP ) analysis of DOCK5 and Raptor expression in Hepa1 - 6 cells . After transfected with indicated plasmids , lysates from cells were immunoprecipitated ( IP ) with anti - DOCK5 antibody or normal IgG , and then immunoblotted ( IB ) with the indicated antibody . DOCK5 binding to Raptor fragments ( 445 - 887 aa ) during transient expression in Hepa1 - 6 cells . The DOCK5 and Raptor binding sites were identified using mutant DOCK5 ( DHR - 2V1559A ) and Raptor fragments ( 445 - 887 aa ) based on a Co - IP assay . VA , mutant DOCK5 DHR - 2 ; His , His - DHR - 2 .

O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Rac1 GEF activation in MPHs from WT and DOCK5-/- mice.SEW2871 treatment promotes Rac1 GEF activation and eliminates the influence of a DOCK5 deficiency on Raptor / S6K1 signaling in the mouse primary hepatocytes (MPHs) of DOCK5-/- mice.Total and phosphorylated mTOR and S6K1 and the level of Raptor protein expression in plasmids expressing a WT DOCK5 or mutant DOCK5 treated-Hep1-6 cells.Coimmunoprecipitation (Co-IP) analysis of DOCK5 and Raptor expression in Hepa1-6 cells. After transfected with indicated plasmids, lysates from cells were immunoprecipitated (IP) with anti-DOCK5 antibody or normal IgG, and then immunoblotted (IB) with the indicated antibody.DOCK5 binding to Raptor fragments (445 - 887 aa) during transient expression in Hepa1-6 cells.The DOCK5 and Raptor binding sites were identified using mutant DOCK5 (DHR-2V1559A) and Raptor fragments (445 - 887 aa) based on a Co-IP assay. VA, mutant DOCK5 DHR-2; His, His-DHR-2.


Figure 1A ) Maximum intensity projections of pediatric ( 0 - 12 months ) small intestine showing the distribution of CD56 , CD90 and CD24 within the ENS . The first image of each row shows a low magnification overview and an inset depicting the DAPI channel in high magnification . The remaining panels correspond to the high magnification inset . Scale bar in overview images = 200 µm . Scale bar in high magnification panels = 40 µm . ( F ) RT - qPCR from CD56 + CD90 + sorted cells vs control non - ENS cells for representative neuronal and glial markers , confirms high enrichment of both neurons and glial cells ( mean ± SD , N = 4 biological replicates , P * < 0 . 05 , P * * * < 0 . 001 , multiple Student ' s t test , FDR correction for multiple comparisons ) .

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Figure 1A) Maximum intensity projections of pediatric (0-12 months) small intestine showing the distribution of CD56, CD90 and CD24 within the ENS. The first image of each row shows a low magnification overview and an inset depicting the DAPI channel in high magnification. The remaining panels correspond to the high magnification inset. Scale bar in overview images = 200 µm. Scale bar in high magnification panels = 40 µm.(F) RT-qPCR from CD56+CD90+ sorted cells vs control non- ENS cells for representative neuronal and glial markers, confirms high enrichment of both neurons and glial cells (mean±SD, N=4 biological replicates, P * < 0.05, P *** < 0.001, multiple Student's t test, FDR correction for multiple comparisons).


Figure 2B ) Representative plot showing full co - expression of TUBB3 ( neuronal marker ) and SOX10 ( glial marker ) , on ENS cells ( bottom ) and only minimal and low - intensity background on non - ENS cells ( Top ) ( N = 1 representative sample , for stats see Figure 2C ) . Intracellular staining was performed on formalin fixed cells from ileum , after pre - staining for the extracellular markers . Selection of the ENS cluster in fixed cells is shown in Appendix Figure S2F . C ) Percentage of TUBB3 + and SOX10 + cells in ENS and non - ENS cells ( mean ± SD , TUBB3 : N = 4 biological replicates , SOX10 : N = 3 biological replicates ) . The single markers TUBB3 and SOX10 were gated vs SSC - A as shown in Appendix Figure S2G .

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Figure 2B) Representative plot showing full co-expression of TUBB3 (neuronal marker) and SOX10 (glial marker), on ENS cells (bottom) and only minimal and low-intensity background on non-ENS cells (Top) (N=1 representative sample, for stats see Figure 2C). Intracellular staining was performed on formalin fixed cells from ileum, after pre-staining for the extracellular markers. Selection of the ENS cluster in fixed cells is shown in Appendix Figure S2F.C) Percentage of TUBB3+ and SOX10+ cells in ENS and non-ENS cells (mean±SD, TUBB3: N=4 biological replicates, SOX10: N=3 biological replicates). The single markers TUBB3 and SOX10 were gated vs SSC-A as shown in Appendix Figure S2G.


Figure 3B ) The distribution of ENS cells and non - nucleated particles is represented in a CD56 / CD90 plot . Left : initial selection of ENS cells . Right : zoom on the ENS cluster , where ENS events are shown as non - nucleated , nucleated , merge . For additional details and stats see Appendix Figure S3E . C ) ENS cells and non - nucleated particles are shown in a CD56 / CD24 plot , as in Figure 3B . Nucleated and non - nucleated events largely overlap and define the same subclusters in CD56 / CD24 and CD56 / CD90 plots , although the average staining intensity of the three markers is lower for non - nucleated events . For additional details and stats see Appendix Figure S3E .

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Figure 3B) The distribution of ENS cells and non-nucleated particles is represented in a CD56/CD90 plot. Left: initial selection of ENS cells. Right: zoom on the ENS cluster, where ENS events are shown as non-nucleated, nucleated, merge. For additional details and stats see Appendix Figure S3E.C) ENS cells and non-nucleated particles are shown in a CD56/CD24 plot, as in Figure 3B. Nucleated and non-nucleated events largely overlap and define the same subclusters in CD56/CD24 and CD56/CD90 plots, although the average staining intensity of the three markers is lower for non-nucleated events. For additional details and stats see Appendix Figure S3E.


Figure 4Confocal images of ENS events sorted as nucleated and non - nucleated , as shown in Figure . 3 . ( A ) Most of the staining belongs to what appears to be non - nucleated ( DAPI - ) cellular debris ( putative neuronal terminations ) attached to a nucleated cell ( putative glial cell ) . This is particularly true for TUBB3 and CD24 , which show ( as expected for neuronal selective markers ) a starker distinction than CD56 ( also expressed by glial cells ) . At a very low frequency among sorted ENS cells , we could observe putative neurons as represented in ( B ) , with a more homogenous distribution of the three markers . ( C ) Examples of sorted Hoechst - negative ENS debris , which are confirmed to be non - nucleated ( thus validating our gates on Hoechst 33342 - A ) , but positive for all neuronal markers .

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Figure 4Confocal images of ENS events sorted as nucleated and non-nucleated, as shown in Figure. 3. (A) Most of the staining belongs to what appears to be non-nucleated (DAPI-) cellular debris (putative neuronal terminations) attached to a nucleated cell (putative glial cell). This is particularly true for TUBB3 and CD24, which show (as expected for neuronal selective markers) a starker distinction than CD56 (also expressed by glial cells). At a very low frequency among sorted ENS cells, we could observe putative neurons as represented in (B), with a more homogenous distribution of the three markers. (C) Examples of sorted Hoechst-negative ENS debris, which are confirmed to be non-nucleated (thus validating our gates on Hoechst 33342-A), but positive for all neuronal markers.


Figure 5C ) RT - qPCR for neuronal markers on cells sorted for CD24TIP vs all the other ENS cells , confirms strong neuronal enrichment ( mean ± SD , N = 5 biological replicates , P * * * < 0 . 001 , multiple Student ' s t test , FDR correction for multiple comparisons ) . E ) Graph showing the proportion of the CD24 - based subclusters in the nucleated vs non - nucleated fraction of ganglionic and aganglionic colon ( mean ± SD , N = 4 biological replicates , P * * * < 0 . 001 , multiple Student ' s t test , FDR correction for multiple comparisons ) . Additional details and stats are shown in Appendix Figure S4E - J .

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Figure 5C) RT-qPCR for neuronal markers on cells sorted for CD24TIP vs all the other ENS cells, confirms strong neuronal enrichment (mean±SD, N=5 biological replicates, P *** < 0.001, multiple Student's t test, FDR correction for multiple comparisons).E) Graph showing the proportion of the CD24-based subclusters in the nucleated vs non-nucleated fraction of ganglionic and aganglionic colon (mean±SD, N=4 biological replicates, P *** < 0.001, multiple Student's t test, FDR correction for multiple comparisons). Additional details and stats are shown in Appendix Figure S4E-J.


Figure 6G ) Stacked violin plots showing enriched genes in specific neuronal subtypes .

Figure 6G) Stacked violin plots showing enriched genes in specific neuronal subtypes.


Figure 1A . Western blot analysis of Tace using lysates from WT and TaceFl / Fl ; P0 - Cre mousesciatic nerves at P10 ( 65ug of total protein lysate loaded ) . The arrowhead indicates the pro - protein and the arrow shows the cleaved active form of Tace . Representative of two independent experiments . B . Western blot analysis ( 50ug of total protein lysate loaded ) shows that Tace expression in axons is maximal at P5 - P10 and then progressively declines in the adult . Representative of two independent experiments . C . Western blot analysis of Tace in Mtmr2 - / - sciatic nerves at P5 , P10 , and P20 . Representative of two independent experiments . D . Western blot analysis of Tace using lysates from Mtmr2 - / - mouse sciatic nerves at 5 months , with quantification , p = 0 . 10 , two - tailed Mann Whitney U test . Representative of two independent experiments . E . Phosphorylation of ErbB2 in Mtmr2 - / - sciatic nerves at P2 , P10 and P15 . At P2 , each lane is a pool of n = 7 animals per genotype , representative of six independent experiments using n = 6 different nerve pools per genotype , and quantification using WT values arbitrary set to1 , p = 0 . 0313 , Wilcoxon Rank Sum test . At P10 and P15 , mean values of two samples are shown . At P10 , each lane is a pool of n = 3 / 4 animals per genotype , representative of three independent experiments using n = 6 different pools per genotype . Nf - l is the neurofilament light chain and Clx is calnexin . F . Phosphorylation of ErbB2 in Mtmr2 - / - Schwann cell / DRG neuron co - cultures after 4 days of ascorbic acid treatment . Each lane is a pool from at least 10 coverslips / DRG per genotype . Representative of two independent experiments using n = 4 different pools of coverslips / DRG per genotype , p = 0 . 20 , two - tailed Mann Whitney U test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Western blot analysis of Tace using lysates from WT and TaceFl/Fl;P0-Cre mousesciatic nerves at P10 (65ug of total protein lysate loaded). The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments.B. Western blot analysis (50ug of total protein lysate loaded) shows that Tace expression in axons is maximal at P5-P10 and then progressively declines in the adult. Representative of two independent experiments.C. Western blot analysis of Tace in Mtmr2-/- sciatic nerves at P5, P10, and P20. Representative of two independent experiments.D. Western blot analysis of Tace using lysates from Mtmr2-/- mouse sciatic nerves at 5 months, with quantification, p=0.10, two-tailed Mann Whitney U test. Representative of two independent experiments.E. Phosphorylation of ErbB2 in Mtmr2-/- sciatic nerves at P2, P10 and P15. At P2, each lane is a pool of n=7 animals per genotype, representative of six independent experiments using n=6 different nerve pools per genotype, and quantification using WT values arbitrary set to1, p=0.0313, Wilcoxon Rank Sum test. At P10 and P15, mean values of two samples are shown. At P10, each lane is a pool of n=3/4 animals per genotype, representative of three independent experiments using n=6 different pools per genotype. Nf-l is the neurofilament light chain and Clx is calnexin.F. Phosphorylation of ErbB2 in Mtmr2-/- Schwann cell/DRG neuron co-cultures after 4 days of ascorbic acid treatment. Each lane is a pool from at least 10 coverslips/DRG per genotype. Representative of two independent experiments using n=4 different pools of coverslips/DRG per genotype, p=0.20, two-tailed Mann Whitney U test.


Figure 2A . ShRNA targeting Nrg1 type III were validated in rat purified neurons . Western blot analysis for Nrg1 shows reduction of its expression ( pro - protein at 150 KDa ) using two different harpins , representative of two independent experiments . Clx is calnexin . B . ShRNA # 1 and 2 downregulate Nrg1 type III expression also in myelin - forming Schwann cell / DRG neuron co - cultures analyzed after 7 days of ascorbic acid treatment , as shown by western blot analysis , representative of two independent experiments . Clx is calnexin . C , D . Titration of shRNA # 1 ( C ) and of shRNA # 2 ( D ) LVs ( lentiviral vectors ) on WT Schwann cell / DRG neuron co - cultures analyzed after 7 days of ascorbic acid treatment , with quantification of the mean number of Mbp positive segments and Schwann cell number . In ( C ) n = 20 ( SCR , Scramble ) , n = 11 ( LV # sh1 , 12 . 5 % ) , n = 16 ( LV # sh1 , 25 % ) , n = 9 ( LV # sh1 , 50 % ) DRG / coverslips from two independent experiments . Results are mean ± SEM , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test . In ( D ) n = 14 ( SCR ) , n = 10 ( LV # sh2 , 12 . 5 % ) , n = 12 ( LV # sh2 , 25 % ) , n = 79 ( LV # sh2 , 50 % ) DRG / coverslips , representative of two independent experiments . Results are mean ± SEM , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test . E . Representative confocal images of Mtmr2 - / - co - cultures transduced using LV expressing Nrg1 type III shRNA # 1 , with quantification of the percentage of myelin outfoldings , n = number of DRG / coverslips , n = 9 , 382 fibers ( SCR ) , n = 9 , 368 fibers ( LV # sh1 , 12 . 5 % ) , n = 8 , 327 fibers ( LV # sh1 , 25 % ) . Asterisks indicate fibers with myelin outfoldings . Results are mean ± SEM , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test , representative of two independent experiments . E . Western blot analysis of Nrg1 and Akt phosphorylation ( S473 ) shows that activation of Akt decreases following Nrg1 type III downregulation ( p - Akt / Akt / Actin , ratio between Mtmr2 - / - Scramble - treated and Mtmr2 - / - sh # 1 - treated using 12 . 5 % LV is 1 : 0 . 85 ) . Each lane is a pool of at least 10 different DRG / coverslips , representative of two independent experiments . Bar in ( C , D ) is 100 μm ; in ( E ) is 20 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. ShRNA targeting Nrg1 type III were validated in rat purified neurons. Western blot analysis for Nrg1 shows reduction of its expression (pro-protein at 150 KDa) using two different harpins, representative of two independent experiments. Clx is calnexin.B. ShRNA#1 and 2 downregulate Nrg1 type III expression also in myelin-forming Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, as shown by western blot analysis, representative of two independent experiments. Clx is calnexin.C, D. Titration of shRNA #1 (C) and of shRNA #2 (D) LVs (lentiviral vectors) on WT Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, with quantification of the mean number of Mbp positive segments and Schwann cell number. In (C) n= 20 (SCR, Scramble), n= 11 (LV #sh1, 12.5%), n= 16 (LV #sh1, 25%), n= 9 (LV #sh1, 50%) DRG/coverslips from two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. In (D) n= 14 (SCR), n= 10 (LV #sh2, 12.5%), n= 12 (LV #sh2, 25%), n= 79 (LV #sh2, 50%) DRG/ coverslips, representative of two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.E. Representative confocal images of Mtmr2-/- co-cultures transduced using LV expressing Nrg1 type III shRNA #1, with quantification of the percentage of myelin outfoldings, n= number of DRG/coverslips, n= 9, 382 fibers (SCR), n=9, 368 fibers (LV #sh1, 12.5%), n= 8, 327 fibers (LV #sh1, 25%). Asterisks indicate fibers with myelin outfoldings. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test, representative of two independent experiments.E. Western blot analysis of Nrg1 and Akt phosphorylation (S473) shows that activation of Akt decreases following Nrg1 type III downregulation (p-Akt/Akt/Actin, ratio between Mtmr2-/- Scramble-treated and Mtmr2-/- sh#1-treated using 12.5% LV is 1 : 0.85). Each lane is a pool of at least 10 different DRG/coverslips, representative of two independent experiments. Bar in (C, D) is 100 μm; in (E) is 20 μm.


Figure 3A . Titration of rhTACE on wild - type ( WT ) co - cultures stained for Mbp with quantification of Mbp positive fibers and Schwann cell nuclei : n = 18 ( NT , not treated ) , n = 6 ( 5ng / ml ) , n = 20 ( 10ng / ml ) , n = 7 ( 50ng / ml ) DRGs / covers from two independent experiments , p = 0 . 0053 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test . B . Immunohistochemistry and confocal microscopy of Mtmr2 - / - cultures treated with 5ng / ml and 10ng / ml rhTACE , with quantification of the percentage of myelin outfoldings ; n = 10 ( NT ) , n = 10 ( 5ng / ml ) , n = 11 ( 10ng / ml ) , DRGs / coverslips . A total of 547 , 557 , and 571 Mbp fibers were quantified , respectively , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test . B . Western blot analysis of lysates from Mtmr2 - / - cultures treated using 10ng / ml rhTACE showing Nrg1 expression and Akt activation ( S473 phosphorylation ) . Each lane is a lysate of at least n = 10 DRGs / coverslips . In Mtmr2 - / - ( 10ng / ml rhTACE ) as compared to Mtmr2 - / - ( NT ) , Nrg1 ( 150 KDa ) / actin ratio is 0 . 8 : 1 ; Nrg1 ( 65 KDa ) / actin ratio is 0 . 48 : 1 ; and p - Akt / Akt / actin ratio is 0 . 6 : 1 . C . Titration of WT co - cultures using different amounts of Niacin , nicotinic acid , with quantification of Mbp positive fibers and Schwann cellnuclei , n = 9 ( NT ) , n = 8 ( 1mM ) , n = 9 ( 5mM ) , n = 8 ( 10mM ) , DRGs / coverslips . Representative of three independent experiments , p = 0 . 0055 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test . D . Tace activity was measured from lysates of Niacin - treated cultures , n = 7 ( KO NT ) and n = 8 ( KO 5mM Niacin ) number of independent plates , each containing from 10 - 15 DRGs plated . Note that Tace activity was similar between WT and Mtmr2 - / - cultures , one - tailed Mann Whitney U test , p = 0 . 037 . D . Immunohistochemistry and confocal microscopy of Mtmr2 - / - cultures treated with 1 mM and 5mM Niacin , with quantification of the percentage of myelin outfoldings ; n = 31 ( NT ) , n = 14 ( 1mM ) , and n = 29 ( 5mM ) DRGs / coverslips from two independent experiments . A total of 1256 , 489 , and 1305 fibers , respectively , were quantified , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test . E . Niacin treatment of Tace - / - explants does not rescue hypermyelination , with quantification , n = 8 DRGs / coverslips per condition , p = 0 . 0056 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test . Representative of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Titration of rhTACE on wild-type (WT) co-cultures stained for Mbp with quantification of Mbp positive fibers and Schwann cell nuclei: n=18 (NT, not treated), n=6 (5ng/ml), n=20 (10ng/ml), n=7 (50ng/ml) DRGs/covers from two independent experiments, p=0.0053, nonparametric one-way ANOVA, followed by Dunn's post hoc test.B. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 5ng/ml and 10ng/ml rhTACE, with quantification of the percentage of myelin outfoldings; n=10 (NT), n=10 (5ng/ml), n=11 (10ng/ml), DRGs/coverslips. A total of 547, 557, and 571 Mbp fibers were quantified, respectively, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.B. Western blot analysis of lysates from Mtmr2-/- cultures treated using 10ng/ml rhTACE showing Nrg1 expression and Akt activation (S473 phosphorylation). Each lane is a lysate of at least n=10 DRGs/coverslips. In Mtmr2-/- (10ng/ml rhTACE) as compared to Mtmr2-/- (NT), Nrg1(150 KDa)/actin ratio is 0.8:1; Nrg1(65 KDa)/actin ratio is 0.48:1; and p-Akt/Akt/actin ratio is 0.6:1.C. Titration of WT co-cultures using different amounts of Niacin, nicotinic acid, with quantification of Mbp positive fibers and Schwann cellnuclei, n=9 (NT), n=8 (1mM), n=9 (5mM), n=8 (10mM), DRGs/ coverslips. Representative of three independent experiments, p=0.0055, nonparametric one-way ANOVA, followed by Dunn's post hoc test.D. Tace activity was measured from lysates of Niacin-treated cultures, n=7 (KO NT) and n=8 (KO 5mM Niacin) number of independent plates, each containing from 10-15 DRGs plated. Note that Tace activity was similar between WT and Mtmr2-/- cultures, one-tailed Mann Whitney U test, p=0.037.D. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 1 mM and 5mM Niacin, with quantification of the percentage of myelin outfoldings; n=31 (NT), n=14 (1mM), and n=29 (5mM) DRGs/coverslips from two independent experiments. A total of 1256, 489, and 1305 fibers, respectively, were quantified, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.E. Niacin treatment of Tace-/- explants does not rescue hypermyelination, with quantification, n=8 DRGs/coverslips per condition, p=0.0056, nonparametric one-way ANOVA, followed by Dunn's post hoc test. Representative of three independent experiments.


Figure 4A . Representative images of semithin section analysis of sciatic nerves from Mtmr2 - / - and Mtmr2 - / - ; Nrg1 ( III ) + / - mice at 6 months . Myelin outfoldings ( arrowheads ) are reduced in Mtmr2 - / - ; Nrg1 ( III ) + / - nerves ( n = 9 ) as compared to Mtmr2 - / - ( n = 8 ) , results are mean ± SEM , p = 0 . 0037 , two - tailed Mann Whitney U test . B . G - ratio analysis on sciatic nerves from Mtmr2 - / - , Mtmr2 - / - ; Nrg1 ( III ) + / - , and Nrg1 ( III ) + / - mice at 6 months indicates that myelin thickness of Mtmr2 - / - ; Nrg1 ( III ) + / - is similar to Nrg1 ( III ) + / - nerves and different from Mtmr2 - / - , which is in the normal range as already reported ( Bonneick et al , 2005 ) , n = 5 animals per genotype . Mean g - ratios : Mtmr2 - / - 0 . 67 ± 0 . 03 , 2129 fibers , n = 5 animals ; Mtmr2 - / - ; Nrg1 ( III ) + / - 0 . 71 ± 0 . 03 , 2139 fibers , n = 5 animals ; Nrg1 ( III ) + / - 0 . 72 ± 0 . 03 , 2152 fibers , n = 4 animals . Mtmr2 - / - as compared to Mtmr2 - / - ; Nrg1 ( III ) + / - , p = 0 . 0145 ; Mtmr2 - / - as compared to Nrg1 ( III ) + / - , p = 0 . 0030 , and Mtmr2 - / - ; Nrg1 ( III ) + / - as compared to Nrg1 ( III ) + / - p = 0 . 3193 , repeated measures ANOVA . C . Western blot analysis on lysates from Mtmr2 - / - , Mtmr2 - / - ; Nrg1 ( III ) + / - , and Nrg1 ( III ) + / - sciatic nerves at P10 indicates that Akt activation ( S473 phosphorylation ) is similar between Mtmr2 - / - ; Nrg1 ( III ) + / - and Nrg1 ( III ) + / - consistent with g - ratio findings , representative of three independent experiments . Phosphorylation of Akt is relative to total Akt normalized on calnexin , Clx .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Representative images of semithin section analysis of sciatic nerves from Mtmr2-/- and Mtmr2-/-;Nrg1 (III)+/- mice at 6 months. Myelin outfoldings (arrowheads) are reduced in Mtmr2-/-;Nrg1 (III)+/- nerves (n=9) as compared to Mtmr2-/- (n=8), results are mean ± SEM, p=0.0037, two-tailed Mann Whitney U test.B. G-ratio analysis on sciatic nerves from Mtmr2-/-, Mtmr2-/-;Nrg1 (III)+/-, and Nrg1 (III)+/- mice at 6 months indicates that myelin thickness of Mtmr2-/-;Nrg1 (III)+/- is similar to Nrg1 (III)+/- nerves and different from Mtmr2-/-, which is in the normal range as already reported (Bonneick et al, 2005), n=5 animals per genotype.Mean g-ratios: Mtmr2-/- 0.67 ± 0.03, 2129 fibers, n=5 animals; Mtmr2-/-;Nrg1 (III)+/- 0.71 ± 0.03, 2139 fibers, n=5 animals; Nrg1 (III)+/- 0.72 ± 0.03, 2152 fibers, n=4 animals. Mtmr2-/- as compared to Mtmr2-/-;Nrg1 (III)+/-, p=0.0145; Mtmr2-/- as compared to Nrg1 (III)+/-, p=0.0030, and Mtmr2-/-;Nrg1 (III)+/- as compared to Nrg1 (III)+/- p=0.3193, repeated measures ANOVA.C. Western blot analysis on lysates from Mtmr2-/-, Mtmr2-/-;Nrg1 (III)+/-, and Nrg1 (III)+/- sciatic nerves at P10 indicates that Akt activation (S473 phosphorylation) is similar between Mtmr2-/-;Nrg1 (III)+/- and Nrg1 (III)+/- consistent with g-ratio findings, representative of three independent experiments. Phosphorylation of Akt is relative to total Akt normalized on calnexin, Clx.


Figure 5A . Western blot analysis on lysates from treated and not treated explants ( at least 10 DRGs / coverslips per lane ) shows that increased Akt activation ( S473 phosphorylation ) in Vim - / - explants is restored to normal levels following 5 mM Niacin treatment . A . Niacin treatment of Vim - / - explants rescues hypermyelination , n = 10 DRGs / coverslips per condition , with quantification , representative of three independent experiments , p = 0 . 0048 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test . B . Niaspan administration ( daily i . p . injection of 160mg / Kg Niaspan starting at P15 for 15 days ) enhances Tace activity in Vim - / - nerves at P30 , n = 6 animals per genotype , p = 0 . 0325 , one - tailed Mann Whitney U test . B . Niaspan treatment of Vim - / - mice does not affect mouse growth , as the growth rate in WT ( n = 8 , saline ; n = 6 Niaspan ) and Vim - / - ( n = 10 saline and n = 10 Niaspan ) mice either saline or Niaspan - treated is not significantly different , Linear Mixed Effect ( LME ) models . C . Semithin section and g - ratio analyses of sciatic nerves at P30 shows that Niaspan does not alter myelin thickness in WT nerves , whereas restores myelin thickness to normal values in Vim - / - nerves . G - ratio values : WT saline , 0 . 71 ± 0 . 003 , 1872 fibers , n = 5 animals ; WT Niaspan , 0 . 71 ± 0 . 004 , 1882 fibers , n = 5 animals ; Vim - / - saline 0 . 69 ± 0 . 004 , 2313 fibers , n = 6 animals ; Vim - / - Niaspan 0 . 72 ± 0 . 01 , 1616 fibers , n = 5 animals . WT saline as compared to Vim - / - saline , p = 0 . 0588 ; Vim - / - saline as compared to Vim - / - Niaspan , p = 0 . 0431 , repeated measures ANOVA , representative of two independent experiments . The number of myelinated fibers is similar between the 4 groups as shown , p = 0 . 3042 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Western blot analysis on lysates from treated and not treated explants (at least 10 DRGs/ coverslips per lane) shows that increased Akt activation (S473 phosphorylation) in Vim-/- explants is restored to normal levels following 5 mM Niacin treatment.A. Niacin treatment of Vim-/- explants rescues hypermyelination, n=10 DRGs/ coverslips per condition, with quantification, representative of three independent experiments, p=0.0048, nonparametric one-way ANOVA, followed by Dunn's post hoc test.B. Niaspan administration (daily i.p. injection of 160mg/Kg Niaspan starting at P15 for 15 days) enhances Tace activity in Vim-/- nerves at P30, n=6 animals per genotype, p=0.0325, one-tailed Mann Whitney U test.B. Niaspan treatment of Vim-/- mice does not affect mouse growth, as the growth rate in WT (n=8, saline; n=6 Niaspan) and Vim-/- (n=10 saline and n=10 Niaspan) mice either saline or Niaspan-treated is not significantly different, Linear Mixed Effect (LME) models.C. Semithin section and g-ratio analyses of sciatic nerves at P30 shows that Niaspan does not alter myelin thickness in WT nerves, whereas restores myelin thickness to normal values in Vim-/- nerves. G-ratio values: WT saline, 0.71 ± 0.003, 1872 fibers, n= 5 animals; WT Niaspan, 0.71 ± 0.004, 1882 fibers, n= 5 animals; Vim-/- saline 0.69 ± 0.004, 2313 fibers, n= 6 animals; Vim-/- Niaspan 0.72 ± 0.01, 1616 fibers, n=5 animals. WT saline as compared to Vim-/- saline, p=0.0588; Vim-/- saline as compared to Vim-/- Niaspan, p=0.0431, repeated measures ANOVA, representative of two independent experiments. The number of myelinated fibers is similar between the 4 groups as shown, p=0.3042, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


Figure 6A . Niaspan administration ( daily i . p . injection of 160mg / Kg starting at P15 for 60 days ) does not affect the growth of Mtmr2 - / - mice . The growth rates of Mtmr2 - / - and WT both saline - treated are significantly different , p < 0 . 0001 , Linear Mixed Effect ( LME ) models , as already reported ( Bolino et al , 2004 ) , n = 8 animals per condition . B . Niaspan administration reduces the percentage of myelin outfoldings ( red asterisks ) in Mtmr2 - / - sciatic nerves without affecting the number of myelinated fibers as assessed by semithin section analysis at P75 , n = 8 mice per genotype , results are mean ± SEM , p = 0 . 0093 , two - tailed Mann Whitney U test , representative of three independent experiments . Niaspan treatment does not alter myelin thickness in WT or Mtmr2 - / - mice as assessed by g - ratio analysis ( WT saline - treated , 0 . 67 ± 0 . 003 , 2014 fibers ; Mtmr2 - / - Niaspan - treated , 0 . 67 ± 0 . 007 , 1924 fibers ; Mtmr2 - / - saline - treated , 0 . 68 ± 0 . 003 , 2244 fibers ; n = 4 animals per condition ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Niaspan administration (daily i.p. injection of 160mg/Kg starting at P15 for 60 days) does not affect the growth of Mtmr2-/- mice. The growth rates of Mtmr2-/- and WT both saline-treated are significantly different, p<0.0001, Linear Mixed Effect (LME) models, as already reported (Bolino et al, 2004), n=8 animals per condition.B. Niaspan administration reduces the percentage of myelin outfoldings (red asterisks) in Mtmr2-/- sciatic nerves without affecting the number of myelinated fibers as assessed by semithin section analysis at P75, n= 8 mice per genotype, results are mean ± SEM, p=0.0093, two-tailed Mann Whitney U test, representative of three independent experiments. Niaspan treatment does not alter myelin thickness in WT or Mtmr2-/- mice as assessed by g-ratio analysis (WT saline-treated, 0.67 ± 0.003, 2014 fibers; Mtmr2-/- Niaspan-treated, 0.67 ± 0.007, 1924 fibers; Mtmr2-/- saline-treated, 0.68 ± 0.003, 2244 fibers; n=4 animals per condition).


Figure 7A . Western blot analysis of lysates from Pmp22 + / - and WT sciatic nerves at P10 shows that Akt phosphorylation ( S473 ) is increased in Pmp22 + / - nerves , results are mean ± SEM , p = 0 . 0317 , two - tailed Mann Whitney U test , representative of two independent experiments . B . Tace activity as measured in Pmp22 + / - and WT sciatic nerves at P30 ( n = 6 mice per genotype , results are mean ± SEM , p = 0 . 0070 , two - tailed Mann Whitney U test ) . B . Western blot analysis of Tace using lysates from Pmp22 + / - and WT sciatic nerves at different stages of development . The arrowhead indicates the pro - protein and the arrow shows the cleaved active form of Tace . Representative of two independent experiments . C . Representative images of semithin section analysis of sciatic nerves at P45 from Pmp22 + / - saline and Pmp22 + / - Niaspan - treated mice . Niaspan was administered daily by i . p . injection at 160mg / Kg starting at P15 for 30 days . C ' . The percentage of tomacula in sciatic nerves was assessed by ultrastructural analysis , results are mean ± SEM , n = 6 animals per condition , p = 0 . 0022 , two - tailed Mann Whitney U test , representative of two independent experiments . D . G - ratio analysis on semithin sections of sciatic nerves shows that increased myelin thickness in Pmp22 + / - sciatic nerves at P45 is restored to normal values following Niaspan treatment . Mean g - ratio values , WT saline - treated : 0 . 69 ± 0 . 002 , 2052 fibers , n = 5 animals ; Pmp22 + / - saline - treated : 0 . 66 ± 0 . 003 , 2643 fibers , n = 6 animals ; Pmp22 + / - Niaspan - treated , 0 . 68 ± 0 . 002 , 2311 fibers , n = 6 animals . WT saline as compared to Pmp22 + / - saline - treated , p < 0 . 0001 ; Pmp22 + / - saline - treated as compared to Pmp22 + / - Niaspan - treated , p = 0 . 0002 ; Pmp22 + / - Niaspan - treated as compared to WT saline , p = 0 . 0899 ; repeated measures ANOVA . E . The number of myelinated fibers is similar between the 3 groups analyzed , p = 0 . 4362 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Western blot analysis of lysates from Pmp22+/- and WT sciatic nerves at P10 shows that Akt phosphorylation (S473) is increased in Pmp22+/- nerves, results are mean ± SEM, p=0.0317, two-tailed Mann Whitney U test, representative of two independent experiments.B. Tace activity as measured in Pmp22+/- and WT sciatic nerves at P30 (n=6 mice per genotype, results are mean ± SEM, p=0.0070, two-tailed Mann Whitney U test).B. Western blot analysis of Tace using lysates from Pmp22+/- and WT sciatic nerves at different stages of development. The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments.C. Representative images of semithin section analysis of sciatic nerves at P45 from Pmp22+/- saline and Pmp22+/- Niaspan-treated mice. Niaspan was administered daily by i.p. injection at 160mg/Kg starting at P15 for 30 days.C'. The percentage of tomacula in sciatic nerves was assessed by ultrastructural analysis, results are mean ± SEM, n= 6 animals per condition, p=0.0022, two-tailed Mann Whitney U test, representative of two independent experiments.D. G-ratio analysis on semithin sections of sciatic nerves shows that increased myelin thickness in Pmp22+/- sciatic nerves at P45 is restored to normal values following Niaspan treatment. Mean g-ratio values, WT saline-treated: 0.69 ± 0.002, 2052 fibers, n=5 animals; Pmp22+/- saline-treated: 0.66 ± 0.003, 2643 fibers, n=6 animals; Pmp22+/- Niaspan-treated, 0.68 ± 0.002, 2311 fibers, n=6 animals. WT saline as compared to Pmp22+/- saline-treated, p<0.0001; Pmp22+/- saline-treated as compared to Pmp22+/- Niaspan-treated, p=0.0002; Pmp22+/- Niaspan-treated as compared to WT saline, p=0.0899; repeated measures ANOVA.E. The number of myelinated fibers is similar between the 3 groups analyzed, p=0.4362, nonparametric one-way ANOVA, followed by Dunn's post hoc test.