Figure 2WAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment . One sister chromatid was labelled per chromosome as in Fig . 1 SCC1 ( WAPL - dTAG cells ) was visualised by immunofluorescence . A , Representative images of control WAPL - dTAG cells synchronised to G2 by RO - 3306 . B , Representative images of ΔWAPL cells synchronised to G2 by RO - 3306 . C , Representative images of ΔSororin cells synchronised to G2 by RO - 3306 . D , Quantification of sister chromatid separation as in Fig . 1D Control WAPL - dTAG G2 ( n = 36 cells ) , ΔWAPL G2 ( n = 53 cells ) and ΔSororin G2 ( n = 45 cells ) cells were analysed . Dots represent individual cells ; red bars indicate the mean . Significance was tested using a two - tailed Mann Whitney U test ; P = 3 . 89 x 10 - 11 ( Control G2 , ΔWAPL G2 ) , P = 5 . 90 x 10 - 7 ( Control G2 , ΔSororin G2 ) , P = 2 . 75 x 10 - 3 ( ΔWAPL G2 , ΔSororin G2 ) . Biological replicates : Control WAPL - dTAG G2 ( n = 3 ) , ΔWAPL G2 ( n = 3 ) , ΔSororin G2 ( n = 3 ) , ΔWAPL ΔSororin G2 ( n = 3 ) , wild type late prophase ( n = 4 ) . Technical replicates Control WAPL - dTAG G2 ( n = 6 ) , ΔWAPL G2 ( n = 6 ) , ΔSororin G2 ( n = 6 ) , ΔWAPL ΔSororin G2 ( n = 6 ) , wild type late prophase ( n = 7 ) . All images show single Z - slices from 3D - stacks . Yellow boxes indicate inset regions . To aid visualisation , the SCC1 channel is not contrast matched . Scale bars large panels : 5 μm , insets : 2 μWAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment . One sister chromatid was labelled per chromosome as in Fig . 1 and SMC4 ( wild type late prophase cells ) or SCC1 ( WAPL - dTAG cells ) was visualised by immunofluorescence . E , Representative images of ΔWAPL ΔSororin cells synchronised to G2 by RO - 3306 . F , Representative images of wild type cells synchronised to late prophase by release from a RO - 3306 - mediated G2 arrest . G , Quantification of sister chromatid separation as in Fig . 1D , E . ΔWAPL ΔSororin G2 cells ( n = 51 cells ) and wild type late prophase ( n = 55 cells ) cells were analysed . Dots represent individual cells ; red bars indicate the mean . Biological replicates : Control WAPL - dTAG G2 ( n = 3 ) , ΔWAPL G2 ( n = 3 ) , ΔSororin G2 ( n = 3 ) , ΔWAPL ΔSororin G2 ( n = 3 ) , wild type late prophase ( n = 4 ) . Technical replicates Control WAPL - dTAG G2 ( n = 6 ) , ΔWAPL G2 ( n = 6 ) , ΔSororin G2 ( n = 6 ) , ΔWAPL ΔSororin G2 ( n = 6 ) , wild type late prophase ( n = 7 ) . All images show single Z - slices from 3D - stacks . Yellow boxes indicate inset regions . To aid visualisation , the SCC1 channel is not contrast matched . Scale bars large panels : 5 μm , insets : 2 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 2WAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment. One sister chromatid was labelled per chromosome as in Fig. 1 SCC1 (WAPL-dTAG cells) was visualised by immunofluorescence. A, Representative images of control WAPL-dTAG cells synchronised to G2 by RO-3306. B, Representative images of ΔWAPL cells synchronised to G2 by RO-3306. C, Representative images of ΔSororin cells synchronised to G2 by RO-3306. D, Quantification of sister chromatid separation as in Fig. 1D Control WAPL-dTAG G2 (n = 36 cells), ΔWAPL G2 (n = 53 cells) and ΔSororin G2 (n = 45 cells) cells were analysed. Dots represent individual cells; red bars indicate the mean. Significance was tested using a two-tailed Mann Whitney U test; P = 3.89 x 10-11 (Control G2, ΔWAPL G2), P = 5.90 x 10-7 (Control G2, ΔSororin G2), P = 2.75 x 10-3 (ΔWAPL G2, ΔSororin G2). Biological replicates: Control WAPL-dTAG G2 (n = 3), ΔWAPL G2 (n = 3), ΔSororin G2 (n = 3), ΔWAPL ΔSororin G2 (n = 3), wild type late prophase (n = 4). Technical replicates Control WAPL-dTAG G2 (n = 6), ΔWAPL G2 (n = 6), ΔSororin G2 (n = 6), ΔWAPL ΔSororin G2 (n = 6), wild type late prophase (n = 7). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. To aid visualisation, the SCC1 channel is not contrast matched. Scale bars large panels: 5 μm, insets: 2 μWAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment. One sister chromatid was labelled per chromosome as in Fig. 1 and SMC4 (wild type late prophase cells) or SCC1 (WAPL-dTAG cells) was visualised by immunofluorescence. E, Representative images of ΔWAPL ΔSororin cells synchronised to G2 by RO-3306. F, Representative images of wild type cells synchronised to late prophase by release from a RO-3306-mediated G2 arrest. G, Quantification of sister chromatid separation as in Fig. 1D, E. ΔWAPL ΔSororin G2 cells (n = 51 cells) and wild type late prophase (n = 55 cells) cells were analysed. Dots represent individual cells; red bars indicate the mean. Biological replicates: Control WAPL-dTAG G2 (n = 3), ΔWAPL G2 (n = 3), ΔSororin G2 (n = 3), ΔWAPL ΔSororin G2 (n = 3), wild type late prophase (n = 4). Technical replicates Control WAPL-dTAG G2 (n = 6), ΔWAPL G2 (n = 6), ΔSororin G2 (n = 6), ΔWAPL ΔSororin G2 (n = 6), wild type late prophase (n = 7). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. To aid visualisation, the SCC1 channel is not contrast matched. Scale bars large panels: 5 μm, insets: 2 μm.


Figure 3A - C , Analysis of sister DNA and SMC protein complex distribution in cross sections perpendicular to the long chromosome axis , as shown in Fig . 2B , E , F . Density of Hoechst minus F - ara - EdU was calculated by subtracting F - ara - EdU fluorescence from Hoechst fluorescence . Line profiles indicate the mean curves of individual line profile measurements aligned to the midpoint between two SMC4 peaks ( wild type late prophase ) , the SCC1 peak ( ΔWAPL G2 cells ) , or the midpoint between two SCC1 peaks ( ΔWAPL ΔSororin G2 cells ) . A , Lines from wild type late prophase ( n = 157 lines from 31 cells ) . B , ΔWAPL G2 cells ( n = 110 lines from 18 cells ) . C , ΔWAPL ΔSororin G2 cells ( n = 138 lines from 18 cells ) were analysed . Wild type late prophase ( n = 4 ) , ΔWAPL G2 ( n = 3 ) , ΔWAPL ΔSororin G2 ( n = 3 ) . Technical replicates : Wild type late prophase ( n = 7 ) , ΔWAPL G2 ( n = 6 ) , ΔWAPL ΔSororin G2 ( n = 5 ) . Scale bars : 2μm .

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

Figure 3A-C, Analysis of sister DNA and SMC protein complex distribution in cross sections perpendicular to the long chromosome axis, as shown in Fig. 2B, E, F. Density of Hoechst minus F-ara-EdU was calculated by subtracting F-ara-EdU fluorescence from Hoechst fluorescence. Line profiles indicate the mean curves of individual line profile measurements aligned to the midpoint between two SMC4 peaks (wild type late prophase), the SCC1 peak (ΔWAPL G2 cells), or the midpoint between two SCC1 peaks (ΔWAPL ΔSororin G2 cells). A, Lines from wild type late prophase (n = 157 lines from 31 cells). B, ΔWAPL G2 cells (n = 110 lines from 18 cells). C, ΔWAPL ΔSororin G2 cells (n = 138 lines from 18 cells) were analysed. Wild type late prophase (n = 4), ΔWAPL G2 (n = 3), ΔWAPL ΔSororin G2 (n = 3). Technical replicates: Wild type late prophase (n = 7), ΔWAPL G2 (n = 6), ΔWAPL ΔSororin G2 (n = 5). Scale bars: 2μm.


Figure 4A , Representative images of one - sister labelled sister chromatids from ΔWAPL , ΔSMC4 ΔWAPL , or ΔSMC4 ΔWAPL ΔSororin prometaphase cells , as indicated . SMC4 was depleted in G1 through the addition of 5 - Ph - IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins . WAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment . B , Quantification of sister chromatid separation as in Fig . 1D , E . ΔWAPL ( n = 21 cells ) , ΔWAPL ΔSMC4 ( n = 25 cells ) , and ΔWAPL ΔSMC4 ΔSororin ( n = 28 cells ) prometaphase cells were analysed . Dots represent individual cells ; red bars indicate the mean . Significance was tested using a two - tailed Mann Whitney U test ; P = 6 . 57 x 10 - 8 ( ΔWAPL ) ; P = 3 . 64 x 10 - 7 ( ΔWAPL ΔSMC4 ΔSororin ) . Biological replicates , ΔWAPL prometaphase ( n = 3 ) , ΔWAPL ΔSMC4 prometaphase ( n = 3 ) , ΔWAPL ΔSMC4 ΔSororin prometaphase ( n = 3 ) . C , Representative images of control prometaphase cells labelled on one sister chromatid and depleted of SMC4 for different amounts of time as indicated . SMC4 was visualised by immunofluorescence using an anti - SMC4 antibody . Cells were synchronised to G2 phase by RO - 3306 , before being arrested in prometaphase by STLC for 60 min . SMC4 was then depleted through the addition of 1 μM 5 - Ph - IAA for 120 or 240 minutes , as indicated . Control ( n = 2 ) , ΔSMC4 120 min ( n = 2 ) , ΔSMC4 240 min ( n = 2 ) . All images show single Z - slices from 3D - stacks . Yellow boxes indicate inset regions . Scale bars large panels : 5 μm , insets : 1 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 4A, Representative images of one-sister labelled sister chromatids from ΔWAPL, ΔSMC4 ΔWAPL, or ΔSMC4 ΔWAPL ΔSororin prometaphase cells, as indicated. SMC4 was depleted in G1 through the addition of 5-Ph-IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins. WAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment. B, Quantification of sister chromatid separation as in Fig. 1D, E. ΔWAPL (n = 21 cells), ΔWAPL ΔSMC4 (n = 25 cells), and ΔWAPL ΔSMC4 ΔSororin (n = 28 cells) prometaphase cells were analysed. Dots represent individual cells; red bars indicate the mean. Significance was tested using a two-tailed Mann Whitney U test; P = 6.57 x 10-8 (ΔWAPL); P = 3.64 x 10-7 (ΔWAPL ΔSMC4 ΔSororin). Biological replicates , ΔWAPL prometaphase (n = 3), ΔWAPL ΔSMC4 prometaphase (n = 3), ΔWAPL ΔSMC4 ΔSororin prometaphase (n = 3).C, Representative images of control prometaphase cells labelled on one sister chromatid and depleted of SMC4 for different amounts of time as indicated. SMC4 was visualised by immunofluorescence using an anti-SMC4 antibody. Cells were synchronised to G2 phase by RO-3306, before being arrested in prometaphase by STLC for 60 min. SMC4 was then depleted through the addition of 1 μM 5-Ph-IAA for 120 or 240 minutes, as indicated. Control (n = 2), ΔSMC4 120 min (n = 2), ΔSMC4 240 min (n = 2). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. Scale bars large panels: 5 μm, insets: 1 μm.


Figure 5C , Genomic resolution analysis for G2 cells . Wild type , ΔNIPBL , ΔSMC4 , ΔWAPL , ΔSororin , and ΔWAPL ΔSororin cells were analysed . Points indicate the values calculated for each replicate , bars indicate the mean and error bars indicate the standard deviation . Significance was tested using a two - tailed Mann Whitney U test ; P = 1 . 24 x 10 - 4 ( ΔNIPBL G2 ) ; P = 1 . 62 x 10 - 4 ( ΔWAPL G2 ) ; P = 5 . 49 x 10 - 3 ( ΔSororin G2 ) , P = 1 . 47 x 10 - 3 ( ΔWAPL ΔSororin G2 ) . C , Wild type G2 ( n = 11 ) , ΔNIPBL G2 ( n = 10 ) , ΔSMC4 G2 ( n = 4 ) , ΔWAPL G2 ( n = 6 ) , ΔSororin G2 ( n = 3 ) , ΔWAPL ΔSororin G2 ( n = 4 ) .

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

Figure 5C, Genomic resolution analysis for G2 cells. Wild type, ΔNIPBL, ΔSMC4, ΔWAPL, ΔSororin, and ΔWAPL ΔSororin cells were analysed. Points indicate the values calculated for each replicate, bars indicate the mean and error bars indicate the standard deviation. Significance was tested using a two-tailed Mann Whitney U test; P = 1.24 x 10-4 (ΔNIPBL G2); P = 1.62 x 10-4 (ΔWAPL G2); P = 5.49 x 10-3 (ΔSororin G2), P = 1.47 x 10-3 (ΔWAPL ΔSororin G2). C, Wild type G2 (n = 11), ΔNIPBL G2 (n = 10), ΔSMC4 G2 (n = 4), ΔWAPL G2 (n = 6), ΔSororin G2 (n = 3), ΔWAPL ΔSororin G2 (n = 4).


Figure 1 ( A ) Tru - Seq RNA sequence analysis of E12 . 5 midbrain floor - plate ( mFP ) , midbrain roof - plate ( mRP ) , anterior ( A , adjacent anterior FP ) and posterior ( P , adjacent posterior FP ) . Pbx1 is enriched in the midbrain FP , together with Th and Pitx3 . Lower levels of Pbx3 are also expressed in the mFP . Gdf7 and Wnt1 are restricted to the mRP at E12 . 5 . ( B ) Pbx1 is expressed in the intermediate zone ( IZ ) and marginal zones ( MZ ) , but not the ventricular zone ( VZ ) , of the mFP at E12 . 5 , as detected by in situ hybridization . ( C ) PBX1 is first detected in the ventro - lateral part of the LMX1a + domain at E10 , preceding the birth of the first ( TH + ) mDA neurons at E10 . 5 . At E12 . 5 , PBX1 is present in all mDA neurons , but not all PBX1 + cells are TH + . White boxes indicate the area showed in higher magnification ( right ) . ( D ) At E11 . 5 , PBX1 protein defines a subpopulation of NURR1 + neuroblasts and labels all NURR1 + TH + mDA neurons . ( E ) PBX1 co - localizes with PITX3 and is also detected in a subpopulation of NURR1 + PITX3 - postmitotic neuroblasts at E12 . 5 . Higher magnification revealed three different populations of postmitotic cells : primary neuroblasts ( NURR1 + PBX1A - PITX3 - cells , green ) , secondary neuroblasts ( NURR1 + PBX1A + PITX3 - cells , yellow / orange ) and tertiary neuroblasts / mDA neurons ( NURR1 + PBX1A + PITX3 + cells , white ) . Nuclear staining , Dapi ( 4 ′ , 6 - diamidino - 2 - phenylindole , blue ) . All scale bars , 20µm .

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 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 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 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

Figure 1(A) Tru-Seq RNA sequence analysis of E12.5 midbrain floor-plate (mFP), midbrain roof-plate (mRP), anterior (A, adjacent anterior FP) and posterior (P, adjacent posterior FP). Pbx1 is enriched in the midbrain FP, together with Th and Pitx3. Lower levels of Pbx3 are also expressed in the mFP. Gdf7 and Wnt1 are restricted to the mRP at E12.5.(B) Pbx1 is expressed in the intermediate zone (IZ) and marginal zones (MZ), but not the ventricular zone (VZ), of the mFP at E12.5, as detected by in situ hybridization.(C) PBX1 is first detected in the ventro-lateral part of the LMX1a+ domain at E10, preceding the birth of the first (TH+) mDA neurons at E10.5. At E12.5, PBX1 is present in all mDA neurons, but not all PBX1+ cells are TH+. White boxes indicate the area showed in higher magnification (right).(D) At E11.5, PBX1 protein defines a subpopulation of NURR1+ neuroblasts and labels all NURR1+TH+ mDA neurons.(E) PBX1 co-localizes with PITX3 and is also detected in a subpopulation of NURR1+PITX3- postmitotic neuroblasts at E12.5. Higher magnification revealed three different populations of postmitotic cells: primary neuroblasts (NURR1+PBX1A-PITX3-cells, green), secondary neuroblasts (NURR1+PBX1A+PITX3-cells, yellow/orange) and tertiary neuroblasts/mDA neurons (NURR1+PBX1A+PITX3+cells, white). Nuclear staining, Dapi (4′,6-diamidino-2-phenylindole, blue). All scale bars, 20µm.


Figure 2 ( A ) PBX1A is the isoform detected in the TH + NURR1 + mDA neurons at E12 . 5 . ( C ) Immunofluorescence microscopy analysis of 7 - week old human VM tissue showing that all mDAn are positive for PBX1 , suggesting a conserved role for this factor in the development of these neurons in mice and humans . ( D ) PBX1 is also present in TH + adult mDA neurons of the substantia nigra ( SN ) and ventral tegmental area ( VTA ) .

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 O O O O O O O O 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

Figure 2(A) PBX1A is the isoform detected in the TH+NURR1+mDA neurons at E12.5.(C) Immunofluorescence microscopy analysis of 7-week old human VM tissue showing that all mDAn are positive for PBX1, suggesting a conserved role for this factor in the development of these neurons in mice and humans.(D) PBX1 is also present in TH+ adult mDA neurons of the substantia nigra (SN) and ventral tegmental area (VTA).


Figure 3 ( A ) To the left , representative immunofluorescence microscopy / confocal images showing that the number of TH + cells is reduced in Pbx1 - / - embryos . Deletion of Pbx1 decreases the proportion of NURR1 + neuroblasts that become TH + neurons at E12 . 5 , indicating that Pbx1 is required for the proper maturation of NURR1 + neuroblasts . To the right , quantification of these experiments . ( B ) Ectopic PBX3 is detected in all rostro - caudal levels of the VM in Pbx1 - / - mice at E12 . 5 , indicating a repressive function of PBX1 on Pbx3 expression . ( C ) Representative immunofluorescence microscopy / confocal images and quantification showing that the conditional deletion of Pbx1 and Pbx3 ( cKO , Pbx1flox / flox ; Pbx3 - / - ; Th - IRES - Cre - ERT ) reduces the number of PITX3 + cells in rostral VM levels , but not of NURR1 + cells , when compared to double heterozygous at E14 . 5 ( cHET , Pbx1flox / + ; Pbx3 - / + ; Th - IRES - Cre - ERT ) . White boxes are magnified to the right . ( D ) At E18 . 5 , a drastic reduction of TH + , PITX3 + and NURR1 + cells is detected from anterior to posterior levels in cKO embryos , but not in cHET . Pyknotic NURR1 + nuclei are observed in cKO embryos ( arrowheads ) . White boxes are magnified to the right . ( E ) The total number of TH + mDA neurons is dramatically reduced and there is an increase in aCASP3 + cells in cKO embryos compared to cHET mice at E18 . 5 .

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 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 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 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

Figure 3(A) To the left, representative immunofluorescence microscopy/confocal images showing that the number of TH+cells is reduced in Pbx1-/- embryos. Deletion of Pbx1 decreases the proportion of NURR1+neuroblasts that become TH+neurons at E12.5, indicating that Pbx1 is required for the proper maturation of NURR1+neuroblasts. To the right, quantification of these experiments.(B) Ectopic PBX3 is detected in all rostro-caudal levels of the VM in Pbx1-/- mice at E12.5, indicating a repressive function of PBX1 on Pbx3 expression.(C) Representative immunofluorescence microscopy/confocal images and quantification showing that the conditional deletion of Pbx1 and Pbx3 (cKO, Pbx1flox/flox;Pbx3-/-;Th-IRES-Cre-ERT) reduces the number of PITX3+cells in rostral VM levels, but not of NURR1+cells, when compared to double heterozygous at E14.5 (cHET, Pbx1flox/+;Pbx3-/+;Th-IRES-Cre-ERT). White boxes are magnified to the right.(D) At E18.5, a drastic reduction of TH+, PITX3+ and NURR1+cells is detected from anterior to posterior levels in cKO embryos, but not in cHET. Pyknotic NURR1+nuclei are observed in cKO embryos (arrowheads). White boxes are magnified to the right.(E) The total number of TH+ mDA neurons is dramatically reduced and there is an increase in aCASP3+ cells in cKO embryos compared to cHET mice at E18.5.


Figure 4 ( B ) Representative immunofluorescence microscopy / confocalimages showing that PBX1 - overexpression increases the number of TH + neurons after 8 days of differentiation of hNES cells ( AF22 line derived from induced pluripotent stem cells ) . ( C ) While the number of cells ( Dapi + ) in both conditions did not change , the number of TH + cells increased 2 . 3 fold in PBX1 - overexpressing cells . ( D ) TH + cells derived from the PBX1 - overexpressing hNES cells were also positive for mDAn markers such as LMX1A and PITX3 . All scale bars , 40µm . Error bars represent mean ± standard deviation . Biological replicates per condition and p - value ( T - test ) are indicated in the graphs .

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Figure 4(B) Representative immunofluorescence microscopy/confocalimages showing that PBX1-overexpression increases the number of TH+neurons after 8 days of differentiation of hNES cells (AF22 line derived from induced pluripotent stem cells). (C) While the number of cells (Dapi+) in both conditions did not change, the number of TH+cells increased 2.3 fold in PBX1-overexpressing cells.(D) TH+cells derived from the PBX1-overexpressing hNES cells were also positive for mDAn markers such as LMX1A and PITX3. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates per condition and p-value (T-test) are indicated in the graphs.


Figure 5 ( B ) Schematic of the track of PBX1 ChIP - seq compared to IgGChIP - seq as a control on different loci with peaks identified by HOMER . Each locus represents a 20kb chromosome region . ( C ) List of the 29 most regulated genes ( t - test q - value < 0 . 05 ) identified by Gene Set Enrichment Analysis ( GSEA ) and ranked by p - value . RPKM stands for Reads Per Kilobase of transcript per Million mapped reads . GSEA was performed on genes identified by RNA - Seq as differentially expressed in the VM at E12 . 5 compared to adjacent brain regions and identified by PBX1Chip - Seq in proximity to transcription starting sites . ( D ) Graphic representation of these top 29 genes ( red dots ) in the GSEA curve showing a high correlation between the cistrome and transcriptome ( p - value < 0 . 001 , q - value < 0 . 005 ) . ( E ) ChIP followed by PCR confirmed that PBX1 binds to genomic regions in close proximity to the TSS of Pitx3 ( Pitx3 - Gbf1 ) , Onecut2and Nfe2l1 genes . No co - immunoprecipitation was detected for the unrelated gene , Lrp1b ( Low Density Lipoprotein Receptor - Related Protein 1B ) . ( F , G ) The regulation of Pitx3 and Onecut2 by PBX1 was confirmed in Pbx1 - / - ; Pbx3 + / - mutant embryos at E12 . 5 , compared to wild - type ( Pbx1 + / + ) littermates . ( F ) Representative immunofluorescence microscopy / confocal images showing that decreased levels of PITX3 and number of PITX3 + cells in E12 . 5 Pbx1 - / - ; Pbx3 + / - embryos . This reduction is more evident in the basal plate ( BP ) and in the midline ( M ) , as shown at higher magnification ( right ) . ( G ) Double ONECUT2 + TH + cells were detected only in the lateral part of the mFP in E12 . 5 Pbx1 - / - ; Pbx3 + / - embryos . Selected areas are magnified to the right . A scheme ( right ) shows the distribution of cells in Pbx1 + / + and Pbx1 - / - ; Pbx3 + / - embryos . All scale bars , 40µm . Error bars represent mean ± standard deviation . Biological replicates ( dots ) per condition and p - value ( T - test ) are indicated in each graph .

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Figure 5(B) Schematic of the track of PBX1 ChIP-seq compared to IgGChIP-seq as a control on different loci with peaks identified by HOMER. Each locus represents a 20kb chromosome region.(C) List of the 29 most regulated genes (t-test q-value <0.05) identified by Gene Set Enrichment Analysis (GSEA) and ranked by p-value. RPKM stands for Reads Per Kilobase of transcript per Million mapped reads. GSEA was performed on genes identified by RNA-Seq as differentially expressed in the VM at E12.5 compared to adjacent brain regions and identified by PBX1Chip-Seq in proximity to transcription starting sites.(D) Graphic representation of these top 29 genes (red dots) in the GSEA curve showing a high correlation between the cistrome and transcriptome (p-value <0.001, q-value<0.005).(E) ChIP followed by PCR confirmed that PBX1 binds to genomic regions in close proximity to the TSS of Pitx3 (Pitx3-Gbf1), Onecut2and Nfe2l1 genes. No co-immunoprecipitation was detected for the unrelated gene, Lrp1b (Low Density Lipoprotein Receptor-Related Protein 1B).(F, G) The regulation of Pitx3 and Onecut2 by PBX1 was confirmed in Pbx1-/-;Pbx3+/- mutant embryos at E12.5, compared to wild-type (Pbx1+/+) littermates. (F) Representative immunofluorescence microscopy/confocal images showing that decreased levels of PITX3 and number of PITX3+cells in E12.5 Pbx1-/-;Pbx3+/- embryos. This reduction is more evident in the basal plate (BP) and in the midline (M), as shown at higher magnification (right). (G) Double ONECUT2+TH+cells were detected only in the lateral part of the mFP in E12.5 Pbx1-/-;Pbx3+/- embryos. Selected areas are magnified to the right. A scheme (right) shows the distribution of cells in Pbx1+/+ and Pbx1-/-;Pbx3+/- embryos. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates (dots) per condition and p-value (T-test) are indicated in each graph.


Figure 6 ( A ) Representative immunofluorescence microscopy / confocal images showing that NFE2L1 is detected in TH + mDAn and neuroblasts at E12 . 5 . ( B ) The expression of NFE2L1 in TH + neurons is dramatically reduced in Pbx1 - / - ; Pbx3 + / - compared to Pbx1 + / + embryos at E12 . 5 ( C ) shRNA lentiviruses against NFE2L1 increased the levels of aCASP3 in H2O2 - treated ( 100µm for 12 hours ) TH + neurons derived from hNES cells differentiated for 8 days ( percentage ) . White arrows indicate double TH + aCASP3 + neurons . ( D ) PBX1 overexpression protected TH + neurons derived from hNES cells from the oxidative stress induced by H2O2 ( 100µm for 12 hours ) , resulting in reduced levels of aCASP3 in TH + neurons ( percentage ) . ( E ) Human postmortem SN tissue of control donors exhibited PBX1 chromogenic staining ( dark - blue / black , indicated by arrowheads ) in the nuclei of neuromelanin + cells ( NM + , cytosolic brown aggregates ) of the SN . In contrast , PBX1 staining was severely reduced in PD patients . ( F ) Graphical representation of PBX1 intensity levels in the SN of NM + neurons in all individual controls and PD patients analyzed . Each dot in the graph represents the level of PBX1 in the nuclei of one single NM + neuron . ( G ) Strong immunoreactivity of the PBX1 direct target , NFE2L1 , was also detected as very well defined nuclear punctuate structures in NM + cells of the SN in control samples ( arrowheads ) , but were dramatically reduced in PD patients . Yellow asterisks indicate the nucleoli . ( H ) Percentage of NM + neurons showing nuclear NFE2L1 staining in the SN of control donors and PD patients . ( I ) Graphical representation of all individual controls and PD patients analyzed , showing that NM + neurons with NFE2L1 + nuclei in the SN of PD patients are less than 10 % or are totally absent .

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Figure 6(A) Representative immunofluorescence microscopy/confocal images showing that NFE2L1 is detected in TH+mDAn and neuroblasts at E12.5.(B) The expression of NFE2L1 in TH+neurons is dramatically reduced in Pbx1-/-;Pbx3+/- compared to Pbx1+/+ embryos at E12.5(C) shRNA lentiviruses against NFE2L1 increased the levels of aCASP3 in H2O2-treated (100µm for 12 hours) TH+neurons derived from hNES cells differentiated for 8 days (percentage). White arrows indicate double TH+aCASP3+neurons.(D) PBX1 overexpression protected TH+neurons derived from hNES cells from the oxidative stress induced by H2O2 (100µm for 12 hours), resulting in reduced levels of aCASP3 in TH+neurons (percentage).(E) Human postmortem SN tissue of control donors exhibited PBX1 chromogenic staining (dark-blue/black, indicated by arrowheads) in the nuclei of neuromelanin+cells (NM+, cytosolic brown aggregates) of the SN. In contrast, PBX1 staining was severely reduced in PD patients. (F) Graphical representation of PBX1 intensity levels in the SN of NM+ neurons in all individual controls and PD patients analyzed. Each dot in the graph represents the level of PBX1 in the nuclei of one single NM+neuron.(G) Strong immunoreactivity of the PBX1 direct target, NFE2L1, was also detected as very well defined nuclear punctuate structures in NM+cells of the SN in control samples (arrowheads), but were dramatically reduced in PD patients. Yellow asterisks indicate the nucleoli. (H) Percentage of NM+ neurons showing nuclear NFE2L1 staining in the SN of control donors and PD patients. (I) Graphical representation of all individual controls and PD patients analyzed, showing that NM+neurons with NFE2L1+nuclei in the SN of PD patients are less than 10% or are totally absent.


Figure 1 ( A ) Diauxic growth of WT E . coli ( NCM3722 ) in minimal media containing glucose and acetate , bacterial density measured as optical density ( OD600 ) . Growth on glucose is captured by the exponential fit ( blue dashed line ) , and proceeds until glucose runs out ( black dashed line ) at time = 0 h . This is followed by a period of growth - lag lasting ~ 3 . 5h before exponential growth resumes on acetate ( red dashed line ) . ( D ) Lag - times for controlled titration of aceBA expression using an inducer construct in strain NQ1350 ( inset ) . Addition of chlorotetracycline ( cTc ) removes the tetR repression and induces aceBA expression . As the expression of aceB / aceA during the response ( inducer added at time = 0h ) is increased , lag times decrease . Bar plot shows mean lag - times ( N = 3 biological repeats ) for different inducer concentration with error bars denoting the Standard Deviations ( SD ) .

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Figure 1(A) Diauxic growth of WT E. coli (NCM3722) in minimal media containing glucose and acetate, bacterial density measured as optical density (OD600). Growth on glucose is captured by the exponential fit (blue dashed line), and proceeds until glucose runs out (black dashed line) at time = 0 h. This is followed by a period of growth-lag lasting ~3.5h before exponential growth resumes on acetate (red dashed line).(D) Lag-times for controlled titration of aceBA expression using an inducer construct in strain NQ1350 (inset). Addition of chlorotetracycline (cTc) removes the tetR repression and induces aceBA expression. As the expression of aceB/aceA during the response (inducer added at time = 0h) is increased, lag times decrease. Bar plot shows mean lag-times (N=3 biological repeats) for different inducer concentration with error bars denoting the Standard Deviations (SD).


Figure 2 ( A ) A plasmid system ( inset ) is used to control the expression of the non - required gene lacZ using the strain NQ1389 . lacZ expression was induced using cTc to varying degrees at the moment of glucose depletion ( time = 0 h ) using the indicated range of cTc concentrations . Diauxic growth conditions with glucose and acetate , same as in Fig . 1 .

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Figure 2(A) A plasmid system (inset) is used to control the expression of the non-required gene lacZ using the strain NQ1389. lacZ expression was induced using cTc to varying degrees at the moment of glucose depletion (time = 0 h) using the indicated range of cTc concentrations. Diauxic growth conditions with glucose and acetate, same as in Fig. 1.


Figure 3 ( B ) Lag - times fall reciprocally with the allocation towards required proteins ( the fraction of mRNA encoding for required proteins ) during the shift . Allocation of translation activity towards different proteins are represented by the weight of red and gray arrows ( top ) .

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Figure 3(B) Lag-times fall reciprocally with the allocation towards required proteins (the fraction of mRNA encoding for required proteins) during the shift. Allocation of translation activity towards different proteins are represented by the weight of red and gray arrows (top).


Figure 4 ( B ) For the various gene categories in panel A , the temporal kinetics of expression during the diauxic shift is plotted . Time 0 indicates time when glucose runs out . The series of RNA - seq through the growth transition was performed once . ( C ) Steady - state growth in glucose and acetate for the WT and the motility deletion strains ΔfliC and Δflh . Growth rate differences between WT and the mutants are not significant in glucose ( p - value > 0 . 05 ) , but are significant for growth in acetate ( p - values < 0 . 02 ) . Additional growth conditions are shown in Fig . EV6 . ( D , E ) Growth transitions for the motility deletion strains ΔfliC and ΔflhD are substantially faster than that for the WT strain , with ( D ) showing representative growth kinetics of the three strains and ( E ) showing mean lag - times and standard error of four independent biological replicates . Lag - times for WT are significantly different from that of ∆ fliC ( p - value 0 . 01 ) and ∆ flhD ( p - value 0 . 004 ) . ( F ) ∆ fliC shows a 2 - fold increase ( p - value 0 . 17 ) and ∆ flhD shows a 3 - fold increase ( p - value 0 . 02 ) in aceB expression compared to WT , as measured by qPCR .

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Figure 4(B) For the various gene categories in panel A, the temporal kinetics of expression during the diauxic shift is plotted. Time 0 indicates time when glucose runs out. The series of RNA-seq through the growth transition was performed once.(C) Steady-state growth in glucose and acetate for the WT and the motility deletion strains ΔfliC and Δflh. Growth rate differences between WT and the mutants are not significant in glucose (p-value >0.05), but are significant for growth in acetate (p-values<0.02). Additional growth conditions are shown in Fig. EV6.(D, E) Growth transitions for the motility deletion strains ΔfliC and ΔflhD are substantially faster than that for the WT strain, with (D) showing representative growth kinetics of the three strains and (E) showing mean lag-times and standard error of four independent biological replicates. Lag-times for WT are significantly different from that of ∆fliC (p-value 0.01) and ∆flhD (p-value 0.004).(F) ∆fliC shows a 2-fold increase (p-value 0.17) and ∆flhD shows a 3-fold increase (p-value 0.02) in aceB expression compared to WT, as measured by qPCR.


Figure 5Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT ( black ) and the motility deletion strains ΔfliC and ΔflhD strain ( magenta and green ) . Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT ( black ) and the motility deletion strains ΔfliC and ΔflhD strain ( magenta and green ) .

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Figure 5Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT (black) and the motility deletion strains ΔfliC and ΔflhD strain (magenta and green).Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT (black) and the motility deletion strains ΔfliC and ΔflhD strain (magenta and green).


Figure 1 ( C ) The quantification of labeled - m6A in mRNA and labeled - 6mdA in genomic DNA . F ) the quantitative results ( F ) of mRNA [ D3 ] - m6A and genomic [ D3 ] - 6mdA of mES , HEK293T , C2C12 , and NIH3T3 cells . The cells were treated with [ D3 ] - L - methionine for 7 days , and [ 15N5 ] - 6mdA nucleoside ( 50 amol ) was spiked in digested DNA samples as external isotopic standard .

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Figure 1(C) The quantification of labeled-m6A in mRNA and labeled-6mdA in genomic DNA.F) the quantitative results (F) of mRNA [D3]-m6A and genomic [D3]-6mdA of mES, HEK293T, C2C12, and NIH3T3 cells. The cells were treated with [D3]-L-methionine for 7 days, and [15N5]-6mdA nucleoside (50 amol) was spiked in digested DNA samples as external isotopic standard.


Figure 2 ( D ) The determination of kinetic constants for recombinant ADAL catabolizing m6rAMP ( red ) and 6mdAMP ( blue ) . The kinetic data were fitted with the Michaelis - Menten equation . ( E ) The catalytic efficiency ( Kcat / Km ) of ADAL to 6mdAMP and m6rAMP . Error bars = s . d . , three technical replicates .

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Figure 2(D) The determination of kinetic constants for recombinant ADAL catabolizing m6rAMP (red) and 6mdAMP (blue). The kinetic data were fitted with the Michaelis-Menten equation.(E) The catalytic efficiency (Kcat/ Km) of ADAL to 6mdAMP and m6rAMP. Error bars = s.d., three technical replicates.


Figure 3B ) quantitative results ( B ) of DNA 6mdA in [ 15N5 ] - rA - treated mES cells after Adal knockdown for 7 days . Two independent biological replicates . D ) quantification ( D ) of genomic 6mdA in Adal - / - mES cells treated with [ 15N5 ] - rA for 3 days . Error bars = s . d . , three independent biological replicates . ( E ) The relative levels of Adal in mES , C2C12 , and NIH3T3 cells against reference gene TUBB . ( F The western blot analysis ( F ) ADAL expression in mES , C2C12 , and NIH3T3 cells . Unpaired t - test , error bars = s . d . , three independent biological replicates . * The standard was not labeled with heavy stable isotope . ND , not detected . G ) quantitative results ( G ) of ADAL expression in mES , C2C12 , and NIH3T3 cells . Unpaired t - test , error bars = s . d . , three independent biological replicates . * The standard was not labeled with heavy stable isotope . ND , not detected .

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Figure 3B) quantitative results (B) of DNA 6mdA in [15N5]-rA-treated mES cells after Adal knockdown for 7 days. Two independent biological replicates.D) quantification (D) of genomic 6mdA in Adal-/- mES cells treated with [15N5]-rA for 3 days. Error bars = s.d., three independent biological replicates.(E) The relative levels of Adal in mES, C2C12, and NIH3T3 cells against reference gene TUBB.(F The western blot analysis (F) ADAL expression in mES, C2C12, and NIH3T3 cells. Unpaired t-test, error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope. ND, not detected.G) quantitative results (G) of ADAL expression in mES, C2C12, and NIH3T3 cells. Unpaired t-test, error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope. ND, not detected.


Figure 4C ) the quantitation ( C ) of intracellular free [ D3 ] - m6rA - related nucleotides in WT and Adal - / - mES cells . Total free [ D3 ] - m6rA = Free [ D3 ] - m6rA + [ D3 ] - m6rAMP + [ D3 ] - m6rADP + [ D3 ] - m6rATP . Error bars = s . d . , three independent biological replicates . * The standard was not labeled with heavy stable isotope . E ) quantitation ( E ) of intracellular free [ D3 ] - 6mdA - related nucleotides in WT and Adal - / - mES cells . The cells were treated with [ D3 ] - methionine for 7 days . Total free [ D3 ] - 6mdA = Free [ D3 ] - 6mdA + [ D3 ] - 6mdAMP + [ D3 ] - 6mdADP + [ D3 ] - 6mdATP , Error bars = s . d . , three independent biological replicates . * The standard was not labeled with heavy stable isotope . ND , not detected .

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Figure 4C) the quantitation (C) of intracellular free [D3]-m6rA-related nucleotides in WT and Adal-/- mES cells. Total free [D3]-m6rA = Free [D3]-m6rA + [D3]-m6rAMP + [D3]-m6rADP + [D3]-m6rATP. Error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope.E) quantitation (E) of intracellular free [D3]-6mdA-related nucleotides in WT and Adal-/- mES cells. The cells were treated with [D3]-methionine for 7 days. Total free [D3]-6mdA = Free [D3]-6mdA + [D3]-6mdAMP + [D3]-6mdADP + [D3]-6mdATP, Error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope. ND, not detected.


Figure 5The western blot analysis of flag - tagged gene overexpression in Adal - / - mES cells ( A ) and NIH3T3 cells ( B ) overexpressing ADK , APRT , AK1 or NDPK gene . ( C ) The quantification of genomic 6mdA in [ 15N5 ] - rA - traced Adal - / - mES cells overexpressing flag - tagged ADK , APRT , AK1 or NDPK gene for 7 days . ( D ) The quantification of genomic 6mdA in NIH3T3 cells overexpressing ADK , APRT , AK1 or NDPK gene . [ 15N5 ] - rA treatment began at 24 h after plasmid transfection and lasted for 72 hours . Two technical replicates . The knockdown of Ak1 leads to a significant decreased i6mdA in NIH3T3 cells . western blot ( F ) were applied to determine the knockdown of Ak1 in NIH3T3 cells . Two independent biological replicates . ( G ) The quantitation of misincorporated 6mdA in Ak1 - knockdown cells . [ 15N5 ] - rA treatment began at 24 h after siRNA transfection and lasted for 48 hours . Error bars = s . d . , three technical replicates .

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Figure 5The western blot analysis of flag-tagged gene overexpression in Adal-/- mES cells (A) and NIH3T3 cells (B) overexpressing ADK, APRT, AK1 or NDPK gene.(C) The quantification of genomic 6mdA in [15N5]-rA-traced Adal-/- mES cells overexpressing flag-tagged ADK, APRT, AK1 or NDPK gene for 7 days. (D) The quantification of genomic 6mdA in NIH3T3 cells overexpressing ADK, APRT, AK1 or NDPK gene. [15N5]-rA treatment began at 24 h after plasmid transfection and lasted for 72 hours. Two technical replicates.The knockdown of Ak1 leads to a significant decreased i6mdA in NIH3T3 cells. western blot (F) were applied to determine the knockdown of Ak1 in NIH3T3 cells. Two independent biological replicates. (G) The quantitation of misincorporated 6mdA in Ak1-knockdown cells. [15N5]-rA treatment began at 24 h after siRNA transfection and lasted for 48 hours. Error bars = s.d., three technical replicates.


Figure 2Sqr mRNA levels ( A , B , C ) , in cerebrum ( A , D ) , kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . SQR protein levels ( D , E , F ) in cerebrum ( A , D ) , kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . Note that SQRwestern - blots were performed in isolated cerebralmitochondria due to the low levels of this protein in cerebrum . In kidneys and muscle the western - blots were performed in tissue homogenates . SQR activity ( G , H ) in kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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Figure 2Sqr mRNA levels (A, B, C), in cerebrum (A, D), kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice.SQR protein levels (D, E, F) in cerebrum (A, D), kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice. Note that SQRwestern-blots were performed in isolated cerebralmitochondria due to the low levels of this protein in cerebrum. In kidneys and muscle the western-blots were performed in tissue homogenates.SQR activity (G, H) in kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


Figure 3Levels of CoQ10 in fibroblasts of controls ( C ) and patients ( P1 - 4 ) with primary CoQ10 deficiency ( A ) . Levels of SQR protein in fibroblasts of controls ( C ) and patients ( P1 - 4 ) with primary CoQ10 deficiency cultured without ( vehicle ) and with 5μM of CoQ10 ( + CoQ10 ) during 1 day or 7 days ( B ) . Total CoQ levels ( CoQ9 + CoQ10 ) in kidneys ( C ) and muscle ( D ) of Coq9 + / + , Coq9R239X and Coq9R239X + ubiquinol - 10 ( Q10H2 ) mice . Levels of SQR protein in kidneys ( E ) and muscle ( F ) of Coq9 + / + , Coq9R239X and Coq9R239X + ubiquinol - 10 ( Q10H2 ) mice .

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Figure 3Levels of CoQ10 in fibroblasts of controls (C) and patients (P1-4) with primary CoQ10 deficiency (A).Levels of SQR protein in fibroblasts of controls (C) and patients (P1-4) with primary CoQ10 deficiency cultured without (vehicle) and with 5μM of CoQ10 (+ CoQ10) during 1 day or 7 days (B).Total CoQ levels (CoQ9 + CoQ10) in kidneys (C) and muscle (D) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.Levels of SQR protein in kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.


Figure 4TST protein levels in cerebrum ( A ) , kidneys ( B ) and muscle ( C ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . TST activity in cerebrum ( D ) , kidneys ( E ) and muscle ( F ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . ETHE1 ( SDO ) protein levels in cerebrum ( G ) , kidneys ( H ) and muscle ( I ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . SUOX protein levels in cerebrum ( J ) , kidneys ( K ) and muscle ( L ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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Figure 4TST protein levels in cerebrum (A), kidneys (B) and muscle (C) of Coq9+/+, Coq9R239X and Coq9Q95X mice.TST activity in cerebrum (D), kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9Q95X mice.ETHE1 (SDO) protein levels in cerebrum (G), kidneys (H) and muscle (I) of Coq9+/+, Coq9R239X and Coq9Q95X mice.SUOX protein levels in cerebrum (J), kidneys (K) and muscle (L) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


Figure 5Quantification of sulfide levels in cerebrum and kidneys of Coq9 + / + , Coq9R239X and Coq9Q95X mice ( A ) . Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9 + / + and Coq9R239X mice ( B ) .

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Figure 5Quantification of sulfide levels in cerebrum and kidneys of Coq9+/+, Coq9R239X and Coq9Q95X mice (A).Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9+/+ and Coq9R239X mice (B).


Figure 6Total GSH in cytosol and mitochondria of cerebrum of Coq9 + / + and Coq9R239X mice ( A ) . Cytosolic GPx and GRd activities in cerebrum of Coq9 + / + and Coq9R239X mice ( B ) . Levels of GPx4 ( C ) and GRd ( D ) protein in cerebral homogenate of Coq9 + / + and Coq9R239X mice . Levels of L - Glutamate ( L - Glu ) , N - Acetyl - Glutamate ( NacGlu ) , L - Tryptophan ( L - Trp ) , 5HIAA , N - Acetyl - Tryptophan ( NALT ) , L - Tyrosine ( L - Tyr ) in cerebrum of Coq9 + / + and Coq9R239X mice ( E ) .

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Figure 6Total GSH in cytosol and mitochondria of cerebrum of Coq9+/+ and Coq9R239X mice(A).Cytosolic GPx and GRd activities in cerebrum of Coq9+/+ and Coq9R239X mice (B).Levels of GPx4 (C) and GRd (D) protein in cerebral homogenate of Coq9+/+ and Coq9R239X mice.Levels of L-Glutamate (L-Glu), N-Acetyl-Glutamate (NacGlu), L-Tryptophan (L-Trp), 5HIAA, N-Acetyl-Tryptophan (NALT), L-Tyrosine (L-Tyr) in cerebrum of Coq9+/+ and Coq9R239X mice (E).


Figure 7SQR ( A ) and TST ( B ) protein levels in human skin fibroblasts supplemented with the H2S donor GYY4137 . TST protein level in kidneys ( C ) and cerebrum ( D ) of Coq9 + / + mice supplemented with the H2S donor GYY4137 . Levels of neurotransmitters in cerebrum of Coq9 + / + mice supplemented with the H2S donor GYY4137 ( E ) .

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Figure 7SQR (A) and TST (B) protein levels in human skin fibroblasts supplemented with the H2S donor GYY4137.TST protein level in kidneys (C) and cerebrum (D) of Coq9+/+ mice supplemented with the H2S donor GYY4137.Levels of neurotransmitters in cerebrum of Coq9+/+ mice supplemented with the H2S donor GYY4137 (E).


Figure 8COX activity in cerebrum , kidneys and muscle ( A ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . Images COX histochemistry ( B ) in gastrocnemious of Coq9 + / + , Coq9R239X and Coq9Q95X mice ; scale bars : 100 μm .

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Figure 8COX activity in cerebrum, kidneys and muscle (A) of Coq9+/+, Coq9R239X and Coq9Q95X mice.Images COX histochemistry (B) in gastrocnemious of Coq9+/+, Coq9R239X and Coq9Q95X mice; scale bars: 100 μm.


Figure 9Systolic , diastolic and mean blood pressure in Coq9 + / + and Coq9R239X mice ( A ) . Heart rate in Coq9 + / + and Coq9R239X mice ( B ) . * * P < 0 . 01 ; Coq9R239X mice versus Coq9 + / + mice ( t test ; n = 5 for each group ) .

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Figure 9Systolic, diastolic and mean blood pressure in Coq9+/+ and Coq9R239X mice (A).Heart rate in Coq9+/+ and Coq9R239X mice (B). **P < 0.01; Coq9R239X mice versus Coq9+/+ mice (t test; n = 5 for each group).


Figure 1 ( A ) Quality of the cell fractionation assays was controlled by western blot by assessing the distribution of nuclear and cytosolic markers in the whole cell extract ( WCE ) , the cytosol and the nucleus . Protein lysates were isolated from mouse primary neocortical cells ( 14 days in culture ) treated for 3hrs with the transcription inhibitor triptolide ( 1µM ) . ( four independent cultures )

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Figure 1(A) Quality of the cell fractionation assays was controlled by western blot by assessing the distribution of nuclear and cytosolic markers in the whole cell extract (WCE), the cytosol and the nucleus. Protein lysates were isolated from mouse primary neocortical cells (14 days in culture) treated for 3hrs with the transcription inhibitor triptolide (1µM). (four independent cultures)


Figure 2Efficiencies of bicuculline ( A ) stimulation were assessed by controlling the induction of ERK phosphorylation by immunostaining . Mouse primary neocortical cells ( 14 days in culture ) were stimulated with bicuculline ( 20µM ) to elevate the neuronal network excitation Cells were stained with anti - phospho - ERK ( green , bottom ) and anti - MAP2 ( white , top ) antibodies and counterstained with Hoechst ( blue , top ) . ( four independent cultures )

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Figure 2Efficiencies of bicuculline (A) stimulation were assessed by controlling the induction of ERK phosphorylation by immunostaining. Mouse primary neocortical cells (14 days in culture) were stimulated with bicuculline (20µM) to elevate the neuronal network excitation Cells were stained with anti-phospho-ERK (green, bottom) and anti-MAP2 (white, top) antibodies and counterstained with Hoechst (blue, top). (four independent cultures)


Figure 3RT - PCR validations of regulated IR - transcripts through splicing upon bicuculline stimulation ( C ) Expression of the IR - isoforms and the spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means and SDs ( standard deviations ) of PIR values are shown beneath each panel . In addition , fold changes in the expression of the IR - transcripts ( red and orange ) and spliced ( dark grey ) isoforms were assessed with real - time qPCR using three different primer sets , as represented in the top scheme . Note that the Gria3 spliced transcript ( C ) does not correspond to the canonical mRNAs and presumably arise from a first step of recursive splicing and thereby likely require splicing completion to generate fully mature Gria3 transcripts ( Sibley et al , 2015 ) . RT - PCR validations of regulated IR - transcripts through splicing upon BDNF stimulation ( D ) Expression of the IR - isoforms and the spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means and SDs ( standard deviations ) of PIR values are shown beneath each panel . In addition , fold changes in the expression of the IR - transcripts ( red and orange ) and spliced ( dark grey ) isoforms were assessed with real - time qPCR using three different primer sets , as represented in the top scheme .

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Figure 3RT-PCR validations of regulated IR-transcripts through splicing upon bicuculline stimulation (C) Expression of the IR-isoforms and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs (standard deviations) of PIR values are shown beneath each panel. In addition, fold changes in the expression of the IR-transcripts (red and orange) and spliced (dark grey) isoforms were assessed with real-time qPCR using three different primer sets, as represented in the top scheme. Note that the Gria3 spliced transcript (C) does not correspond to the canonical mRNAs and presumably arise from a first step of recursive splicing and thereby likely require splicing completion to generate fully mature Gria3 transcripts (Sibley et al, 2015).RT-PCR validations of regulated IR-transcripts through splicing upon BDNF stimulation (D) Expression of the IR-isoforms and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs (standard deviations) of PIR values are shown beneath each panel. In addition, fold changes in the expression of the IR-transcripts (red and orange) and spliced (dark grey) isoforms were assessed with real-time qPCR using three different primer sets, as represented in the top scheme.


Figure 5 ( A ) Heatmap of the IR - transcript expression values in control , bicuculline - stimulated and BDNF - stimulated conditions . IR - transcripts regulated in at least one condition are displayed ( fold change of intron - retaining transcript expression ≥ 20 % and | z - score | ≥ 1 . 5 ) . RT - PCR validations of IR - transcripts commonly regulated ( C ) Expression of the IR - transcripts and the spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means and SDs of PIR values are shown beneath each panel . In addition , fold changes ( FC ) in the expression of the IR - transcripts ( red and orange ) and spliced isoforms ( dark grey ) were assessed with real - time qPCR . RT - PCR validations of IR - transcripts , specifically regulated upon bicuculline stimulation ( D ) , specifically regulated upon BDNF stimulation ( E ) , and bi - directionally regulated ( F ) . Expression of the IR - transcripts and the spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means and SDs of PIR values are shown beneath each panel . In addition , fold changes ( FC ) in the expression of the IR - transcripts ( red and orange ) and spliced isoforms ( dark grey ) were assessed with real - time qPCR .

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Figure 5(A) Heatmap of the IR-transcript expression values in control, bicuculline-stimulated and BDNF-stimulated conditions. IR-transcripts regulated in at least one condition are displayed (fold change of intron-retaining transcript expression ≥ 20% and |z-score| ≥ 1.5).RT-PCR validations of IR-transcripts commonly regulated (C) Expression of the IR-transcripts and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs of PIR values are shown beneath each panel. In addition, fold changes (FC) in the expression of the IR-transcripts (red and orange) and spliced isoforms (dark grey) were assessed with real-time qPCR.RT-PCR validations of IR-transcripts , specifically regulated upon bicuculline stimulation (D), specifically regulated upon BDNF stimulation (E), and bi-directionally regulated (F). Expression of the IR-transcripts and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs of PIR values are shown beneath each panel. In addition, fold changes (FC) in the expression of the IR-transcripts (red and orange) and spliced isoforms (dark grey) were assessed with real-time qPCR.


Figure 2E Irf1 expression in human blastocysts . Integrated single cell RNA - seq data from blastocysts collected at or cultured to indicated time points . Data is derived from a total of 16 embryos which passed quality control metrics .

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Figure 2E Irf1 expression in human blastocysts. Integrated single cell RNA-seq data from blastocysts collected at or cultured to indicated time points. Data is derived from a total of 16 embryos which passed quality control metrics.


Figure 3A Western Blot analysis of Irf1 - / - in ESC and EpiLC , probed with antibodies against IRF1 and VINCULIN as loading control . Shown are two independent clonal cell lines .

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Figure 3A Western Blot analysis of Irf1-/- in ESC and EpiLC, probed with antibodies against IRF1 and VINCULIN as loading control. Shown are two independent clonal cell lines.


Figure 1B . Representative flow cytometry dot plots of CD11b + and CD11b + / Cripto + cells at day 2 , 3 and 5 after injury . PE ( Phycoerythrin / free channel ) . Data are mean ± SEM ( n = 5 biological replicates ; P ≤ 0 . 00008 , Student ' s t - test ) . C . Representative flow cytometry dot plots of CD11b + / F4 / 80 + / Ly6CHigh / Low and CD11b + / Cripto + / F4 / 80 + / Ly6CHigh / Low cells at day 2 , 3 and 5 after injury . D . Quantification of F4 / 80 + / Ly6CHigh / Low cells in the CD11b + and CD11b + / Cripto + cell population from injured muscles at the indicated time points . Data represent mean ± SEM ( n = 5 biological replicates ; * * * P < 0 . 001 , Student ' s t - test ) .

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Figure 1B. Representative flow cytometry dot plots of CD11b+ and CD11b+/Cripto+ cells at day 2, 3 and 5 after injury. PE (Phycoerythrin/free channel). Data are mean±SEM (n=5 biological replicates; P≤0.00008, Student's t-test).C. Representative flow cytometry dot plots of CD11b+/F4/80+/Ly6CHigh/Low and CD11b+/Cripto+/F4/80+/Ly6CHigh/Low cells at day 2, 3 and 5 after injury. D. Quantification of F4/80+/Ly6CHigh/Low cells in the CD11b+ and CD11b+/Cripto+ cell population from injured muscles at the indicated time points. Data represent mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).


Figure 2A . Representative pictures of immunostaining for F4 / 80 ( green ) in Control and CriptoMy - LOF TA sections at day 2 ( top panel ) and 5 ( bottom panel ) after injury . B . Quantification of F4 / 80 staining / damaged area ( μm2 ) at day 2 ( top graph ) and 5 ( bottom graph ) after injury . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 50 μm . Magnification of the boxes is 3 . 5 x . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) . C . Representative pictures of immunostaining for CD206 in Control and CriptoMy - LOF TA sections at day 2 ( top panel ) and 5 ( bottom panel ) after injury , respectively . D - E . Quantification of CD206 + MPs per area ( mm2 ) at day 2 ( D ) and 5 ( E ) after injury . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 50 μm . Magnification of the boxes is 3 . 5 x . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) .

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Figure 2A. Representative pictures of immunostaining for F4/80 (green) in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury. B. Quantification of F4/80 staining/damaged area (μm2) at day 2 (top graph) and 5 (bottom graph) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).C. Representative pictures of immunostaining for CD206 in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury, respectively. D-E. Quantification of CD206+ MPs per area (mm2) at day 2 (D) and 5 (E) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).


Figure 3A . Representative flow cytometry dot plots showing the percentage of GFP + cell population in GFP - Control and GFP - CriptoMy - LOF muscles at day 2 ( top panel ) and 5 ( bottom panel ) after injury . Percentage of GFP + cells expressing F4 / 80 in GFP - Control and GFP - CriptoMy - LOF muscles is shown at day 2 after injury ( middle panel ) . Data are mean ± SEM ( n = 4 biological replicates ; P = ns ; Student ' s t - test ) . B . qRT - PCR analysis of pro - ( Nos2 , Mcp1 and Tnfα ) and anti - ( Arg1 , Fizz1 , Il10 , Il4rα and Tgfβ ) inflammatory markers in GFP - CriptoMy - LOF and GFP - Control MPs at day 2 and 5 after injury . Data represent mean ± SEM of relative mRNA level normalized with Gapdh ( n ≥ 4 biological replicates ; * * * P < 0 . 001 , Student ' s t - test ) . C . Representative pictures of immunostaining for GFP ( green ) , CD206 ( red ) , pSMAD3 ( white ) in GFP - Control and GFP - CriptoMy - LOF TA sections at day 5 after injury . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x . D . Quantification of GFP + / CD206 ± / pSMAD3 ± cell distribution in TA sections from GFP - Control and GFP - CriptoMy - LOF at day 5 after injury . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Data are mean ± SEM ( n = 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) . E . Representative flow cytometry dot plots of F4 / 80 + / Ly6CLow cell population gated on GFP + cells from GFP - Control and GFP - CriptoMy - LOF muscles at day 5 after injury . Data are mean ± SEM ( n = 3 biological replicates ; P = ns ; Student ' s t - test ) . ( F ) Smear plot of RNA - seq data from GFP + / F4 / 80 + / Ly6CLow cells FACS sorted from GFP - Control and GFP - CriptoMy - LOF muscles at day 5 after injury . Data show the expression level as Log2 fold - change ( FC ) [ Log2 ( GFP - Control / GFP - CriptoMy - LOF ) ] , against Log2 ( expression ) [ Log2 ( average of gene expression across all samples ] , for each individual gene . The blue lines correspond to LogFC of 1 and - 1 . ( n = 3 biological replicates ) .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative flow cytometry dot plots showing the percentage of GFP+ cell population in GFP-Control and GFP-CriptoMy-LOF muscles at day 2 (top panel) and 5 (bottom panel) after injury. Percentage of GFP+ cells expressing F4/80 in GFP-Control and GFP-CriptoMy-LOF muscles is shown at day 2 after injury (middle panel). Data are mean±SEM (n=4 biological replicates; P=ns; Student's t-test).B. qRT-PCR analysis of pro- (Nos2, Mcp1 and Tnfα) and anti- (Arg1, Fizz1, Il10, Il4rα and Tgfβ) inflammatory markers in GFP-CriptoMy-LOF and GFP-Control MPs at day 2 and 5 after injury. Data represent mean±SEM of relative mRNA level normalized with Gapdh (n≥4 biological replicates; ***P<0.001, Student's t-test).C. Representative pictures of immunostaining for GFP (green), CD206 (red), pSMAD3 (white) in GFP-Control and GFP-CriptoMy-LOF TA sections at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x. D. Quantification of GFP+/CD206±/pSMAD3± cell distribution in TA sections from GFP-Control and GFP-CriptoMy-LOF at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Data are mean±SEM (n=5 biological replicates; **P<0.01, Student's t-test). E. Representative flow cytometry dot plots of F4/80+/Ly6CLow cell population gated on GFP+ cells from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data are mean±SEM (n=3 biological replicates; P=ns; Student's t-test).(F) Smear plot of RNA-seq data from GFP+/F4/80+/Ly6CLow cells FACS sorted from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data show the expression level as Log2 fold-change (FC) [Log2(GFP-Control/GFP-CriptoMy-LOF)], against Log2(expression) [Log2(average of gene expression across all samples], for each individual gene. The blue lines correspond to LogFC of 1 and -1. (n=3 biological replicates).


Figure 4B . Representative pictures of double immunostaining with Laminin ( green ) and embryonic myosin heavy chain ( eMHC ; red ) at day 5 ( left panel ) and 30 ( right panel ) after cardiotoxin ( CTX ) injection . Data information : Nuclei were counterstained with DAPI ( blue ) . C . Minimal Feret ' s diameter distribution of Control and CriptoMy - LOF myofibers at day 5 ( top panel ) and 30 ( bottom panel ) after CTX injection . Data are mean ± SEM ( n = 5 biological replicates ; P = ns , Student ' s t - test ) . E . Representative pictures of double immunostaining with Laminin ( green ) and eMHC ( red ) at day 5 ( left panel ) and 30 ( right panel ) after re - injury ( CTX - II ) . Data information : Nuclei were counterstained with DAPI ( blue ) . F . Minimal Feret ' s diameter distribution of Control and

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Figure 4B. Representative pictures of double immunostaining with Laminin (green) and embryonic myosin heavy chain (eMHC; red) at day 5 (left panel) and 30 (right panel) after cardiotoxin (CTX) injection. Data information: Nuclei were counterstained with DAPI (blue).C. Minimal Feret's diameter distribution of Control and CriptoMy-LOF myofibers at day 5 (top panel) and 30 (bottom panel) after CTX injection. Data are mean±SEM (n=5 biological replicates; P=ns, Student's t-test).E. Representative pictures of double immunostaining with Laminin (green) and eMHC (red) at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue).F. Minimal Feret's diameter distribution of Control and Cripto


Figure 5B . Representative pictures of immunostaining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections with GFP ( green ) and CD206 ( white ; left panel ) , pSMAD3 ( red ; middle panel ) and GFP and pSMAD3 ( right panel ) . C - E . Quantification of GFP ( C ) , GFP / CD206 ( D ) and GFP / pSMAD3 ( E ) positive cells per area ( mm2 ) in mdx - Control and mdx - CriptoMy - LOF diaphragms . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x . F - G . Representative pictures of Picrosirius red staining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections ( F ) and quantification of picrosirius red staining ( G ) . Data are expressed as percentage of stained area and are box plots displaying minimum , first quartile , median , third quartile and maximum ( n = 5 biological replicates ; * P < 0 . 05 , Student ' s t - test ) . H . Hydroxyproline ( HOP ) concentration in mdx - Control and mdx - CriptoMy - LOF diaphragm muscle sections . Data are expressed as HOP levels ( μg ) per muscle section volume ( mm3 ) and as box plots displaying minimum , first quartile , median , third quartile and maximum ( n = 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) . Representative images of Laminin ( red ) immunostaining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections of centrally nucleated myofibers . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x . Representative mdx - Control and mdx - CriptoMy - LOF diaphragm sections minimal Feret ' s diameter distribution of centrally nucleated myofibers . Data are expressed as mean ± SEM ( n = 5 biological replicates ; * P < 0 . 05 ; * * * P < 0 . 001 ; Student ' s t - test ) Representative mdx - Control and mdx - CriptoMy - LOF diaphragm sections minimal Feret ' s diameter distribution Variance coefficient of centrally nucleated myofibers . box plots displaying minimum , first quartile , median , third quartile and maximumL - M . Representative images of eMHC ( red ) immunostaining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections ( L ) and quantification of eMHC + myofibers per area ( mm2 , M ) . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n = 5 biological replicates ; * P < 0 . 05 ; Student ' s t - test ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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Figure 5B. Representative pictures of immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections with GFP (green) and CD206 (white; left panel), pSMAD3 (red; middle panel) and GFP and pSMAD3 (right panel). C-E. Quantification of GFP (C), GFP/CD206 (D) and GFP/pSMAD3 (E) positive cells per area (mm2) in mdx-Control and mdx-CriptoMy-LOF diaphragms. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.F-G. Representative pictures of Picrosirius red staining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (F) and quantification of picrosirius red staining (G). Data are expressed as percentage of stained area and are box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05, Student's t-test).H. Hydroxyproline (HOP) concentration in mdx-Control and mdx-CriptoMy-LOF diaphragm muscle sections. Data are expressed as HOP levels (μg) per muscle section volume (mm3) and as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; **P<0.01, Student's t-test).Representative images of Laminin (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections of centrally nucleated myofibers. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution of centrally nucleated myofibers. Data are expressed as mean±SEM (n=5 biological replicates; *P<0.05; ***P<0.001; Student's t-test)Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution Variance coefficient of centrally nucleated myofibers. box plots displaying minimum, first quartile, median, third quartile and maximumL-M. Representative images of eMHC (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (L) and quantification of eMHC+ myofibers per area (mm2, M). Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05; Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


Figure 6A . Representative pictures of CD31 ( red ) immunostaining of Control and CriptoMy - LOF TA sections at day 5 ( left panel ) and 30 ( right panel ) after single injury ( CTX - I ) . B - D . Quantification of CD31 + capillary number per area ( mm2 ; B ) , average of capillary cross - sectional area ( µm2 ; C ) and percentage of small ( < 20 µm2 ) and large ( > 100 µm2 ) capillaries ( D ) in Control and CriptoMy - LOF injured muscles at indicated time points after single injury ( CTX - I ) . Data are mean ± SEM ( n = 5 biological replicates ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ' s t - test ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x . E . Representative pictures of CD31 ( red ) immunostaining of Control and CriptoMy - LOF TA sections at day 5 ( left panel ) and 30 ( right panel ) after re - injury ( CTX - II ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x . F . Representative pictures of mdx - Control and mdx - CriptoMy - LOF diaphragm sections immunostained with VEcad ( red ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x . G - H . Quantification of CD31 + capillary number per area ( mm2 ; G ) and average of capillary cross - sectional area ( µm2 ; H ) in Control and CriptoMy - LOF injured muscles at the indicated time points after re - injury ( CTX - II ) Data are mean ± SEM ( n = 6 biological replicates ; * P < 0 . 05 ; * * * P < 0 . 001 , Student ' s t - test ) . I - J . Quantification of capillary number per area ( mm2 ; I ) and average of capillary cross - sectional area ( µm2 ; J ) in mdx - Control and mdx - CriptoMy - LOF diaphragm muscles . Data are mean ± SEM ( n = 5 biological replicates ; * * * P < 0 . 001 , Student ' s 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 O O O O O O O O O O O O O O O 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 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 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

Figure 6A. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after single injury (CTX-I). B-D. Quantification of CD31+ capillary number per area (mm2; B), average of capillary cross-sectional area (µm2; C) and percentage of small (<20 µm2) and large (>100 µm2) capillaries (D) in Control and CriptoMy-LOF injured muscles at indicated time points after single injury (CTX-I). Data are mean±SEM (n=5 biological replicates; *P<0.05; **P<0.01; ***P<0.001, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.E. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.F. Representative pictures of mdx-Control and mdx-CriptoMy-LOF diaphragm sections immunostained with VEcad (red). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.G-H. Quantification of CD31+ capillary number per area (mm2; G) and average of capillary cross-sectional area (µm2; H) in Control and CriptoMy-LOF injured muscles at the indicated time points after re-injury (CTX-II) Data are mean±SEM (n=6 biological replicates; *P<0.05; ***P<0.001, Student's t-test).I-J. Quantification of capillary number per area (mm2; I) and average of capillary cross-sectional area (µm2; J) in mdx-Control and mdx-CriptoMy-LOF diaphragm muscles. Data are mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).


Figure 7A . Representative pictures of double immunostaining with VEcad ( red ; left and middle panel ) and KLF4 ( green , left panel ) or TCF4 ( green , middle panel ) , and with CD31 ( red ; right panels , confocal pictures ) and TCF4 ( white , right panel ) on Control and CriptoMy - LOF TA sections at day 5 after injury . B - D . Quantification of VEcad / KLF4 ( B ) , VEcad / TCF4 ( C ) and CD31 / TCF4 ( D ) double positive cells per area ( mm2 ) in Control and CriptoMy - LOF TA sections at day 5 after injury . Data information : Nuclei were counterstained with DAPI ( blue ) . E . Representative Confocal pictures of CD31 + ( red ) and PDGFRα + ( green ) immunostaining on Control and CriptoMy - LOF TA sections at day 5 after injury . F - G . Quantification of CD31 + / PDGFRα + ( F ) and CD31 - / PDGFRα + ( G ) cells per area ( mm2 ) . Data information : Nuclei were counterstained with DAPI ( blue ) . H - I . Representative pictures of triple immunostaining with VEcad ( red ) , KLF4 ( green ) and pSMAD3 ( white ) on Control and CriptoMy - LOF TA sections at day 5 after injury ( H ) and quantification of VEcad / KLF4 / pSMAD3 triple positive cells per area ( mm2 ; I ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Representative pictures of double immunostaining with VEcad ( red ) and KLF4 ( green ) on Control and CriptoMy - LOF TA sections at day 5 after re - injury ( CTX - II Data information : Nuclei were counterstained with DAPI ( blue ) . Representative pictures of double immunostaining with VEcad ( red ) and KLF4 ( green ) on diaphragm sections of mdx - Control and mdx - CriptoMy - LOF Data information : Nuclei were counterstained with DAPI ( blue ) . Quantification of VEcad / KLF4 double positive cells per area ( mm2 ) in Control and CriptoMy - LOF TA sections at day 5 after re - injury ( CTX - IIQuantification of VEcad / KLF4 double positive cells per area ( mm2 ) in diaphragm sections of mdx - Control and mdx - CriptoMy -

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Figure 7A. Representative pictures of double immunostaining with VEcad (red; left and middle panel) and KLF4 (green, left panel) or TCF4 (green, middle panel), and with CD31 (red; right panels, confocal pictures) and TCF4 (white, right panel) on Control and CriptoMy-LOF TA sections at day 5 after injury. B-D. Quantification of VEcad/KLF4 (B), VEcad/TCF4 (C) and CD31/TCF4 (D) double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after injury. Data information: Nuclei were counterstained with DAPI (blue).E. Representative Confocal pictures of CD31+ (red) and PDGFRα+ (green) immunostaining on Control and CriptoMy-LOF TA sections at day 5 after injury. F-G. Quantification of CD31+/PDGFRα+ (F) and CD31-/PDGFRα+ (G) cells per area (mm2). Data information: Nuclei were counterstained with DAPI (blue).H-I. Representative pictures of triple immunostaining with VEcad (red), KLF4 (green) and pSMAD3 (white) on Control and CriptoMy-LOF TA sections at day 5 after injury (H) and quantification of VEcad/KLF4/pSMAD3 triple positive cells per area (mm2; I). Data information: Nuclei were counterstained with DAPI (blue).Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II Data information: Nuclei were counterstained with DAPI (blue).Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on diaphragm sections of mdx-Control and mdx-CriptoMy-LOF Data information: Nuclei were counterstained with DAPI (blue).Quantification of VEcad/KLF4 double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-IIQuantification of VEcad/KLF4 double positive cells per area (mm2) in diaphragm sections of mdx-Control and mdx-CriptoMy-LOF


Figure 1Strep - Tactin pull down of GIDAnt ( strep - tagged at Gid8 C - terminus ) probing binding of Gid1057 - 292 and Gid4117 - 362 to the complex visualized with Coomassie - stained SDS - PAGE . The experiment was performed with WT and C - terminal deletion ( ∆ C ) of the substrate receptors ( ∆ 289 - 292 ( FEIA ) and ∆ 359 - 362 ( FEFA ) for Gid10 and Gid4 , respectively ) and WT and mutant ( mut , Gid5W606A / Y613A / Q649A ) GIDAnt . Lysates from yeast strains expressing endogenously tagged 3xFLAG - Gid4 or 3x - FLAG - Gid10 that were grown in SD complete at 30 ° C ( no stress ) , SE complete for 19 hours ( EtOH ) , SD complete for 1 hour following 19 hour ethanol treatment ( EtOH rec . ) , 42 ° C for 1 hour ( HS ) , SD complete supplemented with 0 . 5M NaCl for 1 hour ( OS ) , or SD - N for 1 hour ( - N ) were run on an SDS - PAGE gel and immunoblotted with αFLAG ( two exposures from the same gel are shown ) and αPGK . Lysates from yeast strains expressing endogenously tagged 3xFLAG - Gid4 or 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG ( two exposures from the same gel are shown ) and αPGK . Lysates from wildtype and ∆ Gid2 yeast strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK . Lysates from wildtype , Gid5W606A , Y613A , Q649A , and Gid10 ∆ C strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK .

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 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 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 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

Figure 1Strep-Tactin pull down of GIDAnt (strep-tagged at Gid8 C-terminus) probing binding of Gid1057-292 and Gid4117-362 to the complex visualized with Coomassie-stained SDS-PAGE. The experiment was performed with WT and C-terminal deletion (∆C) of the substrate receptors (∆289-292 (FEIA) and ∆359-362 (FEFA) for Gid10 and Gid4, respectively) and WT and mutant (mut, Gid5W606A/Y613A/Q649A) GIDAnt.Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3x-FLAG-Gid10 that were grown in SD complete at 30°C (no stress), SE complete for 19 hours (EtOH), SD complete for 1 hour following 19 hour ethanol treatment (EtOH rec.), 42°C for 1 hour (HS), SD complete supplemented with 0.5M NaCl for 1 hour (OS), or SD-N for 1 hour (-N) were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.Lysates from wildtype and ∆Gid2 yeast strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.Lysates from wildtype, Gid5W606A,Y613A,Q649A, and Gid10∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.