Figure 2 A . Arabidopsis root expressing the dual reporter TCSn : : dndT - tNOS DR5v2 : 3nGFP that is sensitive to cytokinin ( CK ) and auxin . CK ( red ) , auxin ( green ) and overlay of both signals detected in nuclei of cells at the root tip ( top , scale bar 50 µm ) . Green , pink , and blue arrowheads point at epidermal cells located in the meristematic ( MZ ) , transition ( TZ ) and elongation ( EZ ) zones , respectively . Magnification of the TZ shown ( bottom , scale bar 10 µm ) . B . Relative fluorescence intensity of TCSn : : ntdT : tNOS signal ( red ) and DR5v2 : 3nGFP signal ( green ) measured in epidermal cells along the longitudinal root growth axis . Cell number 1 corresponds to a meristematic cell placed at position - 4 before the beginning of TZ ( as marked by green arrow at 2A ) . Mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 by Student ' s t - test , n = 18 roots . C - F . CMTs visualized with MAP4 - GFP in root epidermal cells of the TZ and the EZ at time point 0 and 60 min after CK ( C ) , CK and oryzalin ( D ) , auxin ( E ) and CK and auxin ( F ) treatments , and in DZ cells at time point 0 and 60 min after auxin treatment ( E ) . As CK and auxin sources 10 µM BAP and 0 . 1 µM NAA were used , respectively , and 1 µM oryzalin . For the double ( CK / Oryzalin and CK / Auxin ) treatments , roots were pretreated with CK for 60 min prior to transfer to medium supplemented with both compounds . Scale bar 10 µm . H . Analysis of the CMT plus - end growth with the EB1b - GFP reporter . Z - stack maximum image projections of EB1b - GFP tracked over 30 sec in epidermal cells without ( mock , DMSO ) and with CK , auxin or CK and auxin treatment ( 60 min ) . Scale bars 10 µm . I . CMT plus - end growth rates ( µm / min ) measured by tracking of EB1b - GFP reporter over 20 - 30 min in epidermal cells of different growth zones treated for 60 min with mock ( DMSO ) , or media supplemented with CK ( 30 or 60 min ) , auxin and CK plus auxin . In the boxplots , the center lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software ; whiskers span minimum to maximum values , individual data points are represented by dots . The number of CMT plus - end events ( EB1b - GFP ) tracked per cell was 20 - 100 / min , of which the growth rate average is represented as a single dot . Statistical significance evaluated by two - way ANOVA ( n = 3 - 5 cells per root growth zone with 3 - 5 roots per replicate in 3 independent experiments . On the right , single trajectories of EB1b - GFP signal tracked over 60 sec in root epidermal cells at different growth zones and treatment conditions . Scale bar 0 . 5 µm . Hormone concentrations and treatment conditions ( H , I ) were as described ( C , E and F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2 A. Arabidopsis root expressing the dual reporter TCSn::dndT-tNOS DR5v2:3nGFP that is sensitive to cytokinin (CK) and auxin. CK (red), auxin (green) and overlay of both signals detected in nuclei of cells at the root tip (top, scale bar 50 µm). Green, pink, and blue arrowheads point at epidermal cells located in the meristematic (MZ), transition (TZ) and elongation (EZ) zones, respectively. Magnification of the TZ shown (bottom, scale bar 10 µm).  B. Relative fluorescence intensity of TCSn::ntdT:tNOS signal (red) and DR5v2:3nGFP signal (green) measured in epidermal cells along the longitudinal root growth axis. Cell number 1 corresponds to a meristematic cell placed at position -4 before the beginning of TZ (as marked by green arrow at 2A). Mean ± s.d., * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by Student's t-test, n= 18 roots.  C-F. CMTs visualized with MAP4-GFP in root epidermal cells of the TZ and the EZ at time point 0 and 60 min after CK (C), CK and oryzalin (D), auxin (E) and CK and auxin (F) treatments, and in DZ cells at time point 0 and 60 min after auxin treatment (E). As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively, and 1 µM oryzalin. For the double (CK/Oryzalin and CK/Auxin) treatments, roots were pretreated with CK for 60 min prior to transfer to medium supplemented with both compounds. Scale bar 10 µm.  H. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. Z-stack maximum image projections of EB1b-GFP tracked over 30 sec in epidermal cells without (mock, DMSO) and with CK, auxin or CK and auxin treatment (60 min). Scale bars 10 µm. I. CMT plus-end growth rates (µm/min) measured by tracking of EB1b-GFP reporter over 20-30 min in epidermal cells of different growth zones treated for 60 min with mock (DMSO), or media supplemented with CK (30 or 60 min), auxin and CK plus auxin. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. The number of CMT plus-end events (EB1b-GFP) tracked per cell was 20-100/min, of which the growth rate average is represented as a single dot. Statistical significance evaluated by two-way ANOVA (n = 3-5 cells per root growth zone with 3-5 roots per replicate in 3 independent experiments. On the right, single trajectories of EB1b-GFP signal tracked over 60 sec in root epidermal cells at different growth zones and treatment conditions. Scale bar 0.5 µm. Hormone concentrations and treatment conditions (H, I) were as described (C, E and F).


Figure 3 A - D . CMTs visualized with MAP4 - GFP in root epidermal cells of the cre1 - 12 mutant at the transition ( TZ ) and the elongation ( EZ ) zones at time point 0 and 60 min after mock ( DMSO ) ( A ) , cytokinin ( CK ) ( B ) , auxin ( C ) and CK plus auxin ( D ) treatments . As CK and auxin sources 10 µM BAP and 0 . 1 µM NAA were used , respectively . For the double CK plus auxin treatment roots were pretreated for 60 min with CK , whereafter they were transferred to medium supplemented with both compounds . Scale bar 10 µm . F , G . Analysis of the CMT plus - end growth with the EB1b - GFP reporter in cre1 - 12 . F . Z - stack maximum image projection of EB1b - GFP tracked for 30 seconds ( on the left ) and single trajectories of EB1b - GFP signal tracked over 60 sec ( on the right ) in root epidermal cells of the EZ in the wild type and cre1 - 12 treated with CK for 60 min . Scale bars 10 µm and 0 . 5 µm , respectively . G . CMT plus - end growth rates ( µm / min ) quantified from the EB1b - GFP trajectories tracked during 5 min in epidermal cells of the EZ treated with mock ( DMSO ) or CK . In the boxplots , the center lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software ; whiskers span minimum to maximum values , individual data points are represented by dots . ( ns = non - significant , * * * P < 0 . 001 by Student ' s t - test , n = 7 - 9 cells with 7 roots per condition in 3 independent experiments ) . Concentrations and treatment conditions ( F , G ) were as for ( A , B ) . H , I . CMTs visualized with MAP4 - GFP in root epidermal cells of the cre1 - 12 mutant . Cells at the TZ , the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin ( 1 µM ) ( H ) , and CK plus oryzalin ( I ) . As CK source 10 µM BAP was used and 1 µM oryzalin . For double ( CK and oryzalin ) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds . Scale bar 10 µm . J . Quantification of the MAP4 - GFP CMTs reporter signal in cre1 - 12 root epidermal cells at different root zones treated with mock ( DMSO , white box ) , oryzalin ( 1 µM , light grey box ) and CK ( 10 µM BAP ) plus oryzalin ( 1 µM ) ( dark grey box ) . For double treatments , roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds . Boxplots represent ratio between mean fluorescence intensity ( arbitrary units ) measured in epidermal cells at 60 and 0 min . The center lines show the medians and the box limits indicate the 25th and 75th percentiles ; whiskers span the minimal to maximal values , individual data points are represented by dots . Ratio close to 1 ( segmented line ) corresponds to the unchanged MAP - GFP signal for 60 min . ( * P < 0 . 05 , * * * * P < 0 . 0001 by Student ' s t - test , n = 3 - 10 cells per root growth zone with 5 - 8 roots per condition in 4 independent replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 A-D. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant at the transition (TZ) and the elongation (EZ) zones at time point 0 and 60 min after mock (DMSO) (A), cytokinin (CK) (B), auxin (C) and CK plus auxin (D) treatments. As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively. For the double CK plus auxin treatment roots were pretreated for 60 min with CK, whereafter they were transferred to medium supplemented with both compounds. Scale bar 10 µm.  F, G. Analysis of the CMT plus-end growth with the EB1b-GFP reporter in cre1-12. F. Z-stack maximum image projection of EB1b-GFP tracked for 30 seconds (on the left) and single trajectories of EB1b-GFP signal tracked over 60 sec (on the right) in root epidermal cells of the EZ in the wild type and cre1-12 treated with CK for 60 min. Scale bars 10 µm and 0.5 µm, respectively. G. CMT plus-end growth rates (µm/min) quantified from the EB1b-GFP trajectories tracked during 5 min in epidermal cells of the EZ treated with mock (DMSO) or CK. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. (ns = non-significant, *** P < 0.001 by Student's t-test, n= 7-9 cells with 7 roots per condition in 3 independent experiments). Concentrations and treatment conditions (F, G) were as for (A, B).  H, I. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant. Cells at the TZ, the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin (1 µM) (H), and CK plus oryzalin (I). As CK source 10 µM BAP was used and 1 µM oryzalin. For double (CK and oryzalin) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds. Scale bar 10 µm. J. Quantification of the MAP4-GFP CMTs reporter signal in cre1-12 root epidermal cells at different root zones treated with mock (DMSO, white box), oryzalin (1 µM, light grey box) and CK (10 µM BAP) plus oryzalin (1 µM) (dark grey box). For double treatments, roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Boxplots represent ratio between mean fluorescence intensity (arbitrary units) measured in epidermal cells at 60 and 0 min. The center lines show the medians and the box limits indicate the 25th and 75th percentiles; whiskers span the minimal to maximal values, individual data points are represented by dots. Ratio close to 1 (segmented line) corresponds to the unchanged MAP-GFP signal for 60 min. (* P < 0.05, **** P < 0.0001 by Student's t-test, n= 3-10 cells per root growth zone with 5-8 roots per condition in 4 independent replicates).


Figure 4 A . Relative root growth of wild - type ( Col - 0 ) ( grey lines ) and 35S : : ARR1 ∆ DDK - GR ( purple lines ) seedlings grown on mock ( DMSO ; continuous line ) or with dexamethasone ( DEX 10 µM ; dashed line ) supplemented medium . Five days old seedlings ( Day 0 ) were monitored for 3 days . Mean ± s . d . ; * * P < 0 . 01 , * * * * P < 0 . 0001 by Student ' s t - test , referred to wild - type - mock . n = 12 - 14 roots . B . Immunostaining of α - tubulin in epidermal cells of the transition ( TZ ) and the elongation ( EZ ) zone of 35S : : ARR1 ∆ DDK - GR roots after non - , 3h or 16h of treatment with dexamethasone ( DEX 10 µM ) . Scale bar 10 µm . D , E . Analysis of root sensitivity to oryzalin in wild - type ( Col - 0 ) and 35S : : ARR1 ∆ DDK - GR . Seedlings were grown for 5 days on mock ( Murashige and Skoog ) medium and then transferred to medium supplemented with 1 µM oryzalin , CK with oryzalin ( 10 µM BAP and 1 µM oryzalin ) , with or without DEX 10 µM , for 3 days . For the double CK and oryzalin treatment , seedlings were pretreated with 10 µM BAP for 60 min prior to transfer to medium supplemented with both compounds . Representative images ; white arrowheads indicate root length at day of transfer . Scale bar 1 mm ( D ) . Quantifications calculated as percentage ( % ) of root tips exhibiting swelling ( dark grey bars ) , and weak and strong resistance to oryzalin ( grey and white bars , respectively ) . On the right representative images of root phenotype categories . n = 10 - 25 roots per treatment were evaluated . ( E ) . F . Relative root growth of wild - type ( Col - 0 ) ( grey lines ) and 35S : : ARR1 ∆ DDK - GR ( purple lines ) seedlings grown on oryzalin 1µM ( continuous line ) or on oryzalin 1µM and DEX 10 µM ( dashed line ) supplemented media monitored for 3 days . Day 0 , day of transfer . Mean ± s . d . ; n = 12 - 14 roots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4 A. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on mock (DMSO; continuous line) or with dexamethasone (DEX 10 µM; dashed line) supplemented medium. Five days old seedlings (Day 0) were monitored for 3 days. Mean ± s.d.; ** P < 0.01, **** P < 0.0001 by Student's t-test, referred to wild-type - mock. n = 12-14 roots.  B. Immunostaining of α-tubulin in epidermal cells of the transition (TZ) and the elongation (EZ) zone of 35S::ARR1∆DDK-GR roots after non-, 3h or 16h of treatment with dexamethasone (DEX 10 µM). Scale bar 10 µm.  D, E. Analysis of root sensitivity to oryzalin in wild-type (Col-0) and 35S::ARR1∆DDK-GR. Seedlings were grown for 5 days on mock (Murashige and Skoog) medium and then transferred to medium supplemented with 1 µM oryzalin, CK with oryzalin (10 µM BAP and 1 µM oryzalin), with or without DEX 10 µM, for 3 days. For the double CK and oryzalin treatment, seedlings were pretreated with 10 µM BAP for 60 min prior to transfer to medium supplemented with both compounds. Representative images; white arrowheads indicate root length at day of transfer. Scale bar 1 mm (D). Quantifications calculated as percentage (%) of root tips exhibiting swelling (dark grey bars), and weak and strong resistance to oryzalin (grey and white bars, respectively). On the right representative images of root phenotype categories. n=10-25 roots per treatment were evaluated. (E).  F. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on oryzalin 1µM (continuous line) or on oryzalin 1µM and DEX 10 µM (dashed line) supplemented media monitored for 3 days. Day 0, day of transfer. Mean ± s.d.; n = 12-14 roots.


Figure 5 A . Time lapse imaging of Arabidopsis root expressing the plasma membrane marker ( PM ) pUB10 : : EYFP - NPSN12 ( wave line W131Y ) ( Geldner et al , 2009 ) . Root growth recorded for 2 min and overlay of root tip images at time point 0 ( magenta ) and 2 min ( cyan ) used for the calculation of the root growth rate ( RGR ) and the cell elongation rate ( CER ) . Scale bar 25 µm . B . RGR of wild type ( grey bars ) and cre1 - 12 ( blue bars ) treated for 1 and 4 hours with mock ( DMSO ) , CK ( 10 µM BAP ) or auxin ( 0 . 1 µM NAA ) . Mean ± s . d . , ( * P < 0 . 05and * * * * P < 0 . 0001 by the Student ' s t - test , n = 5 - 8 roots per condition in 3 independent replicates ) . C - E . Analysis of CK and auxin effects on CER of epidermal cells . Epidermal cells of the TZ and the EZ in the wild type and cre1 - 12 with PMs visualized with the YFP - NPSN12 marker . Overlay of images of epidermal cells at time 0 ( magenta ) and 2 min ( cyan ) is presented . Scale bar 10 µm ( C ) . CER ( µm / min ) of root epidermal cells of the TZ ( D ) and the EZ ( E ) of the wild type ( grey bars ) and cre1 - 12 ( blue bars ) mutant . Hormone concentrations and conditions of treatment ( D , E ) were as described for ( C ) . Mean ± s . d . , ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 by Student ' s t - test , n = 15 - 20 cells per root growth zone in 3 - 6 roots per condition , in 3 independent ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5 A. Time lapse imaging of Arabidopsis root expressing the plasma membrane marker (PM) pUB10::EYFP-NPSN12 (wave line W131Y)(Geldner et al, 2009). Root growth recorded for 2 min and overlay of root tip images at time point 0 (magenta) and 2 min (cyan) used for the calculation of the root growth rate (RGR) and the cell elongation rate (CER). Scale bar 25 µm. B. RGR of wild type (grey bars) and cre1-12 (blue bars) treated for 1 and 4 hours with mock (DMSO), CK (10 µM BAP) or auxin (0.1 µM NAA). Mean ± s.d., (* P < 0.05and **** P < 0.0001 by the Student's t-test, n= 5-8 roots per condition in 3 independent replicates).  C-E. Analysis of CK and auxin effects on CER of epidermal cells. Epidermal cells of the TZ and the EZ in the wild type and cre1-12 with PMs visualized with the YFP-NPSN12 marker. Overlay of images of epidermal cells at time 0 (magenta) and 2 min (cyan) is presented. Scale bar 10 µm (C). CER (µm/min) of root epidermal cells of the TZ (D) and the EZ (E) of the wild type (grey bars) and cre1-12 (blue bars) mutant. Hormone concentrations and conditions of treatment (D, E) were as described for (C). Mean ± s.d., (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by Student's t-test, n=15-20 cells per root growth zone in 3-6 roots per condition, in 3 independent).


Figure 6 A . MT plus - end growth rates ( µm / min ) in leukocytes expressing the EB3 - mCherry plus - end marker before ( dark grey ) and after ( light grey ) treatment with mock ( DMSO ) , CK ( 10 µM BAP or 10 µM trans - zeatin ) and auxin ( 0 . 1 µM NAA ) . MT plus - end growth rates were monitored for 10 min before and for 10 min after each treatment . The number of MT plus - end events ( EB3 - mCherry ) tracked per cell was 100 - 200 / min . Mean ± s . d . , ( * * * P < 0 . 001 by Student ' s t - test , n = 1 - 3 cells per condition in 4 independent replicate ) . C - E . Immunostaining of α - tubulin ( green ) in leukocytes mounted in media containing DAPI ( blue , nuclear marker ) and quantification of immunolabeled MTs . Cells were not or treated with 300 nM nocodazole ( N ) for 10 min ( C ) . Leukocytes pretreated for 30 min with mock ( DMSO ) , CK ( 10 µM BAP or 10 µM trans - zeatin ) , adenine ( 10 µM ) or auxin ( 10 µM NAA ) prior to a 10 - min treatment with 300 nM nocodazol ( D ) . Scale bar 50 µm ( C , D ) . E . Relative fluorescence intensity of MTs in leukocytes treated with nocodazole , or nocodazole and hormones as described for ( D ) compared to untreated ( black bar ) cells . Mean ± s . d . , ( P < 0 . 05 by two - way Anova , n = 18 - 33 cells per condition in 3 independent replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MT plus-end growth rates (µm/min) in leukocytes expressing the EB3-mCherry plus-end marker before (dark grey) and after (light grey) treatment with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin) and auxin (0.1 µM NAA). MT plus-end growth rates were monitored for 10 min before and for 10 min after each treatment. The number of MT plus-end events (EB3-mCherry) tracked per cell was 100-200/min. Mean ± s.d., (*** P < 0.001 by Student's t-test, n = 1-3 cells per condition in 4 independent replicate). C-E. Immunostaining of α-tubulin (green) in leukocytes mounted in media containing DAPI (blue, nuclear marker) and quantification of immunolabeled MTs. Cells were not or treated with 300 nM nocodazole (N) for 10 min (C). Leukocytes pretreated for 30 min with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin), adenine (10 µM) or auxin (10 µM NAA) prior to a 10-min treatment with 300 nM nocodazol (D). Scale bar 50 µm (C, D). E. Relative fluorescence intensity of MTs in leukocytes treated with nocodazole, or nocodazole and hormones as described for (D) compared to untreated (black bar) cells. Mean ± s.d., (P < 0.05 by two-way Anova, n = 18-33 cells per condition in 3 independent replicates).


Figure 1A , B , and C . Immunostaining of Tom20 ( mitochondria ) in cells positive for β3 - tubulin in DIV6 Ifnb + / + and Ifnb - / - CNs with or without rIFN - β for 24 h . A . Projection of 3D image of Tom20 . Scale bars equal 20 microns . B . Quantification of total mitochondrial mass per neuron . C . Mean individual mitochondria volume in Ifnb + / + and Ifnb - / - CNs . D , E , F , and G . 3D electron microscopy of thalamus in 12 - month - old Ifnb + / + and Ifnb - / - mice . D . Volume rendering of mitochondria ( left panel ) and corresponding mitochondria in 2D electron microscopy image ( right panel ) in cell bodies . Each individual mitochondrion is colored differently . Scale bars equal 2 microns . Voxel size equals 0 . 00878908x0 . 00878908x0 . 03 microns for the Ifnb + / + neuron and 0 . 00439453x0 . 00438453x0 . 04 microns for Ifnb - / - . Nuclei are in grey and marked with ' N . ' E . Reconstituted axonal mitochondria . Images extracted from Movies EV1 and 2 . F . Size of individual mitochondria in cell bodies and non - myelinated and myelinated axons . N > 30 ; error bars are SD . G . Number of branches per mitochondrion . N > 30 ; error bars are SD .

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Figure 1A, B, and C. Immunostaining of Tom20 (mitochondria) in cells positive for β3-tubulin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without rIFN-β for 24 h. A. Projection of 3D image of Tom20. Scale bars equal 20 microns. B. Quantification of total mitochondrial mass per neuron. C. Mean individual mitochondria volume in Ifnb+/+ and Ifnb-/- CNs. D, E, F, and G. 3D electron microscopy of thalamus in 12-month-old Ifnb+/+ and Ifnb-/- mice. D. Volume rendering of mitochondria (left panel) and corresponding mitochondria in 2D electron microscopy image (right panel) in cell bodies. Each individual mitochondrion is colored differently. Scale bars equal 2 microns. Voxel size equals 0.00878908x0.00878908x0.03 microns for the Ifnb+/+ neuron and 0.00439453x0.00438453x0.04 microns for Ifnb-/-. Nuclei are in grey and marked with 'N.' E. Reconstituted axonal mitochondria. Images extracted from Movies EV1 and 2. F. Size of individual mitochondria in cell bodies and non-myelinated and myelinated axons. N>30; error bars are SD. G. Number of branches per mitochondrion. N>30; error bars are SD.


Figure 2D - E . Respiration of isolated mitochondria from Ifnb + / + and Ifnb - / - whole brains . OCR measurements under Complex 1 substrates ( pyruvate / malate ) were obtained at baseline ( B ) and on addition of ADP , oligomycin , FCCP and rotenone / antimycin A to capture S3 , S4o , S3u , and nonmitochondrial respiration . N = 3 - 4 mice per group ; bar graphs show mean + SE . K . Immunofluorescence of 8OHdG in CN cultures . Neurons were labeled with Nissl . Scale bars equal 50 microns . L . Quantification of 8OHdG by ELISA in brain tissues in young ( aged 1 . 5 - 3 months ) and old ( aged 6 - 12 months ) Ifnb + / + and Ifnb - / - mice . M . Immunoblot for optineurin in 6 - week - old midbrains of Ifnb + / + and Ifnb - / - mice . N . Immunoblot for optineurin in DIV6 Ifnb + / + and Ifnb - / - CNs with or without 30 U / mL rIFN - β .

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Figure 2D-E. Respiration of isolated mitochondria from Ifnb+/+ and Ifnb-/- whole brains. OCR measurements under Complex 1 substrates (pyruvate/malate) were obtained at baseline (B) and on addition of ADP, oligomycin, FCCP and rotenone/antimycin A to capture S3, S4o, S3u, and nonmitochondrial respiration. N=3-4 mice per group; bar graphs show mean + SE.K. Immunofluorescence of 8OHdG in CN cultures. Neurons were labeled with Nissl. Scale bars equal 50 microns. L. Quantification of 8OHdG by ELISA in brain tissues in young (aged 1.5-3 months) and old (aged 6-12 months) Ifnb+/+ and Ifnb-/- mice. M. Immunoblot for optineurin in 6-week-old midbrains of Ifnb+/+ and Ifnb-/- mice. N. Immunoblot for optineurin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without 30 U/mL rIFN-β.


Figure 3H . Kinetics of Drp1 dephosphorylation at S643 on rIFN - β treatment in wild - type CN cultures . PD - L1 was used as a positive control of rIFN - β activity . I . MFN2 and OPA1 immunoblots and quantification in Ifnb + / + and Ifnb - / - CN cultures and quantification . Vinculin was used as a loading control . Error bars are SEM from 3 independent experiments . J . Optineurin and Tom20 immunofluorescence in Ifnb + / + and Ifnb - / - MEFs transfected with Drp1 or phosphomimetic Drp1 .

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Figure 3H. Kinetics of Drp1 dephosphorylation at S643 on rIFN-β treatment in wild-type CN cultures. PD-L1 was used as a positive control of rIFN-β activity.I. MFN2 and OPA1 immunoblots and quantification in Ifnb+/+ and Ifnb-/- CN cultures and quantification. Vinculin was used as a loading control. Error bars are SEM from 3 independent experiments.J. Optineurin and Tom20 immunofluorescence in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1.


Figure 4E . Volume rendering from 3D TEM images of Ifnb + / + and Ifnb - / - thalami . Mitochondria are blue , the ER is yellow , and nuclei are grey . Mitochondria undergoing fission are cyan and indicated with a white arrow . 2D pictures are shown in Fig 2 . H . Projections of 3D images from immunostaining of Drp1 ( blue ) , Hsp60 ( mitochondria , green ) , INF2 ( yellow ) , and calnexin ( ER , magenta ) in Ifnb - / - MEFs with or without rIFN - β treatment for 6 h . Scale bar equal 2 microns . Arrows indicate fission foci . I . Quantifications of triple - positive MT - DR + Drp1 + Cnx + in MEFs . Images are shown in Fig EV3 .

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Figure 4E. Volume rendering from 3D TEM images of Ifnb+/+ and Ifnb-/- thalami. Mitochondria are blue, the ER is yellow, and nuclei are grey. Mitochondria undergoing fission are cyan and indicated with a white arrow. 2D pictures are shown in Fig 2.H. Projections of 3D images from immunostaining of Drp1 (blue), Hsp60 (mitochondria, green), INF2 (yellow), and calnexin (ER, magenta) in Ifnb-/- MEFs with or without rIFN-β treatment for 6 h. Scale bar equal 2 microns. Arrows indicate fission foci. I. Quantifications of triple-positive MT-DR+Drp1+Cnx+ in MEFs. Images are shown in Fig EV3.


Figure 5D . Projections of 3D images from immunostaining for phospho - S622 Drp1 ( yellow ) , Hsp60 ( mitochondria , green ) , and calnexin ( ER , magenta ) in N2A cells depleted of STAT5 A and B ( δStat5 ) or IFN - β ( δIfnb ) or with nontargeting control ( NTC ) . Cells with or without rIFN - β treatment for 6 h . Scale bar equals 5 microns . Arrows indicate triple - positive foci . E . Quantification of D . L . Immunostaining of PGAM5 and Hsp60 in NTC and δSTAT5 N2As transfected with PGAM5 or empty vector control and quantification of mitochondrial section area . Scale bars equal 5 microns . Error bars mean SEM from 10 individual cells . M . Optineurin immunostaining in Ifnb + / + and Ifnb - / - MEFs overexpressing PGAM5 and quantification . Empty vector was used as control . Additional controls are shown in Fig EV5L . Error bars are SD from N = 30 cells .

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Figure 5D. Projections of 3D images from immunostaining for phospho-S622 Drp1 (yellow), Hsp60 (mitochondria, green), and calnexin (ER, magenta) in N2A cells depleted of STAT5 A and B (δStat5) or IFN-β (δIfnb) or with nontargeting control (NTC). Cells with or without rIFN-β treatment for 6 h. Scale bar equals 5 microns. Arrows indicate triple-positive foci. E. Quantification of D. L. Immunostaining of PGAM5 and Hsp60 in NTC and δSTAT5 N2As transfected with PGAM5 or empty vector control and quantification of mitochondrial section area. Scale bars equal 5 microns. Error bars mean SEM from 10 individual cells.M. Optineurin immunostaining in Ifnb+/+ and Ifnb-/- MEFs overexpressing PGAM5 and quantification. Empty vector was used as control. Additional controls are shown in Fig EV5L. Error bars are SD from N=30 cells.


Figure 6E - K . Immunofluorescence in the striatal region in mice treated with 6OH - DA and rIFN - β for optineurin ( E , quantified in F ) and Tom20 ( E ) , with quantification of individual mitochondrial size by mitochondrial section area ( G ) . 8OHdG - ( H - I ) , OxDJ1 - ( J - K ) , and DAPI - labeled nuclei . Scale bars equal 10 microns .

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Figure 6E-K. Immunofluorescence in the striatal region in mice treated with 6OH-DA and rIFN-β for optineurin (E, quantified in F) and Tom20 (E), with quantification of individual mitochondrial size by mitochondrial section area (G). 8OHdG- (H-I), OxDJ1- (J-K), and DAPI-labeled nuclei. Scale bars equal 10 microns.


Figure 1 ( H , I ) Representative H & E ( H ) and dystrophin immunofluorescence of images of TA muscle cross - sections in WT and Prmt5MKO mice . Scale bar : 100 μm . ( J , Distribution of myofiber size ( J ) of WT ( n = 4 ) and Prmt5MKO TA muscles ( n = 4 ) . ( L ) The total number of myofibers in EDL and Soleus from WT ( n = 5 ) and Prmt5MKO mice ( n = 6 ) .

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Figure 1(H, I) Representative H&E (H) and dystrophin immunofluorescence of images of TA muscle cross-sections in WT and Prmt5MKO mice. Scale bar: 100 μm.(J, Distribution of myofiber size (J) of WT (n=4) and Prmt5MKO TA muscles (n=4).(L) The total number of myofibers in EDL and Soleus from WT (n=5) and Prmt5MKO mice (n=6).


Figure 2 ( B - D ) Exercise performance of maximum speed ( B ) , running time ( C ) , running distance ( D ) of WT ( n = 11 ) and Prmt5MKO mice ( n = 9 ) measured by treadmill . ( E , F ) Absolute ( E ) and specific force ( F ) ( on the left panel ) and maximal absolute and specific force ( on right panel ) on EDL muscle from WT ( n = 4 ) and Prmt5MKO mice ( n = 3 ) . ( G , H ) Absolute ( G ) and specific force ( H ) ( on the left panel ) and maximal absolute and specific force ( on right panel ) on Soleus muscle from WT ( n = 4 ) and Prmt5MKO mice ( n = 3 ) .

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Figure 2(B-D) Exercise performance of maximum speed (B), running time (C), running distance (D) of WT (n=11) and Prmt5MKO mice (n=9) measured by treadmill.(E, F) Absolute (E) and specific force (F) (on the left panel) and maximal absolute and specific force (on right panel) on EDL muscle from WT (n=4) and Prmt5MKO mice (n=3). (G, H) Absolute (G) and specific force (H) (on the left panel) and maximal absolute and specific force (on right panel) on Soleus muscle from WT (n=4) and Prmt5MKO mice(n=3).


Figure 3 ( A ) Representative immunostaining of Type I , IIA , and IIB myofibers in in EDL and Soleus muscles of 2 - month - old WT and Prmt5MKO mice . ( B , C ) Quantification of abundance of various fiber types in EDL ( B ) and Soleus ( C ) muscles of WT and Prmt5MKO mice ; ( n = 5 ) . ( D ) Representative histochemical staining image of succinate dehydrogenase ( SDH ) enzymatic activity in EDL and Soleus muscles . ( E , F ) Quantification of abundance of SDH - low , - medium and - high myofibers in EDL ( E ) and Soleus ( F ) muscles of WT ( n = 5 ) and Prmt5MKO mice ( n = 4 ) . ( G , H ) Metabolic rate of O2 consumption ( G ) and CO2 production ( H ) normalized to lean mass for a 48 - hour cycle of 4 - 6 - month - old WT ( n = 8 ) and Prmt5MKO ( n = 7 ) mice measured by an indirect calorimetry .

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Figure 3(A) Representative immunostaining of Type I, IIA, and IIB myofibers in in EDL and Soleus muscles of 2-month-old WT and Prmt5MKO mice. (B, C) Quantification of abundance of various fiber types in EDL (B) and Soleus (C) muscles of WT and Prmt5MKO mice; (n=5).(D) Representative histochemical staining image of succinate dehydrogenase (SDH) enzymatic activity in EDL and Soleus muscles. (E, F) Quantification of abundance of SDH-low, -medium and -high myofibers in EDL (E) and Soleus (F) muscles of WT (n=5) and Prmt5MKO mice (n=4).(G, H) Metabolic rate of O2 consumption (G) and CO2 production (H) normalized to lean mass for a 48-hour cycle of 4-6-month-old WT (n=8) and Prmt5MKO (n=7) mice measured by an indirect calorimetry.


Figure 4 ( A , B ) Representative images of ORO staining ( A ) and immunofluorescence ( B ) in TA muscle from WT and Prmt5MKO mice . Scale bar : 100 μm . ( C ) Western blotting analysis for protein markers of lipolysis , lipogenesis , and electron transport chain ( ETC ) complexes in skeletal muscles of WT ( n = 3 ) and Prmt5MKO mice ; ( n = 3 ) . ( F ) OCR was measured at basal state and after sequential addition of Oligomycin , FCCP , and Rotenone / Antimycin A to determine basal respiration ( BS ) , proton leak ( PL ) , ATP respiration ( ATP R ) , maximal respiration ( Max R ) , spare capacity ( SC ) , and non - mitochondrial respiration ( NR ) ; ( n = 4 , technical replicates ) .

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Figure 4(A, B) Representative images of ORO staining (A) and immunofluorescence (B) in TA muscle from WT and Prmt5MKO mice. Scale bar: 100 μm.(C) Western blotting analysis for protein markers of lipolysis, lipogenesis, and electron transport chain (ETC) complexes in skeletal muscles of WT (n=3) and Prmt5MKO mice; (n=3).(F) OCR was measured at basal state and after sequential addition of Oligomycin, FCCP, and Rotenone/Antimycin A to determine basal respiration (BS), proton leak (PL), ATP respiration (ATP R), maximal respiration (Max R), spare capacity (SC), and non-mitochondrial respiration (NR); (n=4, technical replicates).


Figure 5 ( A ) C2C12 cells overexpressing PRMT5 - GFP alone or PRMT5 - GFP + SREBP1a - Flag were immunoprecipitated with Flag antibody and blotted with SYM10 , GFP , Flag , PRMT5 , and Tubulin antibodies . ( B ) C2C12 cells overexpressing SREBP1a - Flag alone or SREBP1a - Flag + PRMT5 - GFP were immunoprecipitated with GFP antibody and blotted with Flag , GFP , PRMT5 , and Tubulin antibodies . ( E ) HEK293 cells were transfected with PRMT5 - GFP alone or PRMT5 - GFP + SREBP1a - Flag for 24 hours , followed by cycloheximide ( 30 μg / ml ) and protein analysis at 0 , 4 , 8 hours . Lysates were immunoblotted with Flag , GFP , and tubulin antibodies . ( G ) Relative expression of lipogenesis genes ( Dgat1 , Dgat2 , Fabp4 ) in C2C12 transfected with PRMT5 and SREBP1a ; ( n = 4 , technical replicates ) .

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Figure 5(A) C2C12 cells overexpressing PRMT5-GFP alone or PRMT5-GFP + SREBP1a-Flag were immunoprecipitated with Flag antibody and blotted with SYM10, GFP, Flag, PRMT5, and Tubulin antibodies. (B) C2C12 cells overexpressing SREBP1a-Flag alone or SREBP1a-Flag + PRMT5-GFP were immunoprecipitated with GFP antibody and blotted with Flag, GFP, PRMT5, and Tubulin antibodies.(E) HEK293 cells were transfected with PRMT5-GFP alone or PRMT5-GFP + SREBP1a-Flag for 24 hours, followed by cycloheximide (30 μg/ml) and protein analysis at 0, 4, 8 hours. Lysates were immunoblotted with Flag, GFP, and tubulin antibodies.(G) Relative expression of lipogenesis genes (Dgat1, Dgat2, Fabp4) in C2C12 transfected with PRMT5 and SREBP1a; (n=4, technical replicates).


Figure 6 ( A , B ) Immunoblots showing symmetric demethylation of H4R3 in skeletal muscles of WT ( n = 4 ) and Prmt5MKO mice ( n = 4 ) ( A ) , and quantification of H4R3Me2s normalized to total H4 ( B ) . ( E ) ChIP - sequencing results showing the Prmt5 binding peak on the Pnpla2 promoter of 3T3 - L1 cells . ( H , I ) Enrichment of PRMT5 ( H ) and H4R3Me2s ( I ) on the proximal promoter region of the Pnpla2 gene in skeletal muscles of WT and Prmt5MKO mice ( n = 4 for H , and I , biological replicates ) .

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 B-PANEL_START 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

Figure 6(A, B) Immunoblots showing symmetric demethylation of H4R3 in skeletal muscles of WT (n=4) and Prmt5MKO mice (n=4) (A), and quantification of H4R3Me2s normalized to total H4 (B).(E) ChIP-sequencing results showing the Prmt5 binding peak on the Pnpla2 promoter of 3T3-L1 cells.(H, I) Enrichment of PRMT5 (H) and H4R3Me2s (I) on the proximal promoter region of the Pnpla2 gene in skeletal muscles of WT and Prmt5MKO mice (n=4 for H, and I, biological replicates).


Figure 7 ( D ) Immunoblotting analysis showing PRMT5 , ATGL and ETCs in muscle tissues ( upper panel ) and quantification of its relative protein levels in muscles of WT , Prmt5MKO , and Prmt5 / Pnpla2MKO mice ( bottom panel ) ( n = 2 , biological replicates ) . ( E , F ) Exercise performance in running distance ( E ) and griping strength test ( F ) in WT ( n = 11 for E , n = 8 for F ) , Prmt5MKO ( n = 10 for E , n = 9 for F ) and Prmt5 / Pnpla2MKO mice ( n = 9 for E , n = 8 for F ) . ( I ) Representative images of ORO staining in TA muscles under brightfield ( upper panel ) and fluorescent imaging ( bottom panel ) from WT , Prmt5MKO , and Prmt5 / Pnpla2MKO mice . Scale bar : 50 μm .

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 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 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

Figure 7(D) Immunoblotting analysis showing PRMT5, ATGL and ETCs in muscle tissues (upper panel) and quantification of its relative protein levels in muscles of WT, Prmt5MKO, and Prmt5/Pnpla2MKO mice (bottom panel) (n=2, biological replicates).(E, F) Exercise performance in running distance (E) and griping strength test (F) in WT (n=11 for E, n=8 for F), Prmt5MKO (n=10 for E, n=9 for F) and Prmt5/Pnpla2MKO mice (n=9 for E, n=8 for F).(I) Representative images of ORO staining in TA muscles under brightfield (upper panel) and fluorescent imaging (bottom panel) from WT, Prmt5MKO, and Prmt5/Pnpla2MKO mice. Scale bar: 50 μm.


Figure 1Polygon distribution of biological tissues compared with Voronoi diagrams . A ) Segmentation of a Drosophila prepupa wing imaginal disc epithelium ( dWP ) . B ) Adult Biceps ( BCA ) biopsy stained with anti - collagen VI ( green ) and anti - Myosin Heavy Chain ( red ) antibodies . C ) Segmentation of the image showed in ( B ) . D ) Segmented image from a chicken embryo neural tube epithelium ( cNT ) . E ) Polygon distribution of the different biological tissues analysed . Two types of distribution can be distinguished : The Poisson Voronoi polygon distribution for Diagram 1 ( D1 ) and cNT , and the conserved polygon distribution for the rest of image categories .

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Figure 1Polygon distribution of biological tissues compared with Voronoi diagrams. A) Segmentation of a Drosophila prepupa wing imaginal disc epithelium (dWP).B) Adult Biceps (BCA) biopsy stained with anti-collagen VI (green) and anti-Myosin Heavy Chain (red) antibodies. C) Segmentation of the image showed in (B).D) Segmented image from a chicken embryo neural tube epithelium (cNT).E) Polygon distribution of the different biological tissues analysed. Two types of distribution can be distinguished: The Poisson Voronoi polygon distribution for Diagram 1 (D1) and cNT, and the conserved polygon distribution for the rest of image categories.


Figure 2The CVT path matches the polygon distribution of natural tissues . A ) Polygon distribution Voronoi diagrams resulting from the iterative application of Lloyd ´ s algorithm . B - E ) Density plot showing the frequency of a value for four , five , seven and eight sided cells respectively , depending of the percentage of hexagons ( P6 ) in the CVT diagrams . The darker region reflects the higher probability of a determine value . Coloured circles represent the average values of each polygon class for the different sets of images analysed in this study . dWL , Drosophila larva wing disc , light green . dMWP , Drosophila mutant wing disc , violet . EYE , Drosophila eye disc , orange . BCA , biceps control adult , dark green . BNA , biceps neurogenic atrophy , dark blue .

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Figure 2The CVT path matches the polygon distribution of natural tissues. A) Polygon distribution Voronoi diagrams resulting from the iterative application of Lloyd´s algorithm.B-E) Density plot showing the frequency of a value for four, five, seven and eight sided cells respectively, depending of the percentage of hexagons (P6) in the CVT diagrams. The darker region reflects the higher probability of a determine value. Coloured circles represent the average values of each polygon class for the different sets of images analysed in this study. dWL, Drosophila larva wing disc, light green. dMWP, Drosophila mutant wing disc, violet. EYE, Drosophila eye disc, orange. BCA, biceps control adult, dark green. BNA, biceps neurogenic atrophy, dark blue.


Figure 3Relationship between area and polygon distribution . A ) Area distribution of several Voronoi diagrams along the CVT path . Cell areas became progressively more homogeneous after each Lloyd iterationB ) Comparison of area distribution for proliferating epithelia cNT ( blue ) , dWL ( green ) and dWP ( red ) . At the right of the image Biceps ( green ) and Diagram 5 ( grey ) images .

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Figure 3Relationship between area and polygon distribution. A) Area distribution of several Voronoi diagrams along the CVT path. Cell areas became progressively more homogeneous after each Lloyd iterationB) Comparison of area distribution for proliferating epithelia cNT (blue), dWL (green) and dWP (red). At the right of the image Biceps (green) and Diagram 5 (grey) images.


Figure 4A ) Segmentation of a prepupal eye imaginal disc from Drosophila ( anterior to the left ) . The analysed developing photoreceptors are labelled in green . The cells adjacent to the photoreceptors are labelled in blue ( these are " cone cells " in the most posterior clusters ) . B ) Segmentation of a prepupa wing imaginal disc epithelium where myosin II levels have been reduced . C ) Adult Biceps biopsy from a patient with neurogenic atrophy pathology . Fibres outlined with collagen VI antibody ( green ) . D ) Segmentation of the image showed in ( C ) highlighting two atrophic elongated fibres that present seven neighbours .

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Figure 4A) Segmentation of a prepupal eye imaginal disc from Drosophila (anterior to the left). The analysed developing photoreceptors are labelled in green. The cells adjacent to the photoreceptors are labelled in blue (these are "cone cells" in the most posterior clusters).B) Segmentation of a prepupa wing imaginal disc epithelium where myosin II levels have been reduced. C) Adult Biceps biopsy from a patient with neurogenic atrophy pathology. Fibres outlined with collagen VI antibody (green). D) Segmentation of the image showed in (C) highlighting two atrophic elongated fibres that present seven neighbours.


Figure 1 ( B ) Flat - mounted retinas from OIR S1pr1f / stop / f and S1pr1 ECTG pups at P17 . Blood vessels are stained with isolectin ( left ) and pathological neovascular tuft area are highlighted in yellow . Avascular area , total and average neovascular tuft areas are quantified ( right ) ( C ) Flat - mounted retinas from OIR S1pr1f / f and S1pr1 ECKO pups , at P17 . Blood vessels are stained with isolectin and neovascular tufts are highlighted in yellow . Avascular area , total and average neovascular tuft areas are quantified . ( D ) Cross sections from OIR S1pr1f / stop / f , S1pr1 ECTG and S1pr1 ECKO P17 pups . Blood vessels are stained with isolectin ( red ) and nuclei with Hoechst ( blue ) .

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Figure 1(B) Flat-mounted retinas from OIR S1pr1f/stop/f and S1pr1 ECTG pups at P17. Blood vessels are stained with isolectin (left) and pathological neovascular tuft area are highlighted in yellow. Avascular area, total and average neovascular tuft areas are quantified (right)(C) Flat-mounted retinas from OIR S1pr1f/f and S1pr1 ECKO pups, at P17. Blood vessels are stained with isolectin and neovascular tufts are highlighted in yellow. Avascular area, total and average neovascular tuft areas are quantified.(D) Cross sections from OIR S1pr1f/stop/f, S1pr1 ECTG and S1pr1 ECKO P17 pups. Blood vessels are stained with isolectin (red) and nuclei with Hoechst (blue).


Figure 2 ( A ) Retinal flat mounts from OIR S1pr1f / stop / f or S1pr1 ECTG or ECKO P17 pups . High - magnification view of neovascular tufts stained for VE - cadherin ( green ) and Claudin - 5 ( red ) . Junctional levels of VE - cadherin and Claudin - 5 were quantified as described , for a minimum of 5 animals per genotype . ( B ) Retinal flat mounts from OIR S1pr1f / stop / f or S1pr1 ECTG or ECKO P17 pups . Sites of vascular leakage were assessed by staining for fibrinogen ( red ) , blood vessels are delineated by isolectin ( green ) . Extravascular fibrinogen was quantified as described for a minimum of 5 animals per genotype . ( C ) Pericytes stained by NG2 on the surface of isolectin - positive neovascular tufts at P17 post - OIR ( left ) . EC - associated pericytes in neovascular tufts were quantified ( right ) . 70 tufts from 3 different animals were assessed . ( D ) Flat - mounted retinas from OIR S1pr1f / f and S1pr1 ECKO pups at P21 stained with isolectin . Avascular area , total and average neovascular tuft areas are quantified . A minimum of 7 pups were analyzed per group .

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Figure 2(A) Retinal flat mounts from OIR S1pr1f/stop/f or S1pr1 ECTG or ECKO P17 pups. High-magnification view of neovascular tufts stained for VE-cadherin (green) and Claudin-5 (red). Junctional levels of VE-cadherin and Claudin-5 were quantified as described, for a minimum of 5 animals per genotype.(B) Retinal flat mounts from OIR S1pr1f/stop/f or S1pr1 ECTG or ECKO P17 pups. Sites of vascular leakage were assessed by staining for fibrinogen (red), blood vessels are delineated by isolectin (green). Extravascular fibrinogen was quantified as described for a minimum of 5 animals per genotype.(C) Pericytes stained by NG2 on the surface of isolectin-positive neovascular tufts at P17 post-OIR (left). EC-associated pericytes in neovascular tufts were quantified (right). 70 tufts from 3 different animals were assessed.(D) Flat-mounted retinas from OIR S1pr1f/f and S1pr1 ECKO pups at P21 stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. A minimum of 7 pups were analyzed per group.


Figure 3 ( A ) Flat - mounted retinas from OIR Apom + / - and Apom - / - pups at P17 , stained with isolectin . Avascular area , total and average neovascular tuft areas are quantified . ( n = 10 pups / group ) ( B ) Flat - mounted retinas from OIR WT and ApomTG pups at P17 , stained with isolectin . Avascular area , total and average neovascular tuft areas are quantified . ( n = 7 pups / group ) ( C ) Flat - mounted retinas from SAR247799 - treated , OIR WT pups at P17 , stained with isolectin . Avascular area , total and average neovascular tuft areas are quantified . ( n = 10 pups / group ) ( D ) Flat - mounted retinas from ApoM - Fc - S1P - treated , OIR S1pr1f / stop / f or S1pr1 ECTG pups at P17 , stained with isolectin . Avascular area , total and average neovascular tuft areas are quantified . ( n = 6 pups / group )

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Figure 3(A) Flat-mounted retinas from OIR Apom+/- and Apom-/- pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 10 pups /group)(B) Flat-mounted retinas from OIR WT and ApomTG pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 7 pups /group)(C) Flat-mounted retinas from SAR247799-treated, OIR WT pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 10 pups /group)(D) Flat-mounted retinas from ApoM-Fc-S1P-treated, OIR S1pr1f/stop/f or S1pr1 ECTG pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 6 pups /group)


figf1b , MEFs were generated from day 12 . 5embryos of Atg16l1 mutant mice , and western blot analysis was used to detect the a and b isoforms of ATG16L1 from whole - cell lysatesc , d , Atg5 - / - and ATG16L1HM MEFs were grown in the presence of the chemical inducer of autophagy rapamycin ( Rap , 50 µg ml - 1 ) and cyclohexamide ( CHX , 5 µg ml - 1 ) for 0 , 4 , 8 and 12 h . Cell lysates were analysed by western blot for loss of P62 expression ( c ) . P62 levels were quantified by densitometry normalized to actin ( n = 3 ; d ) . * P 0 . 04 ; * * P 0 . 008 ; * * * P = 0 . 0003e , f , Western blot analysis of LC3 expression in MEFs grown in the presence of rapamycin ( 50 µg ml - 1 ) or dimethylsulphoxide ( DMSO ) control for 4 h ( e ) . LC3 - II expression in the presence of rapamycin was quantified and normalized to actin ( n = 6 ; f ) . g , Detection of ATG16L1 , LC3 and P62 by western blot of ileal lysates from ATG16L1HM mice . ATG16L1 can be detected in both mutant lines on longer exposures . h , i , Quantification of ATG16L1 ( h ) and P62 ( i ) normalized to actin detected in ileal lysates ( n = 3 ) . P values were calculated using two - tailed Student ' s t - test . Error bars , s . e . m .

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figf1b, MEFs were generated from day 12.5embryos of Atg16l1 mutant mice, and western blot analysis was used to detect the a and b isoforms of ATG16L1 from whole-cell lysatesc, d, Atg5-/- and ATG16L1HM MEFs were grown in the presence of the chemical inducer of autophagy rapamycin (Rap, 50 µg ml-1) and cyclohexamide (CHX, 5 µg ml-1) for 0, 4, 8 and 12 h. Cell lysates were analysed by western blot for loss of P62 expression (c). P62 levels were quantified by densitometry normalized to actin (n = 3; d). *P 0.04; **P 0.008; ***P = 0.0003e, f, Western blot analysis of LC3 expression in MEFs grown in the presence of rapamycin (50 µg ml-1) or dimethylsulphoxide (DMSO) control for 4 h (e). LC3-II expression in the presence of rapamycin was quantified and normalized to actin (n = 6; f).g, Detection of ATG16L1, LC3 and P62 by western blot of ileal lysates from ATG16L1HM mice. ATG16L1 can be detected in both mutant lines on longer exposures. h, i, Quantification of ATG16L1 (h) and P62 (i) normalized to actin detected in ileal lysates (n = 3). P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.


figf2a , b , Whole - mount images taken immediately above the ileal mucosal surface from WT ( a ) and ATG16L1HM ( b ) littermate mice stained with Helix pomatia lectin that labels mucus ( green ) and antisera directed against lysozyme ( red ) . Images are representative of 3 WT and 3 ATG16L1HM mice . c , d , Ileal sections from WT ( c ) and ATG16L1HM ( d ) mice were stained with PAS / alcian blue to detect Paneth granules by light microscopy ( dashed line denotes the crypt unit and arrowheads indicate Paneth cells ) . Images are representative of 7 WT , 7 ATG16L1HM1 and 5 ATG16L1HM2 mice ; more than 100 crypts were analysed for eache , f , Representative images of indirect immunofluorescence of sections stained for lysozyme ( red ) in WT ( e ) and ATG16L1HM ( f ) mouse ileal crypts . g , Paneth cells display one of four patterns of lysozyme expression ( represented in red ; white represents areas that exclude lysozyme ) : normal ( D0 ) , disordered ( D1 ) , depleted ( D2 ) and diffuse ( D3 ) . h , i , Number of Paneth cells from ATG16L1HM ( h ) and Atg5flox / floxvillin - Cre ( i ) mice displaying each pattern of lysozyme expression ( n = 6 , 660 cells from 5 ATG16L1HM mice and 5 , 634 cells from 5 WT mice ; n = 1 , 756 cells from 2 Atg5flox / floxvillin - Cre mice and 1 , 649 from 2 Atg5flox / flox mice ) . Scale bars : a , b , 200 µm ; c - f , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . P values were calculated using two - tailed Student ' s t - test . Error bars , s . e . m .

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figf2a, b, Whole-mount images taken immediately above the ileal mucosal surface from WT (a) and ATG16L1HM (b) littermate mice stained with Helix pomatia lectin that labels mucus (green) and antisera directed against lysozyme (red). Images are representative of 3 WT and 3 ATG16L1HM mice.c, d, Ileal sections from WT (c) and ATG16L1HM (d) mice were stained with PAS/alcian blue to detect Paneth granules by light microscopy (dashed line denotes the crypt unit and arrowheads indicate Paneth cells). Images are representative of 7 WT, 7 ATG16L1HM1 and 5 ATG16L1HM2 mice; more than 100 crypts were analysed for eache, f, Representative images of indirect immunofluorescence of sections stained for lysozyme (red) in WT (e) and ATG16L1HM (f) mouse ileal crypts.g, Paneth cells display one of four patterns of lysozyme expression (represented in red; white represents areas that exclude lysozyme): normal (D0), disordered (D1), depleted (D2) and diffuse (D3).h, i, Number of Paneth cells from ATG16L1HM (h) and Atg5flox/floxvillin-Cre (i) mice displaying each pattern of lysozyme expression (n = 6,660 cells from 5 ATG16L1HM mice and 5,634 cells from 5 WT mice; n = 1,756 cells from 2 Atg5flox/floxvillin-Cre mice and 1,649 from 2 Atg5flox/flox mice). Scale bars: a, b, 200 µm; c-f, 10 µm. *P 0.05, **P 0.01, ***P 0.001. P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.


figf3a - d , Electron micrograph of littermate WT ( a , c ) and ATG16L1HM ( b , d ) Paneth cells ( for a , b , dashed lines denote individual cells , asterisk indicates normal progenitor cell ; for c , d , blue stars indicate normal mitochondria in WT Paneth cells and ATG16L1HM progenitor cells , red diamonds indicate degenerating mitochondria , the dashed box shows normal Golgi compartments , and yellow asterisks indicate granules ; n = 3 mice per genotype ) . e , Mitochondrial integrity was quantified on the basis of the percentage of the visible mitochondrial section displaying intact cisternae ( n = 49 WT and 71 ATG16L1HM cells ) . g , Chart of selected genes for which mRNA is enriched in either ATG16L1 - deficient Paneth cells ( baseline is WT Paneth cells ) or ATG16L1 - deficient thymocytes ( baseline is WT thymocytes ) . Only haemoglobin is in common in these two sets of analyses . NS , not significant . Scale bars : a , b , 10 µm ; c , d , 500 nm .

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figf3a-d, Electron micrograph of littermate WT (a, c) and ATG16L1HM (b, d) Paneth cells (for a, b, dashed lines denote individual cells, asterisk indicates normal progenitor cell; for c, d, blue stars indicate normal mitochondria in WT Paneth cells and ATG16L1HM progenitor cells, red diamonds indicate degenerating mitochondria, the dashed box shows normal Golgi compartments, and yellow asterisks indicate granules; n = 3 mice per genotype). e, Mitochondrial integrity was quantified on the basis of the percentage of the visible mitochondrial section displaying intact cisternae (n = 49 WT and 71 ATG16L1HM cells).g, Chart of selected genes for which mRNA is enriched in either ATG16L1-deficient Paneth cells (baseline is WT Paneth cells) or ATG16L1-deficient thymocytes (baseline is WT thymocytes). Only haemoglobin is in common in these two sets of analyses. NS, not significant. Scale bars: a, b, 10 µm; c, d, 500 nm.


figf4a , b , Haematoxylin - and - eosin - stained sections of uninvolved areas from ileo - colic resections from patients with Crohn ' s disease homozygous for the safe ( a ) or risk ( b ) allele of ATG16L1 ( blue dashed line denotes crypt unit ) . c - f , Immunofluorescence images of Paneth cells from control patients ( c , e ) and patients with the risk allele ( d , f ) stained for lysozyme ( red ; c , d ) and double - labelled additionally for leptin ( green ; e , f ; yellow dashed line denotes the crypt unit ) . g , Aberrant lysozyme expression was quantified using the same criteria as that used for mouse sections in Fig . 3 ( n = 6 , 829 cells for at risk patients and 8 , 182 for control , n = 5 patients for each genotype ) . Leptin - positive D3 cells were quantified in patient samples ( n = 4 patients per genotype ) homozygous for the risk allele ( 76 out of 322 D3 cells were positive from a total of 580 crypts examined ) and homozygous for the safe allele ( 11 out of 93 D3 cells were positive from a total 749 crypts examined ) . Scale bars : a - f , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . P values were calculated using two - tailed Student ' s t - test . Error bars , s . e . m .

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figf4a, b, Haematoxylin-and-eosin-stained sections of uninvolved areas from ileo-colic resections from patients with Crohn's disease homozygous for the safe (a) or risk (b) allele of ATG16L1 (blue dashed line denotes crypt unit).c-f, Immunofluorescence images of Paneth cells from control patients (c, e) and patients with the risk allele (d, f) stained for lysozyme (red; c, d) and double-labelled additionally for leptin (green; e, f; yellow dashed line denotes the crypt unit).g, Aberrant lysozyme expression was quantified using the same criteria as that used for mouse sections in Fig. 3 (n = 6,829 cells for at risk patients and 8,182 for control, n = 5 patients for each genotype). Leptin-positive D3 cells were quantified in patient samples (n = 4 patients per genotype) homozygous for the risk allele (76 out of 322 D3 cells were positive from a total of 580 crypts examined) and homozygous for the safe allele (11 out of 93 D3 cells were positive from a total 749 crypts examined). Scale bars: a-f, 10 µm. *P 0.05, **P 0.01, ***P 0.001. P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.


Figure 3a . Vero E6 cells were infected with an moi of 0 . 05 with the fluorescent mCherry D614 strains , wild - type ISA D614 or mock infected . Pictures were taken at 48h pi ( 20x ) . Scale bar , 100 µM . b . After 5 passages ( p2 , p3 , p4 and p5 ) on VeroE6 cells , the mCherry reporter gene in ISA mCherry D614 supernatant medium was RT - PCR amplified and analysed using gel electrophoresis . ( d ) Representative neutralizing curves of the nAb fluorescence - based assay . The four - parameter dose - response curve was fitted using the nonlinear regression method and nAbs were calculated in the software Prism 7 . 0 . For negative serum samples , an arbitrary value of 10 was assigned ( detection threshold for both methods ) .

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Figure 3a.Vero E6 cells were infected with an moi of 0.05 with the fluorescent mCherry D614 strains, wild-type ISA D614 or mock infected. Pictures were taken at 48h pi (20x). Scale bar, 100 µM.b. After 5 passages (p2, p3, p4 and p5) on VeroE6 cells, the mCherry reporter gene in ISA mCherry D614 supernatant medium was RT-PCR amplified and analysed using gel electrophoresis.(d) Representative neutralizing curves of the nAb fluorescence-based assay. The four-parameter dose-response curve was fitted using the nonlinear regression method and nAbs were calculated in the software Prism 7.0. For negative serum samples, an arbitrary value of 10 was assigned (detection threshold for both methods).


Figure 4a : Dose response curve for the ISA D614 and for the mCherry D614 strains obtained by fluorescence or viral RNA measurement in VeroE6 cells from one representative experiment . Data are represented as mean ± SD ( indicated by the error bars ) . Each experiment was performed in technical triplicates ( N = 3 ) . b : Table of EC50 values obtained for the two different strains from two technical replicates . and their respective mean ± SD .

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Figure 4a: Dose response curve for the ISA D614 and for the mCherry D614 strains obtained by fluorescence or viral RNA measurement in VeroE6 cells from one representative experiment. Data are represented as mean ± SD (indicated by the error bars). Each experiment was performed in technical triplicates (N=3). b: Table of EC50 values obtained for the two different strains from two technical replicates. and their respective mean ± SD. c


Figure 1 ( B ) Islet graft survival of mice receiving vehicle ( PBS ) or IL - 33 was assessed by monitoring blood glucose and calculated using the Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical analysis was performed with a log - rank test . * * * P < 0 . 001 vs islet + vehicle . ( C ) Blood glucose level of mice treated with IL - 33 ( the horizontal black line indicates a BGL of 16 mmol / liter , the threshold for rejection ) . Each line represents one mouse . ( D ) Intraperitoneal glucose tolerance test ( IPGTT ) was assessed in normal mice , islet transplant mice receiving vehicle ( on the day when grafts were considered rejected ) , and islet transplant mice treated with IL - 33 ( at day 30 and day 80 post - islet transplantation ) . Data shown are the mean ± SEM ( n = 6 - 9 per group ) . ( E ) Area under the curve ( AUC ) for IPGTT was assessed . Data shown are the mean ± SEM ( n = 6 - 9 per group ) and a one - way ANOVA was performed , * * * P < 0 . 001 . ( F ) Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL - 33 . Scale bar = 100 µm .

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 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

Figure 1(B) Islet graft survival of mice receiving vehicle (PBS) or IL-33 was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001 vs islet+vehicle. (C) Blood glucose level of mice treated with IL-33 (the horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection). Each line represents one mouse.(D) Intraperitoneal glucose tolerance test (IPGTT) was assessed in normal mice, islet transplant mice receiving vehicle (on the day when grafts were considered rejected), and islet transplant mice treated with IL-33 (at day 30 and day 80 post-islet transplantation). Data shown are the mean ± SEM (n=6-9 per group). (E) Area under the curve (AUC) for IPGTT was assessed. Data shown are the mean ± SEM (n=6-9 per group) and a one-way ANOVA was performed, ***P<0.001. (F) Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL-33. Scale bar = 100 µm.


Figure 2C57BL / 6 mice were treated with mouse recombinant IL - 33 or PBS daily for 5 consecutive days . ( A , B ) Representative FACS analysis showing the proportion of Tregs ( CD4 + Foxp3 + ) in the CD4 + T cell compartment from the spleens ( A ) and kidneys ( B ) at day 3 after treatment in C57BL / 6 mice receiving PBS ( n = 4 ) or IL - 33 ( n = 6 ) . C57BL / 6 mice were treated with mouse recombinant IL - 33 or PBS daily for 5 consecutive days . ( C , D ) Representative FACS analysis showing the proportion of ILC2s ( Lin - GATA - 3 + ) in the CD45 + leukocyte compartment from the spleens ( C ) and kidneys ( D ) at day 3 after treatment in C57BL / 6 mice receiving PBS ( n = 4 ) or IL - 33 ( n = 6 ) . ( E , F ) proportion of Tregs ( E ) and ILC2s ( F ) in the spleen and kidney in PBS - injected controls ( n = 4 ) and at weeks 1 - 12 after IL - 33 treatment ( n = 4 - 6 per IL - 33 - treated group ) . Data information : Data shown are the mean ± SEM ; Statistical analysis was performed with an unpaired t - test , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 2C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (A, B) Representative FACS analysis showing the proportion of Tregs (CD4+Foxp3+) in the CD4+ T cell compartment from the spleens (A) and kidneys (B) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6). C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (C, D) Representative FACS analysis showing the proportion of ILC2s (Lin-GATA-3+) in the CD45+ leukocyte compartment from the spleens (C) and kidneys (D) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).(E, F) proportion of Tregs (E) and ILC2s (F) in the spleen and kidney in PBS-injected controls (n=4) and at weeks 1-12 after IL-33 treatment (n=4-6 per IL-33-treated group). Data information: Data shown are the mean ± SEM; Statistical analysis was performed with an unpaired t-test, **P<0.01, ***P<0.001.


Figure 3 ( B - D ) Proportion or numbers of CD4 + Foxp3 + Tregs in the spleens , kidneys and islet grafts of normal , islet transplant mice receiving vehicle and islet transplant mice with IL - 33 treatment ( at day 7 , 30 and 80 post - islet transplantation ) . Data shown are the mean ± SEM ( n = 4 - 6 per group ) and a one - way ANOVA was performed ; * * * P < 0 . 001 . ( E ) Representative confocal microscopy images of immunostaining for CD4 , Foxp3 and insulin in islet grafts . Scale bar = 50 µm . ( F - H ) Proportion or numbers of ILC2s in the spleens , kidneys and islet graft of mice with or without IL - 33 treatment . Data shown are the mean ± SEM ( n = 4 - 6 per group ) and a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 . ( I ) Representative confocal microscopy images of immunostaining for CD127 , ST2 , CD3 and insulin in islet grafts . Scale bar = 50 µm . ( J ) The mRNA expression of IL - 25 , IL - 33 and TSLP in islet grafts of mice with or without IL - 33 treatment was examined by qPCR , and expressed relative to the control of each experiment . Data shown are the mean ± SEM ( n = 4 - 6 per group ) and a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 3(B-D) Proportion or numbers of CD4+Foxp3+ Tregs in the spleens, kidneys and islet grafts of normal, islet transplant mice receiving vehicle and islet transplant mice with IL-33 treatment (at day 7, 30 and 80 post-islet transplantation). Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.(E) Representative confocal microscopy images of immunostaining for CD4, Foxp3 and insulin in islet grafts. Scale bar = 50 µm.(F-H) Proportion or numbers of ILC2s in the spleens, kidneys and islet graft of mice with or without IL-33 treatment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.(I) Representative confocal microscopy images of immunostaining for CD127, ST2, CD3 and insulin in islet grafts. Scale bar = 50 µm.(J) The mRNA expression of IL-25, IL-33 and TSLP in islet grafts of mice with or without IL-33 treatment was examined by qPCR, and expressed relative to the control of each experiment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


Figure 4Proportion of CD4 + Foxp3 + Tregs ( B ) from the spleens and kidneys of islet transplant mice receiving vehicle , IL - 33 , IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 at day 5 post - islet transplantation . Data shown are the mean ± SEM ( n = 4 - 5 per group ) and a one - way ANOVA was performed ; NS : non significant , * * * P < 0 . 001 . Proportion of Lin - GATA - 3 + ILC2s ( C ) from the spleens and kidneys of islet transplant mice receiving vehicle , IL - 33 , IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 at day 5 post - islet transplantation . Data shown are the mean ± SEM ( n = 4 - 5 per group ) and a one - way ANOVA was performed ; NS : non significant , * * * P < 0 . 001 . ( D ) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical analysis was performed with a log - rank test . * P < 0 . 05 , * * P < 0 . 01 . ( E ) Data are shown as blood glucose measurement in islet transplant mice treated with IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 . The horizontal black line indicates a BGL of 16 mmol / liter , the threshold for rejection . Each line represents one mouse .

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Figure 4Proportion of CD4+Foxp3+ Tregs (B) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.Proportion of Lin-GATA-3+ ILC2s (C) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.(D) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. *P<0.05, **P<0.01.(E) Data are shown as blood glucose measurement in islet transplant mice treated with IL-33/DT, IL-33/PC61, or IL-33/DT/PC61. The horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection. Each line represents one mouse.


Figure 5 ( A , B ) ILC210 and non - IL - 10 producing ILC2s were assessed in islet graft ( A ) and kidney ( B ) by intracellular IL - 10 staining at day 5 post islet transplantation . Data shown are the mean ± SEM ( n = 5 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 . ST2 : suppression of tumorigenicity 2 . ( C ) IL - 10 reporter C57BL / 6 mice were treated with PBS , IL - 33 alone or IL - 33 and IL - 2C daily for 5 consecutive days . Proportion of lin - CD127 + ST2 + IL - 10 - GFP ILC210 from the kidneys at day 3 after treatment in IL - 10 reporter C57BL / 6 mice . Data shown are the mean ± SEM ( n = 4 - 6 per group ) and a one - way ANOVA was performed ; * * * P < 0 . 001 . ( D ) Kidney ILC2s isolated from normal IL - 10 reporter C57BL / 6 mice were cultured with Medium only or Medium with IL - 33 and IL - 2C for six days . Proportion of ILC210 was assessed by flow cytometry . Data shown are the mean ± SEM ( n = 6 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 . ST2 : suppression of tumorigenicity 2 . ( E ) The secreted cytokine IL - 10 was analyzed via ELISA . Data shown are the mean ± SEM ( n = 6 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 . ( F ) Kidney ILC2s isolated from C57BL / 6 mice were cultured with Medium only or Medium with IL - 33 and IL - 2C for 30 minutes . The expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry . p - STAT5 ( red lines ) and isotype controls ( gray - filled areas ) are shown . Data represent the mean ± SEM of evaluations of MFI from each group ( n = 6 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 . MFI , mean fluorescence intensity . ( G ) The kidney ILC2s were cultured with Medium , IL - 33 / IL - 2C , or IL - 33 / IL - 2C and STAT5 inhibitor ( STAT5 - IN ) for three days . The secreted cytokine IL - 10 was analyzed via ELISA . Data shown are the mean ± SEM ( n = 6 per group ) and a one - way ANOVA was performed ; * * * P < 0 . 001 .

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Figure 5(A,B) ILC210 and non-IL-10 producing ILC2s were assessed in islet graft (A) and kidney (B) by intracellular IL-10 staining at day 5 post islet transplantation. Data shown are the mean ± SEM (n=5 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.(C) IL-10 reporter C57BL/6 mice were treated with PBS, IL-33 alone or IL-33 and IL-2C daily for 5 consecutive days. Proportion of lin-CD127+ST2+IL-10-GFP ILC210 from the kidneys at day 3 after treatment in IL-10 reporter C57BL/6 mice. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.(D) Kidney ILC2s isolated from normal IL-10 reporter C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for six days. Proportion of ILC210 was assessed by flow cytometry. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.(E) The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.(F) Kidney ILC2s isolated from C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for 30 minutes. The expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry. p-STAT5 (red lines) and isotype controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=6 per group) and an unpaired t-test was performed; ***P<0.001. MFI, mean fluorescence intensity.(G) The kidney ILC2s were cultured with Medium, IL-33/IL-2C, or IL-33/IL-2C and STAT5 inhibitor (STAT5-IN) for three days. The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and a one-way ANOVA was performed; ***P<0.001.


Figure 6 ( A ) ILC210 and non - ILC210 were isolated from ex vivo - expanded kidney ILC2 by flow sorting . ILC210 were transfected with control ( ILC210 - C ) or IL - 10 CRISPR - Cas9 ( ILC210 - IL - 10 ) . IL - 10 was measured in culture supernatant of ILC210 - C and ILC210 - IL - 10 via ELISA . Data shown are the mean ± SEM ( n = 6 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 . ( C ) Transfused ILC2s ( CFSE labeled , Green ) were observed in islet graft at day 5 post islet transplantation . Scale bar = 50 µm . The numbers of CFSE - labeled ILC2s in the islet graft were counted . Data shown are the mean ± SEM and a one - way ANOVA was performed ( n = 4 per group ) . NS , not significant . ( D ) The mRNA expression of IL - 10 in islet grafts at day 5 post islet transplantation was examined by qPCR . Data shown are the mean ± SEM ( n = 4 per group ) and a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 . ( E ) Islet graft survival of four groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical analysis was performed with a log - rank test . * * * P < 0 . 001 . ( F ) CD4 + T cells isolated from C57BL / 6 splenocytes were cultured with irradiated splenocytes derived from BALB / c in the presence of ILC210 at the indicated ratios for 4 days . CD4 + T cells proliferation was assessed using [ 3H ] - thymidine incorporation assays . Data shown are the mean ± SEM ( n = 4 - 6 per group ) . ( G ) Neutralizing anti - IL - 10 antibodies or genetic ablation of IL - 10 were used to block the effect of ILC210 on CD4 + T cells proliferation . Data shown are the mean ± SEM ( n = 6 per group ) and an unpaired t - test was performed . * * * P < 0 . 001 .

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Figure 6(A) ILC210 and non-ILC210 were isolated from ex vivo-expanded kidney ILC2 by flow sorting. ILC210 were transfected with control (ILC210-C) or IL-10 CRISPR-Cas9 (ILC210-IL-10). IL-10 was measured in culture supernatant of ILC210-C and ILC210-IL-10 via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.(C) Transfused ILC2s (CFSE labeled, Green) were observed in islet graft at day 5 post islet transplantation. Scale bar = 50 µm. The numbers of CFSE-labeled ILC2s in the islet graft were counted. Data shown are the mean ± SEM and a one-way ANOVA was performed (n=4 per group). NS, not significant.(D) The mRNA expression of IL-10 in islet grafts at day 5 post islet transplantation was examined by qPCR. Data shown are the mean ± SEM (n=4 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.(E) Islet graft survival of four groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.(F) CD4+ T cells isolated from C57BL/6 splenocytes were cultured with irradiated splenocytes derived from BALB/c in the presence of ILC210 at the indicated ratios for 4 days. CD4+ T cells proliferation was assessed using [3H]-thymidine incorporation assays. Data shown are the mean ± SEM (n=4-6 per group).(G) Neutralizing anti-IL-10 antibodies or genetic ablation of IL-10 were used to block the effect of ILC210 on CD4+ T cells proliferation. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed. ***P<0.001.


Figure 7 ( B ) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical analysis was performed with a log - rank test . * * * P < 0 . 001 . ( C ) To track ILC210 in vivo , CD45 . 2 + ILC210 were co - transplanted with islets in diabetic CD45 . 1 + C57BL / 6 mice . Representative FACS analysis showing the proportion of locally transplanted ILC210 ( CD45 . 2 + ST2 + ) in the total CD45 + cell compartment from islet graft at day 5 and 80 post - islet transplantation or at the day when grafts were considered rejected . ( D , E ) Proportion and numbers of locally transplanted CD45 . 2 + ST2 + ILC210 in islet grafts of islet transplant mice over time . Data shown are the mean ± SEM ( n = 4 - 5 per group ) and a one - way ANOVA was performed , * * P < 0 . 01 , * * * P < 0 . 001 . ( F ) The expression of IL - 10 - GFP was examined in locally transplanted CD45 . 2 + ST2 + ILC210 by flow cytometry . IL - 10 - GFP ( red lines ) and controls ( gray - filled areas ) are shown . Data represent the mean ± SEM of evaluations of MFI from each group ( n = 4 - 5 per group ) and a one - way ANOVA was performed ; * * * P < 0 . 001 . MFI , mean fluorescence intensity .

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Figure 7(B) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.(C) To track ILC210 in vivo, CD45.2+ ILC210 were co-transplanted with islets in diabetic CD45.1+ C57BL/6 mice. Representative FACS analysis showing the proportion of locally transplanted ILC210 (CD45.2+ST2+) in the total CD45+ cell compartment from islet graft at day 5 and 80 post-islet transplantation or at the day when grafts were considered rejected.(D,E) Proportion and numbers of locally transplanted CD45.2+ST2+ ILC210 in islet grafts of islet transplant mice over time. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed, **P<0.01, ***P<0.001.(F) The expression of IL-10-GFP was examined in locally transplanted CD45.2+ST2+ ILC210 by flow cytometry. IL-10-GFP (red lines) and controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=4-5 per group) and a one-way ANOVA was performed; ***P<0.001. MFI, mean fluorescence intensity.


Figure 1C . LITATS1 expression upon SMAD4 knockdown ( as detected by RT ‒ qPCR ) in MDA - MB - 231 cells . Cells were serum starved for 16 h and TGF - β was added for 4 h . Representative results from a minimum of three independent experiments are shown . D . LITATS1 expression ( as detected by RT ‒ qPCR ) in HEK293T cells . Cells were transfected without ( Co . vec ) or with the constitutively active TGF - β type I receptor ( caTβRI ) ectopic expression construct . Representative results from a minimum of three independent experiments are shown . EJ . RNA fluorescence in situ hybridization was performed to evaluate LITATS1 expression and subcellular localization in A549 cells . Cells were treated with or without TGF - β for 2 h . Representative images are shown in the left panel , and signal quantification data are shown in the right panel . Scale bar = 10 μm . Representative results from two independent experiments are shown .

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 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

Figure 1C. LITATS1 expression upon SMAD4 knockdown (as detected by RT‒qPCR) in MDA-MB-231 cells. Cells were serum starved for 16 h and TGF-β was added for 4 h. Representative results from a minimum of three independent experiments are shown. D. LITATS1 expression (as detected by RT‒qPCR) in HEK293T cells. Cells were transfected without (Co.vec) or with the constitutively active TGF-β type I receptor (caTβRI) ectopic expression construct. Representative results from a minimum of three independent experiments are shown. EJ. RNA fluorescence in situ hybridization was performed to evaluate LITATS1 expression and subcellular localization in A549 cells. Cells were treated with or without TGF-β for 2 h. Representative images are shown in the left panel, and signal quantification data are shown in the right panel. Scale bar=10 μm. Representative results from two independent experiments are shown.