J HEK - 293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time . LSD1 were examined by western blotting analysis .

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

J HEK-293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time. LSD1 were examined by western blotting analysis.


K HEK - 293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h . Cell lysates were immunoprecipitated using anti - Flag antibody and then subjected to immunoblotting .

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

K HEK-293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-Flag antibody and then subjected to immunoblotting.


L HEK - 293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h . Co - IP assay was performed using anti - Flag and then subjected to western blotting .

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

L HEK-293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h. Co-IP assay was performed using anti-Flag and then subjected to western blotting.


A Immunoprecipitation assay was performed using anti - Flag antibody in MM - 231 - shLSD1 - WT / R838A cells . The interaction of LSD1 and DUBs were detected by immunoblotting with indicated antibodies .

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

A Immunoprecipitation assay was performed using anti-Flag antibody in MM-231-shLSD1-WT/R838A cells. The interaction of LSD1 and DUBs were detected by immunoblotting with indicated antibodies.


B MDA - MB - 231 cells were treated with the indicated amounts of P5091 for 48 h , LSD1 protein levels were assessed by immunoblotting .

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

B MDA-MB-231 cells were treated with the indicated amounts of P5091 for 48 h, LSD1 protein levels were assessed by immunoblotting.


C MDA - MB - 231 cells were treated with P5091 ( 10 μM ) for 48 h and MG132 for 10 h . The LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti - LSD1 antibody .

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

C MDA-MB-231 cells were treated with P5091 (10 μM) for 48 h and MG132 for 10 h. The LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti-LSD1 antibody.


D HEK - 293T cells were transfected with USP7 WT or USP7 C223S , then treated with MG132 for 10 h . Cell lysates were immunoprecipitated using anti - LSD1 antibody followed by immunoblotting analysis .

D HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-LSD1 antibody followed by immunoblotting analysis.


E MDA - MB - 231 cells overexpressing Flag - tagged LSD1 WT / R838A after knockdown of endogenous LSD1 and then treated with or without P5091 for 48 h and MG132 for 10 h . Cell lysates were immunoprecipitated by anti - Flag antibody and then analysed by immunoblotting .

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

E MDA-MB-231 cells overexpressing Flag-tagged LSD1 WT/R838A after knockdown of endogenous LSD1 and then treated with or without P5091 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated by anti-Flag antibody and then analysed by immunoblotting.


F MDA - MB - 231 cells were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h . And cell lysates were immunoprecipitated with anti - LSD1 then subjected to immunoblotting .

F MDA-MB-231 cells were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h. And cell lysates were immunoprecipitated with anti-LSD1 then subjected to immunoblotting.


G MDA - MB - 231 cells were treated with or without MS049 for 48 h and MG132 for 10 h , Cell lysates were immunoprecipitated using anti - LSD1 antibody and then analysed by immunoblotting .

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

G MDA-MB-231 cells were treated with or without MS049 for 48 h and MG132 for 10 h, Cell lysates were immunoprecipitated using anti-LSD1 antibody and then analysed by immunoblotting.


H HEK - 293T cells were transfected with USP7 WT or USP7 C223S , then treated with or without MS049 for 48 h and MG132 for 10 h . Cell lysates were immunoprecipitated with anti - LSD1 antibody followed by immunoblotting analysis .

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

H HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with or without MS049 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated with anti-LSD1 antibody followed by immunoblotting analysis.


Migration assays ( 24 h ) of MM - 231 - shLSD1 - WT / R838A / R838K / R838F cells ( A ) with or without MS049 treatment . Representative micrographs of migrated cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars indicate the mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) .

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

Migration assays (24 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (A) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).


invasion assays ( 48 h ) of MM - 231 - shLSD1 - WT / R838A / R838K / R838F cells ( B ) with or without MS049 treatment . Representative micrographs of invaded cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars represent mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) .

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

invasion assays (48 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (B) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).


Migration assays ( 24 h ) of MCF7 - LSD1 - WT / R838A / R838K / R838F cells ( C ) with or without MS049 treatment . Representative micrographs of migrated cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars indicate the mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) .

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

Migration assays (24 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (C) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).


D invasion assays ( 48 h ) of MCF7 - LSD1 - WT / R838A / R838K / R838F cells ( D ) with or without MS049 treatment . Representative micrographs of invaded cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars represent mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) .

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

D invasion assays (48 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (D) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).


E MM - 231 - shLSD1 - WT / R838A / R838K / R838F cells ( 1 × 106 ) were injected into the tail veins of 5 - week - old female nude mice , and 7 weeks later , mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions . The number of visible surface metastatic lesions in lungs was counted ( mean of 7 mice ) ( Data are mean ± SD , n = 7 mice for each group , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) .

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

E MM-231-shLSD1-WT/R838A/R838K/R838F cells (1×106) were injected into the tail veins of 5-week-old female nude mice, and 7 weeks later, mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions. The number of visible surface metastatic lesions in lungs was counted (mean of 7 mice) (Data are mean ± SD, n = 7 mice for each group, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).


F Lung metastatic nodules confirmed by haematoxylin and eosin staining . Scale bars , 100 μm .

F Lung metastatic nodules confirmed by haematoxylin and eosin staining. Scale bars, 100 μm.


MDA - MB - 231 cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E - cadherin ( A ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - test ) .

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

MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (A) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).


B MDA - MB - 231 cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin ( B ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - test ) .

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

B MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (B) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).


MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 , real - time PCR was performed to analysis the E - cadherin ( C ) mRNA levels ( Data are are presented as mean ± SD , n = 3 experimental replicates , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) .

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

MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the E-cadherin (C) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test).


D MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 , real - time PCR was performed to analysis the vimentin ( D ) mRNA levels ( Data are are presented as mean ± SD , n = 3 experimental replicates , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) .

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

D MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the vimentin (D) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test).


MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 or anti - H3K4me2 or anti - H3K9me2 antibody , and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E - cadherin ( E ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) .

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

MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (E) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).


F MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 or anti - H3K4me2 or anti - H3K9me2 antibody , and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin ( F ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) .

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

F MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (F) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).


A Typical pictures of the immunohistochemical staining of LSD1 R838me2a , LSD1 and CARM1 in breast cancer samples . Scale bars , 100 μm .

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

A Typical pictures of the immunohistochemical staining of LSD1 R838me2a, LSD1 and CARM1 in breast cancer samples. Scale bars, 100 μm.


The percentage of samples with high level of LSD1 R838me2a ( B ) expression in different TNM stages of breast cancer ( n = 70 breast tumor samples ; B , P = 0 . 0095 ; C , P = 0 . 002 ; D , P = 0 . 0024 ; χ2 test ) .

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

The percentage of samples with high level of LSD1 R838me2a (B) expression in different TNM stages of breast cancer (n = 70 breast tumor samples; B, P = 0.0095; C, P = 0.002; D, P = 0.0024; χ2 test).


C , D The percentage of samples with high level of , LSD1 ( C ) and CARM1 ( D ) expression in different TNM stages of breast cancer ( n = 70 breast tumor samples ; B , P = 0 . 0095 ; C , P = 0 . 002 ; D , P = 0 . 0024 ; χ2 test ) .

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

C, D The percentage of samples with high level of , LSD1 (C) and CARM1 (D) expression in different TNM stages of breast cancer (n = 70 breast tumor samples; B, P = 0.0095; C, P = 0.002; D, P = 0.0024; χ2 test).


The correlation of LSD1 R838me2a with LSD1 ( E ) was statistically significant among breast cancer specimens ( n = 70 breast tumor samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation test ) . Note that some samples have overlapping scores . IHC score was evaluated according to Feng et al .

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

The correlation of LSD1 R838me2a with LSD1 (E) was statistically significant among breast cancer specimens (n = 70 breast tumor samples; E, R2 = 0.8835, P < 0.0001; F, R2 = 0.889, P < 0.0001; G, R2 = 0.8478, P < 0.0001; Pearson correlation test). Note that some samples have overlapping scores. IHC score was evaluated according to Feng et al.


F , G The correlation of , CARM1 with LSD1 R838me2a ( F ) and CARM1 with LSD1 ( G ) was statistically significant among breast cancer specimens ( n = 70 breast tumor samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation test ) . Note that some samples have overlapping scores . IHC score was evaluated according to Feng et al .

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

F, G The correlation of , CARM1 with LSD1 R838me2a (F) and CARM1 with LSD1 (G) was statistically significant among breast cancer specimens (n = 70 breast tumor samples; E, R2 = 0.8835, P < 0.0001; F, R2 = 0.889, P < 0.0001; G, R2 = 0.8478, P < 0.0001; Pearson correlation test). Note that some samples have overlapping scores. IHC score was evaluated according to Feng et al.


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 . NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment . B , Representative images from wild type prometaphase cells labelled on one or two sister chromatids as indicated . C , Representative images from wild type G2 cells labelled on one or two sister chromatids as indicated . D , Quantification of sister chromatid separation in one - sister labelled prometaphase cells for experimental conditions as indicated . Dots represent individual cells ; red bars indicate the mean . Wild type ( n = 83 cells ) , ΔSMC4 ( n = 75 cells ) and ΔNIPBL ( n = 48 cells ) were analysed . Significance was tested using a two - tailed Mann Whitney U test ; P = 2 . 33 x 10 - 27 . Two - sister labelled wild type prometaphase ( n = 4 ) , one - sister labelled wild type prometaphase ( n = 6 ) , ΔSMC4 prometaphase ( n = 4 ) , ΔNIPBL prometaphase ( n = 3 ) , wild type G2 ( n = 4 ) , ΔSMC4 G2 ( n = 3 ) , ΔNIPBL G2 ( n = 5 ) . Technical replicates Two - sister labelled wild type prometaphase ( n = 9 ) , one sister - labelled wild type prometaphase ( n = 14 ) , ΔSMC4 prometaphase ( n = 9 ) , ΔNIPBL prometaphase ( n = 6 ) , wild type G2 ( n = 8 ) , ΔSMC4 G2 ( n = 5 ) , ΔNIPBL G2 ( n = 8 ) . All images show single Z - slices from 3D - stacks . Yellow boxes indicate inset regions . Scale bars large panels : 5 μm , insets : 1

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

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. NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment. B, Representative images from wild type prometaphase cells labelled on one or two sister chromatids as indicated. C, Representative images from wild type G2 cells labelled on one or two sister chromatids as indicated. D, Quantification of sister chromatid separation in one-sister labelled prometaphase cells for experimental conditions as indicated. Dots represent individual cells; red bars indicate the mean. Wild type (n = 83 cells), ΔSMC4 (n = 75 cells) and ΔNIPBL (n = 48 cells) were analysed. Significance was tested using a two-tailed Mann Whitney U test; P = 2.33 x 10-27. Two-sister labelled wild type prometaphase (n = 4), one-sister labelled wild type prometaphase (n = 6), ΔSMC4 prometaphase (n = 4), ΔNIPBL prometaphase (n = 3), wild type G2 (n = 4), ΔSMC4 G2 (n = 3), ΔNIPBL G2 (n = 5). Technical replicates Two-sister labelled wild type prometaphase (n = 9), one sister-labelled wild type prometaphase (n = 14), ΔSMC4 prometaphase (n = 9), ΔNIPBL prometaphase (n = 6), wild type G2 (n = 8), ΔSMC4 G2 (n = 5), ΔNIPBL G2 (n = 8). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. Scale bars large panels: 5 μm, insets: 1 μ


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 . NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment . F , Representative images from ΔSMC4 prometaphase cells labelled on one sister chromatid . G , Representative images from ΔSMC4 G2 cells labelled on one sister chromatid . H , Representative images from ΔNIPBL prometaphase cells labelled on one sister chromatid . I , Representative images from ΔNIPBL G2 cells labelled on one sister chromatid . Two - sister labelled , ΔSMC4 prometaphase ( n = 4 ) , ΔNIPBL prometaphase ( n = 3 ) , ΔSMC4 G2 ( n = 3 ) , ΔNIPBL G2 ( n = 5 ) . Technical replicates : Two - sister labelled , ΔSMC4 prometaphase ( n = 9 ) , ΔNIPBL prometaphase ( n = 6 ) , ΔSMC4 G2 ( n = 5 ) , ΔNIPBL G2 ( n = 8 ) .

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

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. NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment. F, Representative images from ΔSMC4 prometaphase cells labelled on one sister chromatid. G, Representative images from ΔSMC4 G2 cells labelled on one sister chromatid. H, Representative images from ΔNIPBL prometaphase cells labelled on one sister chromatid. I, Representative images from ΔNIPBL G2 cells labelled on one sister chromatid. Two-sister labelled , ΔSMC4 prometaphase (n = 4), ΔNIPBL prometaphase (n = 3) , ΔSMC4 G2 (n = 3), ΔNIPBL G2 (n = 5). Technical replicates: Two-sister labelled , ΔSMC4 prometaphase (n = 9), ΔNIPBL prometaphase (n = 6) , ΔSMC4 G2 (n = 5), ΔNIPBL G2 (n = 8).


WAPL 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

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

WAPL 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 μ


WAPL 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 .

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WAPL 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.


A - 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 .

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A-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.


A , 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 ) .

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

A, Representative 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 .

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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.


C , 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 ) .

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C, 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).


( 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 .

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(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 .

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(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 ) .

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(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 .

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(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 .

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(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.


( A ) PBX1A is the isoform detected in the TH + NURR1 + mDA neurons at E12 . 5 .

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(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 .

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(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 ) .

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(D) PBX1 is also present in TH+ adult mDA neurons of the substantia nigra (SN) and ventral tegmental area (VTA).


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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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(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.


( 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 .

(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 .

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(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 ) .

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(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 ) .

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(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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(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.


( A ) Representative immunofluorescence microscopy / confocal images showing that NFE2L1 is detected in TH + mDAn and neuroblasts at E12 . 5 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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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(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.


( 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 ) .

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(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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(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).


( 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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(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.


( 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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(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).


( 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 .

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(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 .

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(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 ) .

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(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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(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.


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 ) .

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).


( C ) The quantification of labeled - m6A in mRNA and labeled - 6mdA in genomic DNA .

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(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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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.


( 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 .

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(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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(E) The catalytic efficiency (Kcat/ Km) of ADAL to 6mdAMP and m6rAMP. Error bars = s.d., three technical replicates.


B ) quantitative results ( B ) of DNA 6mdA in [ 15N5 ] - rA - treated mES cells after Adal knockdown for 7 days . Two independent biological replicates .

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B) 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 .

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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 .

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(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 .

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(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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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.


C ) 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 .

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C) 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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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.


The western blot analysis of flag - tagged gene overexpression in Adal - / - mES cells ( A ) and NIH3T3 cells ( B ) overexpressing ADK , APRT , AK1 or NDPK gene .

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The 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 .

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(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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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.


Sqr mRNA levels ( A , B , C ) , in cerebrum ( A , D ) , kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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Sqr 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 .

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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 .

SQR activity (G, H) in kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


Levels of CoQ10 in fibroblasts of controls ( C ) and patients ( P1 - 4 ) with primary CoQ10 deficiency ( A ) .

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Levels 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 ) .

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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 .

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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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Levels of SQR protein in kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.


TST protein levels in cerebrum ( A ) , kidneys ( B ) and muscle ( C ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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TST 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 .

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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 .

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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 .

SUOX protein levels in cerebrum (J), kidneys (K) and muscle (L) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


Quantification of sulfide levels in cerebrum and kidneys of Coq9 + / + , Coq9R239X and Coq9Q95X mice ( A ) .

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Quantification 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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Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9+/+ and Coq9R239X mice (B).


Total GSH in cytosol and mitochondria of cerebrum of Coq9 + / + and Coq9R239X mice ( A ) .

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Total 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 ) .

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 .

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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 ) .

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).


SQR ( A ) and TST ( B ) protein levels in human skin fibroblasts supplemented with the H2S donor GYY4137 .

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SQR (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 .

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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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Levels of neurotransmitters in cerebrum of Coq9+/+ mice supplemented with the H2S donor GYY4137 (E).


COX activity in cerebrum , kidneys and muscle ( A ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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COX 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 .

Images COX histochemistry (B) in gastrocnemious of Coq9+/+, Coq9R239X and Coq9Q95X mice; scale bars: 100 μm.


Systolic , diastolic and mean blood pressure in Coq9 + / + and Coq9R239X mice ( A ) .

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


( 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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(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)


Efficiencies 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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Efficiencies 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)


RT - 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 ) .

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RT-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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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.


( 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 ) .

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(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 .

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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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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.


E 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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E 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.


A 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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A 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.


B . 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 ) .

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B. 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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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).


A . 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 ) .

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A. 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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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).


A . 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 ) .

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A. 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 ) .

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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 ) .

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

C. 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 ) .

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

E. 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).