( L ) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1 - PAKI cells on day 16 of dorsal ( left , p = 0 . 0007 ( V1 - PAKI ) , p = 0 . 0002 ( + Buf ) ) and ventral ( right , p = 0 . 0083 ( V1 - PAKI ) , p = 0 . 0276 ( + Buf ) ) neural differentiation . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ; ( one - way ANOVA ) .

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

(L) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal (left, p=0.0007 (V1-PAKI), p=0.0002 (+Buf)) and ventral (right, p=0.0083 (V1-PAKI), p=0.0276 (+Buf)) neural differentiation. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).


( B and C ) RIP analysis for endogenous interaction between HDAC10 and SOX1 - OT V1 on day 16 of dorsal neural differentiation ( B ) , p = 0 . 0085 and ventral neural differentiation ( C ) , p = 0 . 0152 . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus IgG group ( unpaired two - tailed 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

(B and C) RIP analysis for endogenous interaction between HDAC10 and SOX1-OT V1 on day 16 of dorsal neural differentiation (B), p=0.0085 and ventral neural differentiation (C), p=0.0152. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus IgG group (unpaired two-tailed Student's t-test).


( D and E ) Enrichment of HDAC10 on SOX1 promoter region in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( D ) , p = 0 . 0052 and ventral neural differentiation ( E ) , p = 0 . 0001 . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed 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

(D and E) Enrichment of HDAC10 on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (D), p=0.0052 and ventral neural differentiation (E), p=0.0001. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( G and H ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 , SOX1 - OT V1 - 5P or SOX1 - OT V1 - 3P overexpression in V1 - PAKI cells on day 16 of dorsal neural differentiation ( G ) , and quantiﬁcation of SOX1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptV1 + dox ) , p = 0 . 0001 ( pt5P + dox ) . Scale bar , 100 μm . ( I and J ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 , SOX1 - OT V1 - 5P or SOX1 - OT V1 - 3P overexpression in V1 - PAKI cells on day 16 of ventral neural differentiation ( I ) , and quantiﬁcation of SOX1 + cells ( J ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptV1 + dox ) , p = 0 . 0001 ( pt5P + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and H) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of ventral neural differentiation (I), and quantiﬁcation of SOX1+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus -dox group (one-way ANOVA).


( A and B ) Immunostaining assay of ASCL1 ( green ) in control , V1 - PAKI and SOX1KO cells on day 25 of dorsal neural differentiation ( A ) , and quantiﬁcation of ASCL1 + cells ( B ) , p = 0 . 0021 ( V1 - PAKI ) , p = 0 . 0024 ( SOX1KO ) . Scale bar , 100 μm . ( C and D ) Immunostaining assay of ASCL1 ( green ) in control , V1 - PAKI and SOX1KO cells on day 25 of ventral neural differentiation ( C ) , and quantiﬁcation of ASCL1 + cells ( D ) , p = 0 . 0003 ( V1 - PAKI ) , p = 0 . 0004 ( SOX1KO ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of dorsal neural differentiation (A), and quantiﬁcation of ASCL1+ cells (B), p=0.0021 (V1-PAKI), p=0.0024 (SOX1KO). Scale bar, 100 μm. (C and D) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of ventral neural differentiation (C), and quantiﬁcation of ASCL1+ cells (D), p=0.0003 (V1-PAKI), p=0.0004 (SOX1KO). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( E and F ) Immunostaining assay of ASCL1 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 25 of dorsal neural differentiation ( E ) , and quantiﬁcation of ASCL1 + cells ( F ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptSOX1 + dox ) . Scale bar , 100 μm . ( G and H ) Immunostaining assay of ASCL1 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 25 of ventral neural differentiation ( G ) , and quantiﬁcation of ASCL1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptSOX1 + dox ) . Scale bar , 100 μm . ( I and J ) Immunostaining assay of MAP2 ( green ) after ASCL1 overexpression in V1 - PAKI cells on day 35 of dorsal neural differentiation ( I ) , and quantiﬁcation of MAP2 + cells ( J ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptASCL1 + dox ) . Scale bar , 100 μm . ( K and L ) Immunostaining assay of GABA ( green ) after ASCL1 overexpression in V1 - PAKI cells on day 35 of ventral neural differentiation ( K ) , and quantiﬁcation of GABA + cells ( L ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptASCL1 + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ; # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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

(E and F) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of dorsal neural differentiation (E), and quantiﬁcation of ASCL1+ cells (F), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (G and H) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of ventral neural differentiation (G), and quantiﬁcation of ASCL1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of MAP2 (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (I), and quantiﬁcation of MAP2+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. (K and L) Immunostaining assay of GABA (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (K), and quantiﬁcation of GABA+ cells (L), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).


( A and B ) Immunostaining assay of SOX1 ( red ) in control , V1 - PAKI and SOX1KO cells on day 40 of dorsal ( A ) and ventral ( B ) organoids differentiation . Scale bar , 100 μm . ( C ) Immunostaining assay of MAP2 ( green ) in control , V1 - PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation . Scale bar , 100 μm . ( D ) Immunostaining assay of GABA ( green ) in control , V1 - PAKI and SOX1KO cells on day 40 of ventral organoids differentiation . Scale bar , 100 μm .

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

(A and B) Immunostaining assay of SOX1 (red) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal (A) and ventral (B) organoids differentiation. Scale bar, 100 μm. (C) Immunostaining assay of MAP2 (green) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (D) Immunostaining assay of GABA (green) in control, V1-PAKI and SOX1KO cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.


( E and F ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of dorsal ( E ) and ventral ( F ) organoids differentiation . Scale bar , 100 μm . ( G ) Immunostaining assay of MAP2 ( green ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of dorsal organoids differentiation . Scale bar , 100 μm . ( H ) Immunostaining assay of GABA ( green ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of ventral organoids differentiation . Scale bar , 100 μm .

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

(E and F) Immunostaining assay of SOX1 (red) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal (E) and ventral (F) organoids differentiation. Scale bar, 100 μm. (G) Immunostaining assay of MAP2 (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (H) Immunostaining assay of GABA (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.


Flow cytometric evaluation of CXCR4 surface expression using an anti - CXCR4 - PE antibody . Left : positive patient ; right : negative patient ; representative data are shown . The gating strategy is depicted in Supplementary Fig S1 .

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

Flow cytometric evaluation of CXCR4 surface expression using an anti-CXCR4-PE antibody. Left: positive patient; right: negative patient; representative data are shown. The gating strategy is depicted in Supplementary Fig S1.


Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype .

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

Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype.


Median fluorescence intensity of surface CXCR4 expression relative to isotype control ( n = 7 - 10 patients ) . Horizontal bars indicate mean of all individual patient values ; asterisks indicate statistical significance ( plasma cells vs B cells : P = 0 . 0082 ; plasma cells vs T cells : P = 0 . 0091 ; plasma cells vs CD34 + cells : P = 0 . 011 ; plasma cells vs monocytes : P = 0 . 0423 ; Student ' s t - test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype ) . MM patients judged positive for CXCR4 expression were selected for this analysis .

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

Median fluorescence intensity of surface CXCR4 expression relative to isotype control (n = 7-10 patients). Horizontal bars indicate mean of all individual patient values; asterisks indicate statistical significance (plasma cells vs B cells: P = 0.0082; plasma cells vs T cells: P = 0.0091; plasma cells vs CD34+ cells: P = 0.011; plasma cells vs monocytes: P = 0.0423; Student's t-test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype). MM patients judged positive for CXCR4 expression were selected for this analysis.


Representative MM bone marrow staining of a patient positive for MM CXCR4 expression : hematoxylin and eosin ( H & amp ; amp ; E ) staining . Immunohistochemistry for CD138 , CXCR4 , light chain kappa , and light chain lambda .

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

Representative MM bone marrow staining of a patient positive for MM CXCR4 expression: hematoxylin and eosin (H&amp;E) staining. Immunohistochemistry for CD138, CXCR4, light chain kappa, and light chain lambda.


Real - time PCR analysis of cxcr4 transcript expression levels in HeLa ( negative control ) and in MM cell lines MM . 1S and OPM - 2 . Shown is the mean relative expression ± SEM ( n = 3 independent experiments ) . Values are normalized to the expression of ubiquitin ( Ub ) . The asterisk indicates statistically significant differences ( HeLa vs MM . 1S : P < 0 . 001 ; HeLa vs OPM - 2 : P < 0 . 0001 ; 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

Real-time PCR analysis of cxcr4 transcript expression levels in HeLa (negative control) and in MM cell lines MM.1S and OPM-2. Shown is the mean relative expression ± SEM (n = 3 independent experiments). Values are normalized to the expression of ubiquitin (Ub). The asterisk indicates statistically significant differences (HeLa vs MM.1S: P < 0.001; HeLa vs OPM-2: P < 0.0001; Student's t-test).


CXCR4 protein expression assessed by immunoblotting ( one representative blot out of 3 is shown ) .

CXCR4 protein expression assessed by immunoblotting (one representative blot out of 3 is shown).


Binding of [ 68Ga ] Pentixafor to MM . 1S and OPM - 2 cells after the indicated incubation periods ( n = 3 per cell line and time point ) . Shown is the mean ± SEM . The difference between the OPM - 2 groups is statistically significant ; * P < 0 . 0017 ( one - way ANOVA ) .

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

Binding of [68Ga]Pentixafor to MM.1S and OPM-2 cells after the indicated incubation periods (n = 3 per cell line and time point). Shown is the mean ± SEM. The difference between the OPM-2 groups is statistically significant; *P < 0.0017 (one-way ANOVA).


Mean tumor - to - background ratio ( TBR ) for [ 18F ] FDG ( left ) and for [ 68Ga ] Pentixafor ( right ) in MM . 1S and OPM - 2 xenograft - bearing NOD SCID mice . Shown is the mean ± SEM , n = 8 tumors ( 4 mice ) ; * P = 0 . 0111 for [ 18F ] FDG and * P = 0 . 0113 for [ 68Ga ] Pentixafor ( Student ' s t - test ) . One - way ANOVA revealed significant differences between the groups ; P < 0 . 0001 ( not graphically shown ) .

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

Mean tumor-to-background ratio (TBR) for [18F]FDG (left) and for [68Ga]Pentixafor (right) in MM.1S and OPM-2 xenograft-bearing NOD SCID mice. Shown is the mean ± SEM, n = 8 tumors (4 mice); *P = 0.0111 for [18F]FDG and *P = 0.0113 for [68Ga]Pentixafor (Student's t-test). One-way ANOVA revealed significant differences between the groups; P < 0.0001 (not graphically shown).


Representative [ 68Ga ] Pentixafor PET images of three mice bearing MM . 1S ( right shoulder ) and OPM - 2 ( left shoulder ) tumors .

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

Representative [68Ga]Pentixafor PET images of three mice bearing MM.1S (right shoulder) and OPM-2 (left shoulder) tumors.


Flow cytometric quantification of CXCR4 cell surface expression on resected MM . 1S and OPM - 2 tumors . Data are the mean ± SEM , n = 4 . * P = 0 . 0286 ; Mann - Whitney U - 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

Flow cytometric quantification of CXCR4 cell surface expression on resected MM.1S and OPM-2 tumors. Data are the mean ± SEM, n = 4. *P = 0.0286; Mann-Whitney U-test.


Correlation of [ 68Ga ] Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry . n = 8 tumors were analyzed .

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

Correlation of [68Ga]Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry. n = 8 tumors were analyzed.


Mice ( n = 4 ) bearing OPM - 2 and MM . 1S xenografts were coinjected with AMD3100 ( right image , one representative mouse ) or not pretreated ( left image ) before undergoing [ 68Ga ] PentixaforPET . The white arrows point to the bladder . Quantification is shown in Supplementary Fig S3A . Source data are available online for this figure .

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 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

Mice (n = 4) bearing OPM-2 and MM.1S xenografts were coinjected with AMD3100 (right image, one representative mouse) or not pretreated (left image) before undergoing [68Ga]PentixaforPET. The white arrows point to the bladder. Quantification is shown in Supplementary Fig S3A.Source data are available online for this figure.


[ 68Ga ] PentixaforPET / CT and [ 18F ] FDGPET / CTA - D Maximum intensity projections ( MIP ) of [ 68Ga ] Pentixafor ( A ) and [ 18F ] FDGPET / CT ( B ) of a 68 - year - old male with histologically proven multiple myeloma indicating the better lesion - to - background contrast for [ 68Ga ] Pentixafor in the corresponding myeloma manifestations . Trans - axial views of the upper thorax ( C ) and the pelvis ( D ) underline the higher uptake values of the bone manifestations ( yellow arrows ) of [ 68Ga ] Pentixafor compared to [ 18F ] FDG .

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

[68Ga]PentixaforPET/CT and [18F]FDGPET/CTA-D Maximum intensity projections (MIP) of [68Ga]Pentixafor (A) and [18F]FDGPET/CT (B) of a 68-year-old male with histologically proven multiple myeloma indicating the better lesion-to-background contrast for [68Ga]Pentixafor in the corresponding myeloma manifestations. Trans-axial views of the upper thorax (C) and the pelvis (D) underline the higher uptake values of the bone manifestations (yellow arrows) of [68Ga]Pentixafor compared to [18F]FDG.


Visual comparison of [ 18F ] FDG - and [ 68Ga ] Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer ( total : n = 14 ) . Number of patients ( total n = 14 ) for whom imaging with [ 18F ] FDG PET ( FDG , n = 2 ) or [ 68Ga ] Pentixafor PET ( Pentixafor , n = 7 ) was superior , with comparable positivity ( comparable , n = 3 ) , and with dual imaging providing complementary visual information ( complementary , n = 2 ) .

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

Visual comparison of [18F]FDG- and [68Ga]Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer (total: n = 14).Number of patients (total n = 14) for whom imaging with [18F]FDG PET (FDG, n = 2) or [68Ga]Pentixafor PET (Pentixafor, n = 7) was superior, with comparable positivity (comparable, n = 3), and with dual imaging providing complementary visual information (complementary, n = 2).


[ 68Ga ] PentixaforPET / MRimagesA - C Coronal views of [ 68Ga ] Pentixafor ( A ) , T2 STIR weighted MRI ( B ) and CTbone window ( C ) of a maleuptake with histologically proven multiple myeloma . The increased [ 68Ga ] Pentixaforbone correlates with the hyperintense T2 STIR signal ; however , the myeloma manifestations are underestimated in the corresponding bone window CT .

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

[68Ga]PentixaforPET/MRimagesA-C Coronal views of [68Ga]Pentixafor (A), T2 STIR weighted MRI (B) and CTbone window (C) of a maleuptake with histologically proven multiple myeloma. The increased [68Ga]Pentixaforbone correlates with the hyperintense T2 STIR signal; however, the myeloma manifestations are underestimated in the corresponding bone window CT.


( A ) The activities ( mUnits / g of protein ) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and ∆ 4 - CYB cells and normalized by the percentage of citrate synthase ( CS ) activity . Results are expressed as mean ± SD ( n = 4 - 6 biological replicates ) . Unpaired Student ' s t - test * * p = 0 . 0100 ; * * * p = 0 . 0002 ; * * * * p < 0 . 0001 .

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(A) The activities (mUnits/g of protein) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and ∆4-CYB cells and normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=4-6 biological replicates). Unpaired Student's t-test **p=0.0100; ***p=0.0002; ****p<0.0001.


( B ) Complex I in gel activity assays ( IGA ) after Blue - native gel electrophoresis ( BNGE ) of WT and ∆ 4 - CYB samples solubilized either with 1 . 6 mg DDM / mg protein or 4 mg digitonin / mg protein . The gels were incubated in the reaction mixture for 1 . 5 hours ( lighter signals in DDM gels ) or were left to continue the reaction for 24 hours to obtain darker signals ( DDM and Digitonin gels ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Complex I in gel activity assays (IGA) after Blue-native gel electrophoresis (BNGE) of WT and ∆4-CYB samples solubilized either with 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 hours (lighter signals in DDM gels) or were left to continue the reaction for 24 hours to obtain darker signals (DDM and Digitonin gels).


( C ) BNGE , Western blot and immunodetection , with anti - NDUFS1 ( cI ) , anti - ATP5A ( cV ) and anti - SDHB ( cII ) antibodies , of samples from the WT cybrids and from ∆ 4 - CYB clones E ( # 17 . 3E ) and B ( # 17 . 3B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BNGE, Western blot and immunodetection, with anti-NDUFS1 (cI), anti-ATP5A (cV) and anti-SDHB (cII) antibodies, of samples from the WT cybrids and from ∆4-CYB clones E (#17.3E) and B (#17.3B).


( A ) Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy to light ( H / L ) and ( L / H ) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and ∆ 4 - CYB cells The logarithmic ratios were calculated using MaxQuant and the statistical significance of the differences for the enrichment or depletion of the proteins were determined with

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy to light (H/L) and (L/H) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and ∆4-CYB cells The logarithmic ratios were calculated using MaxQuant and the statistical significance of the differences for the enrichment or depletion of the proteins were determined with Perseus


( B ) Labeling of the thirteen mtDNA - encoded MRC structural subunits . Cells were incubated with [ 35S ] - L - Met for one hour in the presence of emetine 100 μg / ml to inhibit cytoplasmic translation .

O O O O O O O O O O O O O O O O O O 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) Labeling of the thirteen mtDNA-encoded MRC structural subunits. Cells were incubated with [35S]-L-Met for one hour in the presence of emetine 100 μg/ml to inhibit cytoplasmic translation.


( C ) Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * * p < 0 . 0001 ; * * * p = 0 . 0007 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001; ***p=0.0007.


( D ) Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * * p < 0 . 0001 ; * * p = 0 . 0024 ( NDUFS3 ) ; * * p = 0 . 0061 ( NDUFB8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001; **p=0.0024 (NDUFS3); **p=0.0061 (NDUFB8).


( E ) Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * p = 0 . 0011 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0011.


( F ) Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001.


( G ) Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . There were no differences in the steady - state levels of the tested subunits ( 2 - way ANOVA with Sidak ' s multiple comparisons 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

(G) Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). There were no differences in the steady-state levels of the tested subunits (2-way ANOVA with Sidak's multiple comparisons test).


( A ) Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised The graphs plot the relative peptide peak intensities along the lane , setting the maximum to 1 . 0 , vs . the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve . The relative amounts of the proteins between the two cell lines was determined by calculating the H / L ratios of peptides that were present in both WT ( blue traces ) and ∆ 4 - CYB samples ( red traces ) . The represented values are the mean ± SEM of the two reciprocal labeling experiments .

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

(A) Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, vs. the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines was determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and ∆4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Second - dimension BNGE of digitonin - solubilized samples from WT and ∆ 4 - CYB cells , western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies . The immunodetection patterns were equivalent to the complexome profiles .

O O O O O O O O O O O O O O O O O O O O O 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) Second-dimension BNGE of digitonin-solubilized samples from WT and ∆4-CYB cells, western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles.


( C ) Quantification of the total peak area under the curves ( AUC ) defined by the peptide intensity peaks for the indicated cIII2 subunits . The x - axis values were the slice number ( 1 - 64 ) and the y - axis values were the relative peptide intensity . The graph shows the mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * p = 0 . 0083 ( UQCRC2 ) ; * * p = 0 . 0033 ( UQCRFS1 ) ; * p = 0 . 0224 ; n . s . = non - significant .

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(C) Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII2 subunits. The x-axis values were the slice number (1-64) and the y-axis values were the relative peptide intensity. The graph shows the mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0083 (UQCRC2); **p=0.0033 (UQCRFS1); *p=0.0224; n.s.=non-significant.


( D ) Complexome profiles of two cIII2 assembly factors ( BCS1L and LYRM7 or MZM1L ) generated The represented values are the mean ± SEM of the two reciprocal labeling experiments .

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

(D) Complexome profiles of two cIII2 assembly factors (BCS1L and LYRM7 or MZM1L) generated The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( A ) SDS - PAGE , Western blot an immunodetection , with the indicated specific antibodies , of ∆ 4 - CYB and WT cells expressing HA - tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert ( Empty ) .

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(A) SDS-PAGE, Western blot an immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing HA-tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).


( B ) BNGE , Western blot and immunodetection , with an anti - HA tag antibody , of samples from the same cell lines as in ( A ) solubilized either with digitonin or DDM .

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(B) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the same cell lines as in (A) solubilized either with digitonin or DDM.


( C ) BNGE , Western blot and immunodetection , with the monoclonal ( M ) anti - UQCRQ antibody ( Abcam ab110255 ) , of non - transduced ∆ 4 - CYB and WT cells . The mitoplast samples were solubilized with

O O O O O O O O O O O O O O O O O O O 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) BNGE, Western blot and immunodetection, with the monoclonal (M) anti-UQCRQ antibody (Abcam ab110255), of non-transduced ∆4-CYB and WT cells. The mitoplast samples were solubilized with DDM


( D ) Scatter plot generated from the analysis of the logarithmic heavy ( H ) to light ( L ) ratios in the x - axis and the reverse in the y - axis , in the two reciprocal labeling SILAC experiments ( 1 and 2 ) and anti - HA immunopurification of ∆ 4 - CYB and WT cells expressing UQCR10HA .

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(D) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments (1 and 2) and anti-HA immunopurification of ∆4-CYB and WT cells expressing UQCR10HA.


( E ) Complexome profiles for the proteins found specifically enriched in ∆ 4 - CYB UQCR10HA , according to the SILAC immunopurification experiments The represented values are the mean ± SEM of the two reciprocal labeling experiments .

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(E) Complexome profiles for the proteins found specifically enriched in ∆4-CYB UQCR10HA, according to the SILAC immunopurification experiments The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( F ) SDS - PAGE , Western blot and immunodetection , with the indicated specific antibodies , of ∆ 4 - CYB and WT cells expressing an HA - tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert ( Empty ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SDS-PAGE, Western blot and immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing an HA-tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).


( G ) BNGE , Western blot and immunodetection , with an anti - HA tag antibody , of samples from the cell lines solubilized either with digitonin .

O O O O O 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) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the cell lines solubilized either with digitonin.


( H ) Scatter plot generated from the analysis of the logarithmic heavy ( H ) to light ( L ) ratios in the x - axis and the reverse in the y - axis , in the two reciprocal labeling SILAC experiments of anti - HA immunopurification of ∆ 4 - CYB and WT cells expressing CYC1HA .

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

(H) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments of anti-HA immunopurification of ∆4-CYB and WT cells expressing CYC1HA.


( A ) Complexome profiles of the different cI structural modules . The graphs were generated but in this case , the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis The represented values are the mean ± SEM of the two reciprocal labeling experiments .

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

(A) Complexome profiles of the different cI structural modules. The graphs were generated but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Complexome profiles of the cI assembly factor NDUFAF2 in WT ( blue ) and ∆ 4 - CYB ( red ) mitochondria The represented values are the mean ± SEM of the two reciprocal labeling experiments .

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

(B) Complexome profiles of the cI assembly factor NDUFAF2 in WT (blue) and ∆4-CYB (red) mitochondria The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( C ) Quantification of the total area under the curve in the profiles corresponding to each cI module ( top graph ) . The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules . The plotted values are the mean mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * p = 0 . 0006 ; * * p = 0 . 0012 ( N - module ) ; * * p = 0 . 0016 ( ND1 - module ) ; * * p = 0 . 0032 ( ND2 - module ) ; * * p = 0 . 0057 ( ND4 - module ) ; * * p = 0 . 0020 ( ND5 - module ) ; * p = 0 . 0140 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the total area under the curve in the profiles corresponding to each cI module (top graph). The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules. The plotted values are the mean mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test ***p=0.0006; **p=0.0012 (N-module); **p=0.0016 (ND1-module); **p=0.0032 (ND2-module); **p=0.0057 (ND4-module); **p=0.0020 (ND5-module); *p=0.0140.


( D ) Quantification of the area of the N - module peak corresponding to the molecular mass of the fully assembled cI : 1 , 172 kDa in ∆ 4 - CYB samples and the respirasome ( cI + cIII2 + cIV ) SCs in # 4 . 1 WT samples ( 2 , 110 kDa ) . The plotted values are the mean mean ± SD ( n = 2 ) . Unpaired Student ' s t - test * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the area of the N-module peak corresponding to the molecular mass of the fully assembled cI: 1,172 kDa in ∆4-CYB samples and the respirasome (cI+cIII2+cIV) SCs in #4.1 WT samples (2,110 kDa). The plotted values are the mean mean ± SD (n=2). Unpaired Student's t-test ****p<0.0001.


( A ) SDS - PAGE resolving the radioactively labeled mitochondrial translation products after a 2 - hour pulse ( P ) . The 35S - Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times ( 2 , 5 and 24 hours ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SDS-PAGE resolving the radioactively labeled mitochondrial translation products after a 2-hour pulse (P). The 35S-Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times (2, 5 and 24 hours).


( B ) First dimension ( 1D ) BNGE analysis of the same cells prepared with digitonin .

(B) First dimension (1D) BNGE analysis of the same cells prepared with digitonin.


( C ) Denaturing second dimension ( 2D ) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species .

O O O O O O O O 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) Denaturing second dimension (2D) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species.


( D ) Complex I IGA ( cI - IGA ) analysis of digitonin - solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days ( 0h ) . After removing the drug and restoring synthesis of the mtDNA - encoded subunits , the cells were collected at the indicated times to follow the appearance of cI reactivity with time . The gels were incubated in the reaction mixture for 24 hours . SS = steady state . The asterisk indicates the presence of a high molecular weight cI - containing band of unknown

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Complex I IGA (cI-IGA) analysis of digitonin-solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0h). After removing the drug and restoring synthesis of the mtDNA-encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 hours. SS = steady state. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature


( E ) 2D BNGE , Western blot and immunodetection analysis of WT and ∆ 4 - CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear - encoded subunits , belonging to different structural modules . The blots shown were either exposed for 16 sec . ( low exposures ) or 160 sec . ( high exposures ) in order to visualize the qualitative signals in the ∆ 4 - CYB samples .

O O O 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

(E) 2D BNGE, Western blot and immunodetection analysis of WT and ∆4-CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear-encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 sec. (low exposures) or 160 sec. (high exposures) in order to visualize the qualitative signals in the ∆4-CYB samples.


( A ) SDS - PAGE , Western blot and immunodetection of AOXHA expression in whole - cell lysates from WT and ∆ 4 - CYB cells transduced with the AOXHA / pWPXLd - ires - HygroR lentiviral vector . The transduction and selection controls were the same cell lines transfected with an empty pWPXLd - ires - HygroR vector ( EV ) .

(A) SDS-PAGE, Western blot and immunodetection of AOXHA expression in whole-cell lysates from WT and ∆4-CYB cells transduced with the AOXHA/pWPXLd-ires-HygroR lentiviral vector. The transduction and selection controls were the same cell lines transfected with an empty pWPXLd-ires-HygroR vector (EV).


( B ) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls . Cell growth was monitored every six hours after substituting the medium in two replicate 24 - well plates , one plate with medium without uridine ( Uridine - ) , and the second plate with medium supplemented with 50 µg / ml uridine ( Uridine + ) . The graphs show the average confluence ± SD at each time point ( n = 6 wells per cell line ) .

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

(B) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls. Cell growth was monitored every six hours after substituting the medium in two replicate 24-well plates, one plate with medium without uridine (Uridine-), and the second plate with medium supplemented with 50 µg/ml uridine (Uridine+). The graphs show the average confluence ± SD at each time point (n=6 wells per cell line).


( C ) Immunodetection of cI structural subunits and NDUFAF2 in the same

(C) Immunodetection of cI structural subunits and NDUFAF2 in the same samples


( D ) 1D BNGE , Western blot and immunodetection analyses of digitonin - solubilized mitochondria from WT and ∆ 4 - CYB cybrids expressing AOXHA and their corresponding EV controls .

(D) 1D BNGE, Western blot and immunodetection analyses of digitonin-solubilized mitochondria from WT and ∆4-CYB cybrids expressing AOXHA and their corresponding EV controls.


( E ) Complex I in gel activity assays ( IGA ) after BNGE The gels were incubated in the IGA reaction mixture for 5 hours . The asterisk indicates the presence of a high molecular weight cI - containing band of unknown

(E) Complex I in gel activity assays (IGA) after BNGE The gels were incubated in the IGA reaction mixture for 5 hours. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature


( F ) Spectrophotometric kinetic measurements of cI activity in WT and ∆ 4 - CYB AOXHA and EV samples , normalized by the percentage of citrate synthase ( CS ) activity . Results are expressed as mean ± SD ( n = 5 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons test * * p = 0 . 0077 ( WT AOXHA vs . ∆ 4 - CYB AOXHA ) ; * * p = 0 . 0044 ( ∆ 4 - CYB EV vs . ∆ 4 - CYB AOXHA ) ; * * * p = 0 . 0003 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Spectrophotometric kinetic measurements of cI activity in WT and ∆4-CYB AOXHA and EV samples, normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=5 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test **p=0.0077 (WT AOXHA vs. ∆4-CYB AOXHA); **p=0.0044 (∆4-CYB EV vs. ∆4-CYB AOXHA); ***p=0.0003; ****p<0.0001.


( G ) High resolution respirometry analyses performed in intact cells in an Oroboros instrument . ROUTINE : cellular basal oxygen consumption rate ( in pmol O2 / sec ) per million cells in DMEM medium . LEAK is the non - phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin . ETS : Maximal respiration rate in the presence of the uncoupler CCCP . AOX : oxygen consumption in the presence of antimycin A , inhibiting cIII2 activity but not that of AOX . ROTENONE : Oxygen consumption in the presence of the cI inhibitor rotenone . In all cases this was equal to the background . Results are expressed as mean ± SD ( n = 2 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons test * * * * p < 0 . 0001 .

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

(G) High resolution respirometry analyses performed in intact cells in an Oroboros instrument. ROUTINE: cellular basal oxygen consumption rate (in pmol O2/sec) per million cells in DMEM medium. LEAK is the non-phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin. ETS: Maximal respiration rate in the presence of the uncoupler CCCP. AOX: oxygen consumption in the presence of antimycin A, inhibiting cIII2 activity but not that of AOX. ROTENONE: Oxygen consumption in the presence of the cI inhibitor rotenone. In all cases this was equal to the background. Results are expressed as mean ± SD (n=2 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.


( H ) Respirometry analyses , in WT EV controls and AOXHA - expressing cells untreated or treated with 2 . 5 µM antimycin A for 7 days ( + AA ) . Results are expressed as mean ± SD ( n = 4 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons test * * * * p < 0 . 0001 .

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

(H) Respirometry analyses , in WT EV controls and AOXHA- expressing cells untreated or treated with 2.5 µM antimycin A for 7 days (+AA). Results are expressed as mean ± SD (n=4 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.


( I ) 1D BNGE followed by cI - IGA ( right ) or Western blot and immunodetection of cIII2 subunit CYC1 ( left ) in digitonin - solubilized samples from WT EV controls and AOXHA - expressing cells untreated ( - ) or treated ( + ) with 2 . 5 µM antimycin A for 7 days .

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

(I) 1D BNGE followed by cI-IGA (right) or Western blot and immunodetection of cIII2 subunit CYC1 (left) in digitonin-solubilized samples from WT EV controls and AOXHA- expressing cells untreated (-) or treated (+) with 2.5 µM antimycin A for 7 days.


( A ) Complexome profiles of the two cV structural and assembly modules . The graphs were generated as in Figure 3 but in this case , the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis . The represented values are the mean ± SEM of the two reciprocal labeling experiments .

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

(A) Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Figure 3 but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Complexome profiles of the three detected cII subunits . The represented values are the mean ± SEM of the two reciprocal labeling experiments .

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

(B) Complexome profiles of the three detected cII subunits. The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( C ) Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated ( NDUFA4 ) . , the peptide intensity values for the individual subunits belonging to each assembly module were averaged to simplify the analysis . The represented values are the mean ± SEM of the two reciprocal labeling experiments . The bar graph represents the quantification of the total area under the curve in the profiles corresponding to each cIV module . The plotted values are mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * p = 0 . 0088 ( Early ) ; * * p = 0 . 0063 ( MT - CO2 ) ; * * p = 0 . 0040 ( NDUFA4 ) ; * p = 0 . 0264 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated (NDUFA4). , the peptide intensity values for the individual subunits belonging to each assembly module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total area under the curve in the profiles corresponding to each cIV module. The plotted values are mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0088 (Early); **p=0.0063 (MT-CO2); **p=0.0040 (NDUFA4); *p=0.0264.


( D ) 1D BNGE , Western blot and immunodetection of two cIV subunits ( MT - CO1 and COX7B ) in samples from WT and ∆ 4 - CYB cells solubilized with DDM and digitonin ( Dig ) .

(D) 1D BNGE, Western blot and immunodetection of two cIV subunits (MT-CO1 and COX7B) in samples from WT and ∆4-CYB cells solubilized with DDM and digitonin (Dig).


( A , B ) qRT - PCR analysis of IL6 expression upon indicated stimulations in presence or absence of NOD1 ( A ) or NOD2 ( B ) . HeLa inducible NOD1 cells ( A ) or NOD2 cells ( B ) were induced in absence or presence of doxycycline overnight and afterwards stimulated with various stimuli for 4 h .

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

(A, B) qRT-PCR analysis of IL6 expression upon indicated stimulations in presence or absence of NOD1 (A) or NOD2 (B). HeLa inducible NOD1 cells (A) or NOD2 cells (B) were induced in absence or presence of doxycycline overnight and afterwards stimulated with various stimuli for 4 h.


( C ) qRT - PCR analysis of Cxcl2 expression upon indicated stimulations in WT , Nod1 / 2 KO and Rip2 KO bone marrow derived macrophages ( BMDMs ) .

O O O O O O O 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) qRT-PCR analysis of Cxcl2 expression upon indicated stimulations in WT, Nod1/2 KO and Rip2 KO bone marrow derived macrophages (BMDMs).


ELISA analysis of IL6 in supernatants of HDFs ( E ) upon indicated stimulations for 20 h in absence or presence of the inhibitor of sphingosine kinases .

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

ELISA analysis of IL6 in supernatants of HDFs (E) upon indicated stimulations for 20 h in absence or presence of the inhibitor of sphingosine kinases.


( H ) qRT - PCR analysis of Cxcl2 expression in WT , Sphk1 KO or Sphk2 KO BMDMs upon indicated stimulations for 4 h .

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

(H) qRT-PCR analysis of Cxcl2 expression in WT, Sphk1 KO or Sphk2 KO BMDMs upon indicated stimulations for 4 h.


( D ) Western blot analysis of MAPKs and NF - κB activation upon S1P stimulation . HeLa NOD1 or NOD2 cells were treated with cytosolic S1P ( 20 µM ) , iE - DAP ( 20 µM ) or MDP ( 20 µM ) for indicated times .

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

(D) Western blot analysis of MAPKs and NF-κB activation upon S1P stimulation. HeLa NOD1 or NOD2 cells were treated with cytosolic S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) for indicated times.


Multiplex analysis of CXCL2 ( I ) , CCL5 ( J ) and IL16 ( K ) production in supernatants of WT , Nod1 / 2 and Rip2 KO BMDMs upon S1P ( 10 µM ) or MDP ( 10 µM ) stimulations together with digitonin ( 2 . 5 µg / ml ) for 20 h .

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

Multiplex analysis of CXCL2 (I), CCL5 (J) and IL16 (K) production in supernatants of WT, Nod1/2 and Rip2 KO BMDMs upon S1P (10 µM) or MDP (10 µM) stimulations together with digitonin (2.5 µg/ml) for 20 h.


( A , B ) Immunoprecipitation with various lipid - coated beads using HeLa NOD1 - GFP ( A ) and NOD2 - GFP cells ( B ) . Cell lysates of HeLa NOD1 or HeLa NOD2 cells were incubated with lipid - coated beads at room temperature for 2 h .

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

(A, B) Immunoprecipitation with various lipid-coated beads using HeLa NOD1-GFP (A) and NOD2-GFP cells (B). Cell lysates of HeLa NOD1 or HeLa NOD2 cells were incubated with lipid-coated beads at room temperature for 2 h.


( E , F ) Single particle tracking of NOD1 - GFP ( E ) or NOD2 - GFP ( F ) with S1P - TAMRA . After doxycycline induction overnight , HeLa NOD1 or NOD2 cells were stimulated with S1P - TAMRA ( 20 µM ) for 1 h together with digitonin . Scale bar : 5 µm .

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

(E, F) Single particle tracking of NOD1-GFP (E) or NOD2-GFP (F) with S1P-TAMRA. After doxycycline induction overnight, HeLa NOD1 or NOD2 cells were stimulated with S1P-TAMRA (20 µM) for 1 h together with digitonin. Scale bar: 5 µm.


( D ) MST analysis of direct binding of NOD1 to S1P and iE - DAP . ( E ) MST analysis of direct binding of NOD2 with S1P and MDP .

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

(D) MST analysis of direct binding of NOD1 to S1P and iE-DAP. (E) MST analysis of direct binding of NOD2 with S1P and MDP.


( F ) ELISA analysis of IL8 in supernatants of HEK293T NOD1 / 2 KO cells transfected with indicated NOD1 or NOD2 mutants . After 24 h transfection , cells were stimulated with S1P ( 20 µM ) , iE - DAP ( 20 µM ) or MDP ( 20 µM ) together with digitonin for 20 h .

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

(F) ELISA analysis of IL8 in supernatants of HEK293T NOD1/2 KO cells transfected with indicated NOD1 or NOD2 mutants. After 24 h transfection, cells were stimulated with S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) together with digitonin for 20 h.


( A , B ) MST analysis of direct binding of NOD1 ( A ) or NOD2 ( B ) with ADP in the presence of S1P ( 2 µM ) .

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

(A, B) MST analysis of direct binding of NOD1 (A) or NOD2 (B) with ADP in the presence of S1P (2 µM).


A - O ( A , F , K ) slices through electron cryo - tomograms of L . pneumophila , P . aeruginosa , and S . oneidensis cells , respectively , highlighting a PL sub - complex in the outer membrane ( dashed yellow circle ) . ( B , G , L ) Enlarged views of the complexes . ( C , H , M ) Sub - tomogram averages of PL sub - complexes from each species . ( D , I , N ) Schematic representations of the sub - tomogram averages with different rings colored and labeled . ( E , J , O ) Sub - tomogram averages of fully - assembled flagella from each species for comparison . Scale bars : ( black ) 50 nm , ( orange ) 25 nm , ( red ) 20 nm .

A-O (A, F, K) slices through electron cryo-tomograms of L. pneumophila, P. aeruginosa, and S. oneidensis cells, respectively, highlighting a PL sub-complex in the outer membrane (dashed yellow circle). (B, G, L) Enlarged views of the complexes. (C, H, M) Sub-tomogram averages of PL sub-complexes from each species. (D, I, N) Schematic representations of the sub-tomogram averages with different rings colored and labeled. (E, J, O) Sub-tomogram averages of fully-assembled flagella from each species for comparison. Scale bars: (black) 50 nm, (orange) 25 nm, (red) 20 nm.


Sub - tomogram average of the IM sub - complex constituting the C - ring , MS - ring and export apparatus . Schematic representation of the sub - tomogram average shown in ( A ) highlighting the different parts of the complex . Sub - tomogram average of the motors of fully - assembled flagella . Slices through electron cryo - tomograms showing neighboring PL and IM sub - complexes . Dashed - yellow circles highlight the IM sub - complex while dashed - blue arrows highlight the PL sub - complex . Dashed - red lines mark the border between two images used to make a composite image when the PL and IM sub - complexes were found at different levels in the tomogram . Schematic representation of an IM sub - complex in the vicinity of a PL sub - complex . Slices through electron cryo - tomograms of L . pneumophila highlighting the presence of a fully - assembled basal body lacking the hook and the filament . Schematic representation of the basal bodies in ( L and M ) . Central slice through an electron cryo - tomogram of a lysed cell . The dashed - yellow circle highlights the flagellar motor . Enlarged view of the same slice shown in ( O ) . The absence of the C - ring and the export apparatus is highlighted by the dashed - orange arrow . Schematic representation of the complex found in ( P ) .

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

Sub-tomogram average of the IM sub-complex constituting the C- ring, MS-ring and export apparatus. Schematic representation of the sub-tomogram average shown in (A) highlighting the different parts of the complex. Sub-tomogram average of the motors of fully-assembled flagella. Slices through electron cryo-tomograms showing neighboring PL and IM sub-complexes. Dashed-yellow circles highlight the IM sub-complex while dashed-blue arrows highlight the PL sub-complex. Dashed-red lines mark the border between two images used to make a composite image when the PL and IM sub-complexes were found at different levels in the tomogram. Schematic representation of an IM sub-complex in the vicinity of a PL sub-complex. Slices through electron cryo-tomograms of L. pneumophila highlighting the presence of a fully-assembled basal body lacking the hook and the filament. Schematic representation of the basal bodies in (L and M). Central slice through an electron cryo-tomogram of a lysed cell. The dashed-yellow circle highlights the flagellar motor. Enlarged view of the same slice shown in (O). The absence of the C-ring and the export apparatus is highlighted by the dashed-orange arrow. Schematic representation of the complex found in (P).


Slices through electron cryo - tomograms showing fully - assembled motors without the hook and filament . The dashed - yellow circles indicate the IM sub - complex . Schematic representation of the P . aeruginosa motors lacking the hook and filament shown in ( A - C ) . Slices through electron cryo - tomograms showing fully - assembled motors with the hook and lacking the filament . Schematic representation of the motors with the hook shown in ( E - G ) . Slices through electron cryo - tomograms of intact P . aeruginosa ∆ flgI cells showing the presence of the inner - membrane sub - complex with the rod . Schematic representation of the inner - membrane sub - complex with the rod shown in ( I - K ) . Slices through electron cryo - tomogram of lysed P . aeruginosa ∆ flgI cells showing the presence of the sub - complex constituting the MS - ring and the rod . Schematic representation of the sub - complex described in ( M - O ) . A slice through an electron cryo - tomogram of a P . aeruginosa ∆ flgG cell highlighting the presence of the inner - membrane sub - complex . Schematic representation of the inner - membrane sub - complex shown in ( Q ) .

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

Slices through electron cryo-tomograms showing fully-assembled motors without the hook and filament. The dashed-yellow circles indicate the IM sub-complex. Schematic representation of the P. aeruginosa motors lacking the hook and filament shown in (A-C). Slices through electron cryo-tomograms showing fully-assembled motors with the hook and lacking the filament. Schematic representation of the motors with the hook shown in (E-G). Slices through electron cryo-tomograms of intact P. aeruginosa ∆flgI cells showing the presence of the inner-membrane sub-complex with the rod. Schematic representation of the inner-membrane sub-complex with the rod shown in (I-K). Slices through electron cryo-tomogram of lysed P. aeruginosa ∆flgI cells showing the presence of the sub-complex constituting the MS-ring and the rod. Schematic representation of the sub-complex described in (M-O). A slice through an electron cryo-tomogram of a P. aeruginosa ∆flgG cell highlighting the presence of the inner-membrane sub-complex. Schematic representation of the inner-membrane sub-complex shown in (Q).


Slices through electron cryo - tomograms of wild type cells showing fully - assembled motors without the hook and filament . Schematic representation of the motors lacking the hook and filament shown in ( A - C ) . Sub - tomogram average of the motors of fully - assembled flagella . Slice through an electron cryo - tomogram of a ΔflgH cell showing an IM sub - complex , indicated by the dashed - yellow circle . Schematic representation of the IM sub - complex shown in ( F ) . Slices through electron cryo - tomograms of ΔflgH cells showing the IM sub - complex with the rod and the P - ring , indicated by the dashed - yellow circles . Schematic representation of the structures shown in ( H and I ) . Slices through electron cryo - tomograms of ΔflaA / B cells highlighting the flagellar motor and the hook ( without the filament ) . Sub - tomogram average of the flagellar motor and the hook structure found in ΔflaA / B cells . Schematic representation of the sub - tomogram average shown in ( N ) . Slices through electron cryo - tomograms of ΔflaA / B cells illustrating a disassembly product constituting the PL sub - complex , the rod and the hook . Sub - tomogram average of the disassembly complex shown in ( P - R ) . The dashed - orange arrow indicates the absence of the IM sub - complex in this structure . Schematic representation of the disassembly product shown in ( P - S ) . Slices through electron cryo - tomograms of ΔflaA / B cells highlighting PL sub - complexes ( dashed - yellow circles ) . Sub - tomogram average of the PL sub - complexes in ΔflaA / B cells . Schematic representation of the sub - tomogram average shown in ( X ) .

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

Slices through electron cryo-tomograms of wild type cells showing fully-assembled motors without the hook and filament. Schematic representation of the motors lacking the hook and filament shown in (A-C). Sub-tomogram average of the motors of fully-assembled flagella. Slice through an electron cryo-tomogram of a ΔflgH cell showing an IM sub-complex, indicated by the dashed-yellow circle. Schematic representation of the IM sub-complex shown in (F). Slices through electron cryo-tomograms of ΔflgH cells showing the IM sub-complex with the rod and the P-ring, indicated by the dashed-yellow circles. Schematic representation of the structures shown in (H and I). Slices through electron cryo-tomograms of ΔflaA/B cells highlighting the flagellar motor and the hook (without the filament). Sub-tomogram average of the flagellar motor and the hook structure found in ΔflaA/B cells. Schematic representation of the sub-tomogram average shown in (N). Slices through electron cryo-tomograms of ΔflaA/B cells illustrating a disassembly product constituting the PL sub-complex, the rod and the hook. Sub-tomogram average of the disassembly complex shown in (P-R). The dashed-orange arrow indicates the absence of the IM sub-complex in this structure. Schematic representation of the disassembly product shown in (P-S). Slices through electron cryo-tomograms of ΔflaA/B cells highlighting PL sub-complexes (dashed-yellow circles). Sub-tomogram average of the PL sub-complexes in ΔflaA/B cells. Schematic representation of the sub-tomogram average shown in (X).


C . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in control and PHSL1 - CDC20 cells . Top , time - lapse series . Bottom , meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . Data information : data are representative of four independent experiments .

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

C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in control and PHSL1-CDC20 cells. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of four independent experiments.


D . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 strains containing the indicated mutations . Meiotic events were analyzed as in ( C ) . Data information : data are representative of two independent experiments .

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

D. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 strains containing the indicated mutations. Meiotic events were analyzed as in (C). Data information: data are representative of two independent experiments.


A . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control cells and spo13 mutants . Top , time - lapse series . Bottom , meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . Data information : data are representative of two independent experiments .

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

A. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and spo13 mutants. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of two independent experiments.


C . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control cells and cells containing clb1 ∆ or cdc28 - as1 , which have been treated with 1NM - PP1 at metaphase I ( 8 h in SPM ) . Meiotic events ( bottom ) were analyzed as in ( A ) . Data information : data are representative of two independent experiments .

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

C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and cells containing clb1∆ or cdc28-as1, which have been treated with 1NM-PP1 at metaphase I (8 h in SPM). Meiotic events (bottom) were analyzed as in (A). Data information: data are representative of two independent experiments.


A . Imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ cells containing SPC72 , spc72 - 7 , or spc72 - 7 plus hrr25 - as . Cells were shifted from 24 to 36 ° C at 4 h in SPM and treated with 1NM - PP1 to inhibit Hrr25 - as . Top , time - lapse series . Bottom , Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Data information : data are representative of three independent experiments .

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

A. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus hrr25-as. Cells were shifted from 24 to 36°C at 4 h in SPM and treated with 1NM-PP1 to inhibit Hrr25-as. Top, time-lapse series. Bottom, Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Data information: data are representative of three independent experiments.


C . Imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ spo13 ∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM . Mpc70 loading ( bottom ) was analyzed as in ( A ) . PEST - SPC72 expression delays the time of Mpc70 loading by 98 min ( 95 % CI , 67 - 129 ; P < 0 . 0001 ; Welch ' s t - test ) . Data information : data are representative of three independent experiments .

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

C. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ spo13∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM. Mpc70 loading (bottom) was analyzed as in (A). PEST-SPC72 expression delays the time of Mpc70 loading by 98 min (95% CI, 67-129; P < 0.0001; Welch's t-test). Data information: data are representative of three independent experiments.


( B ) Analysis of cells containing wild - type Spo13 , Spo13 - mD , or Spo13 - mD - m2 . Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . spo13 - mD delays Spc72 removal and Mpc70 loading by 30 min ( 95 % CI , 26 - 34 ; P < 0 . 0001 ) and 60 min ( 95 % CI , 55 - 66 ; P < 0 . 0001 ; Welch ' s t - test ) , respectively . ( C ) Analysis of cells expressing Clb1 - mDK and different versions of Spo13 . Meiotic events were quantified as in ( B ) . spo13 - mD plus PEST - clb1 - mDK delays Spc72 removal and Mpc70 loading by 72 min ( 95 % CI , 63 - 80 ; P < 0 . 0001 ) and 114 min ( 95 % CI , 99 - 129 ; P < 0 . 0001 ; Welch ' s t - test ) , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of cells containing wild-type Spo13, Spo13-mD, or Spo13-mD-m2. Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. spo13-mD delays Spc72 removal and Mpc70 loading by 30 min (95% CI, 26-34; P < 0.0001) and 60 min (95% CI, 55-66; P < 0.0001; Welch's t-test), respectively. (C) Analysis of cells expressing Clb1-mDK and different versions of Spo13. Meiotic events were quantified as in (B). spo13-mD plus PEST-clb1-mDK delays Spc72 removal and Mpc70 loading by 72 min (95% CI, 63-80; P < 0.0001) and 114 min (95% CI, 99-129; P < 0.0001; Welch's t-test), respectively.


C . Cdc5 interacts with Ime2 . Cdc5 - ha3 , Ime2 , and Spo13 were detected by immunoblotting in whole - cell extracts and α - Ha immunoprecipitates from the indicated strains . Data information : data are representative of two independent experiments .

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

C. Cdc5 interacts with Ime2. Cdc5-ha3, Ime2, and Spo13 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates from the indicated strains. Data information: data are representative of two independent experiments.


D . Cdc5 ' s polo - box domain ( PBD ) binds to Ime2 . Ha3 - tagged versions of the wild - type PBD or the phosphopeptide - binding mutant PBD - FAA were expressed at t = 5 . 5 h in SPM in cells depleted of Cdc20 and Cdc5 . PBD - ha3 and Ime2 were detected by immunoblotting in whole - cell extracts and α - Ha immunoprecipitates . Data information : data are representative of two independent experiments .

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

D. Cdc5's polo-box domain (PBD) binds to Ime2. Ha3-tagged versions of the wild-type PBD or the phosphopeptide-binding mutant PBD-FAA were expressed at t = 5.5 h in SPM in cells depleted of Cdc20 and Cdc5. PBD-ha3 and Ime2 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates. Data information: data are representative of two independent experiments.


E . Cdc5 - dependent modification of Ime2 . PHSL1 - CDC20 ama1 ∆ control cells and cells containing spo13 ∆ or cdc5 - as were treated with CMK ( to inhibit Cdc5 - as ) at t = 8 h in SPM ( arrow heads ) . The panel shows immunoblot analysis of whole - cell extracts . C , sample from proliferating cells . Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5 - as . The arrow marks the modified form of Ime2 . Data information : data are representative of two independent experiments .

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

E. Cdc5-dependent modification of Ime2. PHSL1-CDC20 ama1∆ control cells and cells containing spo13∆ or cdc5-as were treated with CMK (to inhibit Cdc5-as) at t = 8 h in SPM (arrow heads). The panel shows immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5-as. The arrow marks the modified form of Ime2. Data information: data are representative of two independent experiments.


C , D . Immunoblot analysis of whole - cell extracts . C , sample from proliferating cells . ( C ) Protein levels in the cultures shown in ( A ) . ( D ) Protein levels in the cultures shown in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. (C) Protein levels in the cultures shown in (A). (D) Protein levels in the cultures shown in (B).


A , B . Mitotic cdc20 - 3 cells were shifted to 36 ° C for 80 min . At t = 0 , cells were treated with estradiol to induce expression from the PEST promoter . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP . Frame width , 19 μm . Bottom , quantification of cells with Spc72 - GFP at SPBs . ( A ) Expression of Ime2 - ∆ C induces removal of Spc72 from SPBs , which is inhibited by co - expression of Spo13 . ( B ) Expression of Cdc5 plus Ime2 - ∆ C causes rapid removal of Spc72 , which is inhibited by co - expression of Spo13 . Data information : data are representative of three independent experiments .

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

A, B. Mitotic cdc20-3 cells were shifted to 36°C for 80 min. At t = 0, cells were treated with estradiol to induce expression from the PEST promoter. Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Spc72-GFP. Frame width, 19 μm. Bottom, quantification of cells with Spc72-GFP at SPBs. (A) Expression of Ime2-∆C induces removal of Spc72 from SPBs, which is inhibited by co-expression of Spo13. (B) Expression of Cdc5 plus Ime2-∆C causes rapid removal of Spc72, which is inhibited by co-expression of Spo13. Data information: data are representative of three independent experiments.


C . Mitotic cdc20 - 3 cells containing PEST - CDC5 plus PEST - IME2 - ∆ C and / or PEST - NDT80 were shifted to 36 ° C for 30 min and subsequently treated with estradiol ( t = 0 ) . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP . Frame width , 19 μm . Bottom , quantification of cells with Mpc70 - GFP at SPBs . Data information : data are representative of two independent experiments .

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

C. Mitotic cdc20-3 cells containing PEST-CDC5 plus PEST-IME2-∆C and/or PEST-NDT80 were shifted to 36°C for 30 min and subsequently treated with estradiol (t = 0). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Frame width, 19 μm. Bottom, quantification of cells with Mpc70-GFP at SPBs. Data information: data are representative of two independent experiments.


C . PHSL1 - CDC20 ama1 ∆ cdc28 - as2 control cells and cells containing spo13 ∆ or cdc5 - as were treated with 1Na - PP1 at t = 3 h in SPM to inhibit Cdc28 - as2 . At t = 9 h in SPM , Cdc28 - as2 was activated by washing cells with cSPM lacking 1Na - PP1 ( washout ) . cdc5 - as cells were washed with cSPM plus CMK to activate Cdc28 - as2 and inhibit Cdc5 - as . Top , time - lapse series from the imaging of Mpc70 - GFP and RFP - tubulin . The weak , nuclear signal originates from Ndt80 - GFP . Bottom , quantification of spindle formation and Mpc70 loading to SPBs . D . PHSL1 - CDC20 ama1 ∆ cells containing SPC72 , spc72 - 7 , or spc72 - 7 plus cdc5 - as were shifted from 24 to 36 ° C at t = 4 . 2 h in SPM ( to inactivate Spc72 - 7 , arrow heads ) and treated with CMK at t = 6 h in SPM ( to inhibit Cdc5 - as ) . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP . Bottom , quantification of SPB separation and Mpc70 loading to SPBs . Inhibition of Cdc5 - as in spc72 - 7 cells advances the time of Mpc70 loading by 119 min ( 95 % CI , 76 - 162 ; P < 0 . 0001 ; Welch ' s t - test ) . Data are representative of two independent experiments .

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

C. PHSL1-CDC20 ama1∆ cdc28-as2 control cells and cells containing spo13∆ or cdc5-as were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 9 h in SPM, Cdc28-as2 was activated by washing cells with cSPM lacking 1Na-PP1 (washout). cdc5-as cells were washed with cSPM plus CMK to activate Cdc28-as2 and inhibit Cdc5-as. Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal originates from Ndt80-GFP. Bottom, quantification of spindle formation and Mpc70 loading to SPBs. D. PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus cdc5-as were shifted from 24 to 36°C at t = 4.2 h in SPM (to inactivate Spc72-7, arrow heads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5-as). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Bottom, quantification of SPB separation and Mpc70 loading to SPBs. Inhibition of Cdc5-as in spc72-7 cells advances the time of Mpc70 loading by 119 min (95% CI, 76-162; P < 0.0001; Welch's t-test). Data are representative of two independent experiments.


A . Inhibition of Cdk1 at anaphase I . CDC20 - mAR and CDC20 - mAR cdc28 - as1 cells were released from the metaphase I - arrest with CuSO4 at 7 h in SPM ( t = 0 ) and treated with 1NM - PP1 at t = 40 min to inhibit Cdc28 - as1 ( arrows ) . Top , time - lapse series from the imaging of Mpc70 - GFP , SPBs ( Cnm67 - RFP ) , and nuclei ( TetR - RFP ) . Bottom , quantification of meiotic events .

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

A. Inhibition of Cdk1 at anaphase I. CDC20-mAR and CDC20-mAR cdc28-as1 cells were released from the metaphase I-arrest with CuSO4 at 7 h in SPM (t = 0) and treated with 1NM-PP1 at t = 40 min to inhibit Cdc28-as1 (arrows). Top, time-lapse series from the imaging of Mpc70-GFP, SPBs (Cnm67-RFP), and nuclei (TetR-RFP). Bottom, quantification of meiotic events.


Multiple tissue ( eye , brain , adrenal gland , submandibular gland , oesophagus , lung , heart , liver , kidney , spleen , pancreas , stomach , duodenum , jejunum , ileum , caecum , colon , spinal cord , dorsal root ganglia , skeletal muscle , testis , epididymis , female gonad , skull ) northern blot for CAVII . In the P56 mouse , CAVII is prominently expressed in the central nervous system including brain and spinal cord . Gapdh served as the loading control .

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

Multiple tissue (eye, brain, adrenal gland, submandibular gland, oesophagus, lung, heart, liver, kidney, spleen, pancreas, stomach, duodenum, jejunum, ileum, caecum, colon, spinal cord, dorsal root ganglia, skeletal muscle, testis, epididymis, female gonad, skull) northern blot for CAVII. In the P56 mouse, CAVII is prominently expressed in the central nervous system including brain and spinal cord. Gapdh served as the loading control.


( C ) Developmental profile of CAVII protein expression . Western blot analysis of protein lysates of whole hippocampus shows increasing CAVII levels from P3 to P30 . The band of the appropriate size ( ∼ 30 kDa ) was absent in protein lysates from Car7 KO tissue . beta Actin served as the loading control in C and D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Developmental profile of CAVII protein expression. Western blot analysis of protein lysates of whole hippocampus shows increasing CAVII levels from P3 to P30. The band of the appropriate size (∼30 kDa) was absent in protein lysates from Car7 KO tissue.beta Actin served as the loading control in C and D.


( D ) In western blots from protein lysates of cultured cells , CAVII was detected in mixed glial / neuron cultures but not in glia cell cultures . The presence of astrocytes in all cultures was confirmed by detection of GFAP . Actin served as the loading control in C and D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In western blots from protein lysates of cultured cells, CAVII was detected in mixed glial/neuron cultures but not in glia cell cultures. The presence of astrocytes in all cultures was confirmed by detection of GFAP. Actin served as the loading control in C and D.


Original single - cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons ( baseline pHi 7 . 21 , 7 . 13 and 7 . 11 , respectively ) . Superfusion with CO2 / HCO3 − - free HEPES solution ( upper horizontal bars ) evoked an intracellular alkalinization , which in P14 WT was large and suppressed by 100 μM acetazolamide ( AZ , lower horizontal bar ) , indicating the presence of CA activity ( see Supplementary Materials and methods ) . The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide ( a poorly permeant CA inhibitor ) . Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT ( scale bar 10 μm ) .

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

Original single-cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons (baseline pHi 7.21, 7.13 and 7.11, respectively). Superfusion with CO2/HCO3−-free HEPES solution (upper horizontal bars) evoked an intracellular alkalinization, which in P14 WT was large and suppressed by 100 μM acetazolamide (AZ, lower horizontal bar), indicating the presence of CA activity (see Supplementary Materials and methods). The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide (a poorly permeant CA inhibitor). Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT (scale bar 10 μm).