Figure 1A . Frequencies and total numbers ( mean + SEM ) of CD11b + myeloid cells isolated from the spleen and peripheral LN of naïve non - immunized mice . B . Total numbers ( mean + SEM ) of CD11b + cells expressing IL - 1β shown in ( A ) and restimulated with indicated stimuli for 4 h . C . Quantification ( mean + SEM ) of myeloid cell populations expressing IL - 1β , isolated from LN and restimulated with GM - CSF shown in ( B ) . D . Frequencies and total numbers ( mean + SEM ) of CD11b + myeloid cells isolated from the spleen of immunized mice . E . Analysis of IL - 1R1 expression by CD11b + cells isolated from dLN of mice treated as in ( D ) . Data are representative FACS histogram overlays , gated on VD - CD11b + cells with mean frequencies per group . F . Quantification ( mean + SEM ) of IL - 1β expression by CD11b + cells shown in ( E ) . G . Analysis of IL - 1β expressing CD11b + cells shown in ( E ) . Data are representative FACS plots , gated on VD - CD11b + IL - 1β + cells with mean frequencies per group . H . Quantification ( mean + SEM ) of myeloid cell populations expressing IL - 1β shown in ( G ) .

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

Figure 1A. Frequencies and total numbers (mean +SEM) of CD11b+myeloid cells isolated from the spleen and peripheral LN of naïve non-immunized mice.B. Total numbers (mean +SEM) of CD11b+ cells expressing IL-1β shown in (A) and restimulated with indicated stimuli for 4 h.C. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β, isolated from LN and restimulated with GM-CSF shown in (B).D. Frequencies and total numbers (mean +SEM) of CD11b+myeloid cells isolated from the spleen of immunized mice.E. Analysis of IL-1R1 expression by CD11b+cells isolated from dLN of mice treated as in (D). Data are representative FACS histogram overlays, gated on VD-CD11b+cells with mean frequencies per group.F. Quantification (mean +SEM) of IL-1β expression by CD11b+ cells shown in (E).G. Analysis of IL-1β expressing CD11b+cells shown in (E). Data are representative FACS plots, gated on VD- CD11b+IL-1β+cells with mean frequencies per group.H. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β shown in (G).


Figure 2A - D . Analysis of IL - 1R1 expression by different populations of murine ( A ) CD4 T cells and ( B ) Th17 cells isolated from the dLN 9 days after immunization ; and ( C ) CD4T cells and ( D ) Th17 cells isolated from the CNS at the peak of EAE . Cells ( A - D ) were restimulated in the presence of MOG for 6 h . Data consist of n = 4 wild type mice immunized with MOG / CFA / PTx . Data ( A , C ) are representative FACS histogram overlay of VD - TCRβ + CD4 + cells gated on IL - 17A + , IL - 17A - IFNγ + ( IFNγ + ) , IL - 17A - GM - CSF + ( GM - CSF + ) or IL - 17A - FoxP3 + ( FoxP3 + ) cells ( also see Appendix Fig S1 ) and quantifications shown as bar diagram ( mean + SEM ) . Data ( B , D , F ) are FACS plots of Th17 cells and histograms of IL - 1R1 staining of indicated cell populations with mean fluorescent intensity ( MFI ) ± SEM or mean frequencies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Experiments were performed twice with similar results . E - F . Analysis of IL - 1R1 expression by different populations of human ( E ) CD4 T cells and ( F ) Th17 cells isolated from the peripheral blood . Cells ( E - F ) were restimulated with PMA / Ionomycin for 4 h . Data consist of PBMC isolated from n = 4 healthy individuals . Data ( E ) are FACS plots of cells gated on CD14 - CD3 + CD4 + cells with mean frequencies per group and quantifications shown as bar diagram ( mean + SEM ) . Data ( B , D , F ) are FACS plots of Th17 cells and histograms of IL - 1R1 staining of indicated cell populations with mean fluorescent intensity ( MFI ) ± SEM or mean frequencies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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

Figure 2A-D. Analysis of IL-1R1 expression by different populations of murine (A) CD4 T cells and (B) Th17 cells isolated from the dLN 9 days after immunization; and (C) CD4T cells and (D) Th17 cells isolated from the CNS at the peak of EAE.Cells (A-D) were restimulated in the presence of MOG for 6 h. Data consist of n=4 wild type mice immunized with MOG/CFA/PTx.Data (A, C) are representative FACS histogram overlay of VD-TCRβ+CD4+ cells gated on IL-17A+, IL-17A-IFNγ+ (IFNγ+), IL-17A-GM-CSF+ (GM-CSF+) or IL-17A-FoxP3+ (FoxP3+) cells (also see Appendix Fig S1) and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.E-F. Analysis of IL-1R1 expression by different populations of human (E) CD4 T cells and (F) Th17 cells isolated from the peripheral blood.Cells (E-F) were restimulated with PMA/Ionomycin for 4 h. Data consist of PBMC isolated from n=4 healthy individuals.Data (E) are FACS plots of cells gated on CD14-CD3+CD4+cells with mean frequencies per group and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.


Figure 3A . Analysis of cytokine expression by CD4T cells isolated from the spleen . Data are representative FACS plots , gated on VD - TCRβ + CD4 + CD44 + cells ( upper row ) with mean frequencies among CD4T cells per group ± SEM , and ( lower row ) with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results . B . Frequencies and total numbers ( mean + SEM ) of MOG - specific Th17 cells isolated from the dLN . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results . C - D . Frequencies and total numbers ( mean + SEM ) of MOG - specific GM - CSF + Th17 cells isolated from the spleen ( C ) and dLN ( D ) . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results .

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

Figure 3A. Analysis of cytokine expression by CD4T cells isolated from the spleen. Data are representative FACS plots, gated on VD-TCRβ+CD4+CD44+cells (upper row) with mean frequencies among CD4T cells per group ± SEM, and (lower row) with mean frequencies per group.Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.B. Frequencies and total numbers (mean +SEM) of MOG-specific Th17 cells isolated from the dLN.Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.C-D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+ Th17 cells isolated from the spleen (C) and dLN (D).Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.


Figure 4A . Analysis of CD4T cells isolated from the dLN and restimulated with PMA / Ionomycin for 4 h . Data are representative FACS plots , gated on VD - CD4 + cells with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results . B . Analysis of cytokine expression by CD4T cells isolated from the spleen and restimulated with MOG for 6 h . Data are representative FACS plots , gated on VD - TCRβ + CD4 + CD44 + cells ( upper row ) with mean frequencies among CD4T cells per group ± SEM , and ( lower row ) with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results . C . Total cell numbers ( mean + SEM ) of MOG - specific Th17 cells isolated from the spleen , shown in ( B , upper row ) . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results . D . Frequencies and total numbers ( mean + SEM ) of MOG - specific GM - CSF + IFNγ + / - Th17 cells isolated from the spleen , shown in ( B , lower row ) . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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

Figure 4A. Analysis of CD4T cells isolated from the dLN and restimulated with PMA/Ionomycin for 4 h. Data are representative FACS plots, gated on VD-CD4+cells with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.B. Analysis of cytokine expression by CD4T cells isolated from the spleen and restimulated with MOG for 6 h. Data are representative FACS plots, gated on VD-TCRβ+CD4+CD44+cells (upper row) with mean frequencies among CD4T cells per group ± SEM, and (lower row) with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.C. Total cell numbers (mean +SEM) of MOG-specific Th17 cells isolated from the spleen, shown in (B, upper row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+IFNγ+/- Th17 cells isolated from the spleen, shown in (B, lower row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.


Figure 5A - C . ( A ) Mean , ( B ) maximal EAE clinical scores and ( C ) area under the curve ( AUC ) of mice immunized with MOG / CFA / PTx . Data shown as ( A ) mean ( + SEM ) , ( B , C ) individual plots with mean and are combined from at least four independent experiments and consist of n = 41 WT , n = 24 IL - 1R1ΔT , n = 18 IL - 1R1 - / - mice . * p < 0 . 05 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Black ( * ) signs in ( A ) represent statistical analysis of IL - 1R1ΔT vs WT and blue ( * ) of IL - 1R1ΔT vs IL - 1R1 - / - groups . D - E . ( D ) Maximal EAE clinical scores ( individual plots with mean ) of selected miceD - E . ( E ) total cell numbers ( mean + SEM ) of infiltrates isolated from CNS and used for further analysis . F - H . Analysis of cytokine expression by CD4 T cells isolated from the CNS of mice shown in ( D ) . Data ( F ) are representative FACS plots , gated on VD - CD90 + CD4 + CD44 + cells ( upper row ) with mean frequencies among CD4 T cells per group ± SEM , and ( lower row ) with mean frequencies per group . ( G ) Mean fluorescent intensity ( mean + SEM ) of IL - 17A staining by MOG - specific Th17 cells , shown in ( F , upper row ) . ( H ) Quantification of MOG - specific GM - CSF + Th17 cells ( mean + SEM ) shown in ( F , lower row ) . I - K . Analysis of Treg cells isolated from mice shown in ( D ) . ( I ) Total Treg cell numbers ( mean + SEM ) isolated from CNS and dLN . ( J ) Ratio of Treg and Th17cells isolated from the CNS . Data are representative FACS plots , gated on VD - CD90 + CD4 + cells with mean frequencies per group . ( K ) Quantification of Treg / Th17 cell ratio ( mean + SEM ) within CNS and dLN . Cells were restimulated with MOG for 6 h and ( I - N ) in the presence of PMA / Ionomycin for 4 h . L - N . Analysis of CCR6 expression by CD4 T cells isolated from the dLN and CNS of EAE diseased mice . Data ( L ) are representative FACS histogram overlays of ( black ) control , ( blue ) IL - 1R1ΔTcells , gated on CD3 + CD4 + IL - 17A + cells ; and of ( black filled ) control , ( blue filled ) IL - 1R1ΔTcells , gated on CD3 + CD4 + IL - 17A - cells . ( M ) Mean fluorescent intensity ( mean + SEM ) of CCR6 staining by CD4 T cells , shown in ( L ) . ( N ) Mean fluorescent intensity ( mean + SEM ) of CCR6 staining by GM - CSF + and GM - CSF - Th17 cells isolated from CNS , shown in ( L ) .

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

Figure 5A-C. (A) Mean, (B) maximal EAE clinical scores and (C) area under the curve (AUC) of mice immunized with MOG/CFA/PTx. Data shown as (A) mean (+SEM), (B, C) individual plots with mean and are combined from at least four independent experiments and consist of n=41 WT, n=24 IL-1R1ΔT, n=18 IL-1R1-/-mice. *p < 0.05, ***p < 0.001; two-tailed unpaired t-test. Black (*) signs in (A) represent statistical analysis of IL-1R1ΔT vs WT and blue (*) of IL-1R1ΔT vs IL-1R1-/- groups.D-E. (D) Maximal EAE clinical scores (individual plots with mean) of selected miceD-E. (E) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.F-H. Analysis of cytokine expression by CD4 T cells isolated from the CNS of mice shown in (D). Data (F) are representative FACS plots, gated on VD-CD90+CD4+CD44+cells (upper row) with mean frequencies among CD4 T cells per group ± SEM, and (lower row) with mean frequencies per group. (G) Mean fluorescent intensity (mean +SEM) of IL-17A staining by MOG-specific Th17 cells, shown in (F, upper row). (H) Quantification of MOG-specific GM-CSF+ Th17 cells (mean +SEM) shown in (F, lower row).I-K. Analysis of Treg cells isolated from mice shown in (D). (I) Total Treg cell numbers (mean +SEM) isolated from CNS and dLN. (J) Ratio of Treg and Th17cells isolated from the CNS. Data are representative FACS plots, gated on VD-CD90+CD4+cells with mean frequencies per group. (K) Quantification of Treg/Th17 cell ratio (mean +SEM) within CNS and dLN.Cells were restimulated with MOG for 6 h and (I-N) in the presence of PMA/Ionomycin for 4 h.L-N. Analysis of CCR6 expression by CD4 T cells isolated from the dLN and CNS of EAE diseased mice. Data (L) are representative FACS histogram overlays of (black) control, (blue) IL-1R1ΔTcells, gated on CD3+CD4+IL-17A+cells; and of (black filled) control, (blue filled) IL-1R1ΔTcells, gated on CD3+CD4+IL-17A-cells. (M) Mean fluorescent intensity (mean +SEM) of CCR6 staining by CD4 T cells, shown in (L). (N) Mean fluorescent intensity (mean +SEM) of CCR6 staining by GM-CSF+ and GM-CSF-Th17 cells isolated from CNS, shown in (L).


Figure 6A - C . Analysis of ( A ) IL - 17A expression by wild type CD4T cells , followed with the analysis of ( B ) GM - CSF and ( C ) GM - CSF and IFNγ co - expression by Th17 cells . D - G . Analysis of cytokine expression by cells isolated from WT ( black ) , IL - 1R1ΔT ( blue ) and IL - 1R1 - / - ( red ) mice and cultured with or without IL - 23 . Data ( D ) are representative FACS plots , gated on VD - TCRβ + cells with mean frequencies per group . Cells ( A - G ) were isolated from dLN and the spleen of MOG / CFA immunized mice , cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis . Data consist of n = 4 of each genotype . D - G . Analysis of cytokine expression by cells isolated from WT ( black ) , IL - 1R1ΔT ( blue ) and IL - 1R1 - / - ( red ) mice and cultured with or without IL - 23 . Data ( D ) are representative FACS plots , gated on VD - TCRβ + cells with mean frequencies per group . Quantification ( mean + SEM ) of ( E ) MOG - specific Th17 cells , ( F ) GM - CSF + Th17 cells and ( G ) GM - CSF + IFNγ + Th17 cells . Cells ( A - G ) were isolated from dLN and the spleen of MOG / CFA immunized mice , cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis . Data consist of n = 4 of each genotype . H - I . ( H ) Mean EAE clinical scores ( + SEM ) of Rag1 - / - mice which received 1 x 105 IL - 17A + cells of indicated genotypes and ( I ) total cell numbers ( mean + SEM ) of infiltrates isolated from CNS and used for further analysis . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results . H - I . Rag1 - / - mice which received 1 x 105 IL - 17A + cells of indicated genotypes and ( I ) total cell numbers ( mean + SEM ) of infiltrates isolated from CNS and used for further analysis . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results . J - L . Analysis of cytokine expression by CD4 T cells isolated from CNS of mice shown in ( H ) and restimulated with MOG for 6 h . ( C ) Data are representative FACS plots , gated on VD - TCRβ + cells ( upper row ) with mean frequencies ± SEM . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results . J - L . Analysis of cytokine expression by CD4T cells isolated from CNS of mice shown in ( H ) and restimulated with MOG for 6 h . ( C ) Data are representative FACS plots , gated on VD - TCRβ + cells ( upper row ) with mean frequencies ± SEM . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) and IL - 1R1 - / - ( n = 4 ) cells polarized in the presence of IL - 23 prior to transfer . All experiments were performed at least twice with similar results .

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

Figure 6A-C. Analysis of (A) IL-17A expression by wild type CD4T cells, followed with the analysis of (B) GM-CSF and (C) GM-CSF and IFNγ co-expression by Th17 cells.D-G. Analysis of cytokine expression by cells isolated from WT (black), IL-1R1ΔT (blue) and IL-1R1-/- (red) mice and cultured with or without IL-23. Data (D) are representative FACS plots, gated on VD-TCRβ+cells with mean frequencies per group.Cells (A-G) were isolated from dLN and the spleen of MOG/CFA immunized mice, cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis. Data consist of n=4 of each genotype.D-G. Analysis of cytokine expression by cells isolated from WT (black), IL-1R1ΔT (blue) and IL-1R1-/- (red) mice and cultured with or without IL-23. Data (D) are representative FACS plots, gated on VD-TCRβ+cells with mean frequencies per group. Quantification (mean +SEM) of (E) MOG-specific Th17 cells, (F) GM-CSF+Th17 cells and (G) GM-CSF+IFNγ+Th17 cells.Cells (A-G) were isolated from dLN and the spleen of MOG/CFA immunized mice, cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis. Data consist of n=4 of each genotype.H-I. (H) Mean EAE clinical scores (+SEM) of Rag1-/- mice which received 1 x 105 IL-17A+cells of indicated genotypes and (I) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.H-I. Rag1-/- mice which received 1 x 105 IL-17A+cells of indicated genotypes and (I) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.J-L. Analysis of cytokine expression by CD4 T cells isolated from CNS of mice shown in (H) and restimulated with MOG for 6 h. (C) Data are representative FACS plots, gated on VD-TCRβ+cells (upper row) with mean frequencies ± SEM.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.J-L. Analysis of cytokine expression by CD4T cells isolated from CNS of mice shown in (H) and restimulated with MOG for 6 h. (C) Data are representative FACS plots, gated on VD-TCRβ+cells (upper row) with mean frequencies ± SEM.Data (H-L) consist of Rag1-/-mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.


Figure 7A . Mean EAE clinical scores ( + SEM ) . Data consist of ( A ) n = 10 WT , n = 4 IL - 1R1 - / - PBS , n = 4 WT , n = 4 IL - 1R1ΔT and n = 5 IL - 1R1 - / - PC61 treated mice . B . Maximal EAE clinical scores ( individual plots with mean ) of selected mice . C . Total cell numbers ( mean + SEM ) of infiltrates isolated from the CNS of mice , shown in ( B ) . D . Analysis of IL - 1R1 expression by Th17 cells isolated from CNS of EAE diseased mice , shown in ( B ) . Data are representative FACS histogram overlays of cells isolated from ( grey ) wild type mice treated with PBS , ( black ) wild type , ( blue ) IL - 1R1ΔT and ( red ) IL - 1R1 - / - mice treated with PC61 , gated on VD - TCRβ + CD4 + IL - 17A + cells . ( E ) Frequencies of Th17 cells positive for IL - 1R1 , shown in ( D ) .

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Figure 7A. Mean EAE clinical scores (+SEM).Data consist of (A) n=10 WT, n=4 IL-1R1-/- PBS, n = 4 WT, n=4 IL-1R1ΔT and n=5 IL-1R1-/- PC61 treated mice.B. Maximal EAE clinical scores (individual plots with mean) of selected mice.C. Total cell numbers (mean +SEM) of infiltrates isolated from the CNS of mice, shown in (B).D. Analysis of IL-1R1 expression by Th17 cells isolated from CNS of EAE diseased mice, shown in (B). Data are representative FACS histogram overlays of cells isolated from (grey) wild type mice treated with PBS, (black) wild type, (blue) IL-1R1ΔT and (red) IL-1R1-/-mice treated with PC61, gated on VD-TCRβ+CD4+IL-17A+cells.(E) Frequencies of Th17 cells positive for IL-1R1, shown in (D).


Figure 1 ( a ) Synchronization dynamics of a population of HeLa cells within almost 3 complete cell cycles measured via PI staining followed by FACS analysis . The increase in cell number following each mitosis is shown by an overlaid curve ( in orange ; mean and s . d . of n = 3 ) . ( b ) Computational modelling of the synchronization loss , considering 11 % cell - cell variation in doubling time , shows that the simulated fraction of the cells in G1 , S , and G2 / M in the synchronized cells throughout the cell cycle ( straight lines ) match experimental measurements ( marked with asterisk ) . ( c ) The measured average cell volume in the synchronized cell population ( red ; mean and s . d . of n = 3 ; in Equation 6 ) , the deconvoluted signal ( in case of no synchronization loss ; green ; in Equation 6 ) , and the simulated average cell volume considering the loss in synchronization ( black ; matching the measured concentration data ; Equation 6 ) . ( d ) The measured concentration of CTP in synchronized cells shown in red ( mean and s . d . of n = 5 ; ) in Equation 7 ; the deconvoluted concentration dynamics , in case of no synchronization loss ( green ; in Equation 7 ) ; and the expected concentration dynamics based on the deconvoluted concentrations and considering the loss in synchronization , matching the measured concentrations ( black ; Equation 7 ) . The measured concentrations converge towards the steady - state concentrations measured in non - synchronized cells ( horizontal blue line ) .

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

Figure 1(a) Synchronization dynamics of a population of HeLa cells within almost 3 complete cell cycles measured via PI staining followed by FACS analysis. The increase in cell number following each mitosis is shown by an overlaid curve (in orange; mean and s.d. of n=3).(b) Computational modelling of the synchronization loss, considering 11% cell-cell variation in doubling time, shows that the simulated fraction of the cells in G1, S, and G2/M in the synchronized cells throughout the cell cycle (straight lines) match experimental measurements (marked with asterisk).(c) The measured average cell volume in the synchronized cell population (red; mean and s.d. of n=3; in Equation 6), the deconvoluted signal (in case of no synchronization loss; green; in Equation 6), and the simulated average cell volume considering the loss in synchronization (black; matching the measured concentration data; Equation 6).(d) The measured concentration of CTP in synchronized cells shown in red (mean and s.d. of n=5; ) in Equation 7; the deconvoluted concentration dynamics, in case of no synchronization loss (green; in Equation 7); and the expected concentration dynamics based on the deconvoluted concentrations and considering the loss in synchronization, matching the measured concentrations (black; Equation 7). The measured concentrations converge towards the steady-state concentrations measured in non-synchronized cells (horizontal blue line).


Figure 2Figure 2 : Oscillation in metabolite concentrations throughout the cell cycle in HeLa cells . The figure shows metabolites found to significantly oscillate throughout the cell cycle ( concentrations normalized per metabolite ; maximal concentration in red ; minimal concentration in blue ) . The amplitude of the oscillations is shown on the right . Metabolites are color - coded according to metabolic pathways : Energy / redox cofactors ( purple ) ; amino acids ( blue ) ; glycolytic metabolites ( red ) ; TCA cycle metabolites ( green ) .

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Figure 2Figure 2: Oscillation in metabolite concentrations throughout the cell cycle in HeLa cells. The figure shows metabolites found to significantly oscillate throughout the cell cycle (concentrations normalized per metabolite; maximal concentration in red; minimal concentration in blue). The amplitude of the oscillations is shown on the right. Metabolites are color-coded according to metabolic pathways: Energy/redox cofactors (purple); amino acids (blue); glycolytic metabolites (red); TCA cycle metabolites (green).


Figure 3 ( c - e ) Measured relative fraction of the m + 2 labeling of TCA cycle intermediates after feeding [ U - 13C ] - glucose ( red ; mean and s . d . of n = 3 ) , the deconvoluted signal ( green ) , and the expected labeling dynamics considering the loss in synchronization ( black ; representing TCA cycle oxidation of glucose - derived acetyl - CoA ) . ( c - e ) Measured relative fraction of the m + 2 labeling of TCA cycle intermediates after feeding [ U - 13C ] - glucose ( red ; mean and s . d . of n = 3 ) , the deconvoluted signal ( green ) , and the expected labeling dynamics considering the loss in synchronization ( black ; representing TCA cycle oxidation of glucose - derived acetyl - CoA ) . ( c - e ) Measured relative fraction of the m + 2 labeling of TCA cycle intermediates after feeding [ U - 13C ] - glucose ( red ; mean and s . d . of n = 3 ) , the deconvoluted signal ( green ) , and the expected labeling dynamics considering the loss in synchronization ( black ; representing TCA cycle oxidation of glucose - derived acetyl - CoA ) . ( f ) Oscillations in citrate m + 4 labeling after feeding [ U - 13C ] - glutamine ( mean and s . d . of n = 3 ) . ( g ) Oscillations in the total citrate concentration throughout the cell cycle ( mean and s . d . of n = 5 ) . ( h ) The measured lactate secretion flux in synchronized cells shown in red ( mean and s . d . of n = 5 ; in Equation 9 ; ) ; the deconvoluted secretion flux dynamics , in case of no synchronization loss ( green ; in Equation 10 ) ; and the expected secretion flux based on the deconvoluted fluxes and considering the loss in synchronization , matching the measured fluxes ( black ; Equation 10 ) .

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

Figure 3(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).(f) Oscillations in citrate m+4 labeling after feeding [U-13C]-glutamine (mean and s.d. of n=3).(g) Oscillations in the total citrate concentration throughout the cell cycle (mean and s.d. of n=5).(h) The measured lactate secretion flux in synchronized cells shown in red (mean and s.d. of n=5; in Equation 9;); the deconvoluted secretion flux dynamics, in case of no synchronization loss (green; in Equation 10); and the expected secretion flux based on the deconvoluted fluxes and considering the loss in synchronization, matching the measured fluxes (black; Equation 10).


Figure 4Oscillations in aspartate ( a ) concentrations throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) . Oscillations in malate ( b ) concentrations throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) . ( c ) Uniform malate m + 4 labeling throughout the cell cycle ( combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase ) . ( d ) Oscillations in pyrimidines m + 3 labeling throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing de novo pyrimidine biosynthesis ) . ( e ) Oscillations in lactate m + 3 labeling throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing malic enzyme activity ) . ( f ) Oscillations in malate m + 3 throughout the cell cycle when feeding [ U - 13C ] - glucose ( representing pyruvate carboxylase activity ) . ( g ) Oscillations in citrate m + 5 when feeding [ U - 13C ] - glutamine throughout the cell cycle ( representing reductive IDH flux ) . ( h ) Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glucose , representing oxidative glucose metabolism . Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glutamine , representing reductive glutamine metabolism ( i ) . For measurements of metabolite concentrations ( a - b ) , red marks represent mean and s . d . of n = 5 . For measurements of fractional isotopic labeling ( c - i ) , red marks represent mean and s . d . of n = 3 .

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

Figure 4Oscillations in aspartate (a)concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).Oscillations in malate (b) concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).(c) Uniform malate m+4 labeling throughout the cell cycle (combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase).(d) Oscillations in pyrimidines m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing de novo pyrimidine biosynthesis).(e) Oscillations in lactate m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing malic enzyme activity).(f) Oscillations in malate m+3 throughout the cell cycle when feeding [U-13C]-glucose (representing pyruvate carboxylase activity).(g) Oscillations in citrate m+5 when feeding [U-13C]-glutamine throughout the cell cycle (representing reductive IDH flux).(h) Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glucose, representing oxidative glucose metabolism.Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glutamine, representing reductive glutamine metabolism (i). For measurements of metabolite concentrations (a-b), red marks represent mean and s.d. of n=5. For measurements of fractional isotopic labeling (c-i), red marks represent mean and s.d. of n=3.


Figure 5Figure 5 : Complementary oscillations of glucose versus glutamine - derived fluxes in TCA cycle . ( a - i ) Oscillations in metabolic flux throughout the cell cycle ( in mM / h ) , computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling ( red and green marks represent optimal estimates of transient flux with 95 % c . i . ) . Blue lines represent average fluxes inferred in a non - synchronized cell population . As shown , glucose - derived flux into TCA cycle peak in late G1 phase , while oxidative and reductive glutamine metabolism dominates S phase .

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Figure 5Figure 5: Complementary oscillations of glucose versus glutamine-derived fluxes in TCA cycle. (a-i) Oscillations in metabolic flux throughout the cell cycle (in mM/h), computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling (red and green marks represent optimal estimates of transient flux with 95% c.i.). Blue lines represent average fluxes inferred in a non-synchronized cell population. As shown, glucose-derived flux into TCA cycle peak in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase.


Figure 6 ( a - b ) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m + 2 ( a ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) . ( a - b ) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl - CoA m + 2 ( b ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) . It further inhibits oscillations in citrate m + 5 / m + 4 ratio ( c ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) . It further inhibits oscillations in acetyl - CoA m + 2 ( d ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) . ( e ) The fraction of cells in G1 , S , and G2 / M phases in synchronized HeLa cells after 3 hour treatment with DCA ( normalized by measurements in untreated control cells ) . ( f ) The fraction of cells in G1 , S , and G2 / M phases in non - synchronized HeLa and HCT - 116 cells after 24 hour treatment with DCA ( normalized by measurements in untreated control cells ) . As shown , DCA significantly increases the fraction of cells in S phase , inhibiting cellular progression into G2 phase ; showing mean and s . d . of n = 3 for all isotopic labeling forms and FACS measurements ( a - f ) . Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two - tailed , unequal variance t - test ( e - f ) .

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

Figure 6(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m+2 (a) from isotopic glucose (representing glycolytic flux into TCA cycle).(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl-CoA m+2 (b) from isotopic glucose (representing glycolytic flux into TCA cycle).It further inhibits oscillations in citrate m+5/m+4 ratio (c) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).It further inhibits oscillations in acetyl-CoA m+2 (d) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).(e) The fraction of cells in G1, S, and G2/M phases in synchronized HeLa cells after 3 hour treatment with DCA (normalized by measurements in untreated control cells).(f) The fraction of cells in G1, S, and G2/M phases in non-synchronized HeLa and HCT-116 cells after 24 hour treatment with DCA (normalized by measurements in untreated control cells). As shown, DCA significantly increases the fraction of cells in S phase, inhibiting cellular progression into G2 phase; showing mean and s.d. of n=3 for all isotopic labeling forms and FACS measurements (a-f). Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two-tailed, unequal variance t-test (e-f).


Figure 1 ( A ) Representative images of H & E staining of colon from 3m C57Bl ( n = 4 ) and mdx ( n = 4 ) mice . High magnification ( scale bar : 20 μm ) and low magnification ( scale bar : 200 μm ) . ( B ) Mucus layer , area between yellow dash lines ; crypt length , yellow headed arrow . Scale bar : 100 μm . ( C ) Mucus layer thickness and crypt length were quantified for n = 4 mice per group ( with pooled samples of n = 60 for mucus layer thickness and n = 80 for crypt length ) . ( D ) SCFA faecal quantification of 3m C57Bl ( n = 3 ) and mdx ( n = 4 - 5 ) mice . Data information : Data are presented as mean ± SD ( * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Student t - test ) . ( E ) Colon images captured with the iMScope TRIO described altered pattern of expression of different Phosphatidylcholines ( PC ) and Lysophosphatidylcholines ( LysoPC ) ( as indicated by m / z values ) in 3m mdx mice ( n = 3 ) . Scale bar : 50 μm . For each lipid , the mean intensities measured at 12 positions throughout colon images are shown on the right side where bars are mean ± SEM ( n = 3 ) . Data information : Data are presented as mean ± SD ( * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Student t - test ) . ( F ) Cropped images of representative WB showing the expression of proteins involved in inflammation and fibrosis in colon tissues of 3m C57Bl ( n = 3 ) and 3m mdx ( n = 5 ) . Densitometric analyses of protein expression was shown as ratio to actin . Data information : Data are presented as mean ± SD ( * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Student t - test ) .

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

Figure 1(A) Representative images of H&E staining of colon from 3m C57Bl (n=4) and mdx (n=4) mice. High magnification (scale bar: 20 μm) and low magnification (scale bar: 200 μm).(B) Mucus layer, area between yellow dash lines; crypt length, yellow headed arrow. Scale bar: 100 μm. (C) Mucus layer thickness and crypt length were quantified for n=4 mice per group (with pooled samples of n=60 for mucus layer thickness and n=80 for crypt length).(D) SCFA faecal quantification of 3m C57Bl (n=3) and mdx (n=4-5) mice. Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).(E) Colon images captured with the iMScope TRIO described altered pattern of expression of different Phosphatidylcholines (PC) and Lysophosphatidylcholines (LysoPC) (as indicated by m/z values) in 3m mdx mice (n=3). Scale bar: 50 μm. For each lipid, the mean intensities measured at 12 positions throughout colon images are shown on the right side where bars are mean ± SEM (n = 3). Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).(F) Cropped images of representative WB showing the expression of proteins involved in inflammation and fibrosis in colon tissues of 3m C57Bl (n=3) and 3m mdx (n=5). Densitometric analyses of protein expression was shown as ratio to actin. Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).


Figure 4 ( E ) Faecal content quantification of SCFAs in 3m mdx and 3m mdx + ABX mice ( n = 5 per group ) . Data equal to 0 . Data are presented as mean ± SD ( * * p < 0 . 01 , * * * p < 0 . 001 ; Student t - test ) .

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Figure 4(E) Faecal content quantification of SCFAs in 3m mdx and 3m mdx+ABX mice (n=5 per group). Data equal to 0. Data are presented as mean ± SD (**p< 0.01, ***p< 0.001; Student t-test).


Figure 5 ( A - D ) FACS analysis of spleen and muscle homogenates from 3m C57Bl ( n = 4 ) , mdx ( n = 5 ) and mdx + ABX ( n = 7 ) mice demonstrates no significant alteration of CD45 + CD11b + CD4 - CD8 - myeloid cells ( A and C ) and few differences in CD4 + or CD8 + naïve ( CD62L + CD44 - ) , central memory ( CD62L + CD44 + ) and effector ( CD62L - CD44 + ) T cells ( B and D ) . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test . ( E ) FACS analysis of spleen of 3m C57Bl ( n = 7 ) , mdx ( n = 9 ) and GFmdx ( n = 5 ) mice revealed similar proportions of CD4 + and CD8 + T cells but reduced activated CD44 + T cells in GFmdx mice . Representative plots are depicted . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test . ( F ) Graphs show cumulative frequencies of CD4 + and CD8 + T cells on live cells of 3m C57Bl ( n = 7 ) , mdx ( n = 4 ) and GFmdx ( n = 5 ) mice . Representative dot plots and cumulative frequencies of splenic CD4 + GITR + CD25 + Treg . Frequencies of effector CD44 + T cells were significantly decreased in spleen of GFmdx mice . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test . ( G ) Representative dot - plots showing the proportion of muscle - infiltrating CD45 + cells of 3m C57Bl ( n = 6 ) , mdx ( n = 6 ) and GFmdx ( n = 5 ) mice . Cumulative frequencies of muscle - infiltrating CD45 + cells are shown . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test . ( H ) Representative images of TA muscles from 3m mdx and GFmdx mice stained for CD45 ( in green ) , isolectin ( in red ) and phalloidin ( in purple ) . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 10 μm . ( I ) Absolute number of CD3 + inflammatory cells ( white arrows ) were quantified in n = 12 images of TA of 3m C57Bl , 3m mdx and 3m GFmdx mice ( n = 6 each ) . CD3 staining is shown in green and DAPI in blue . Scale bars : 50 μm . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 The comparisons among the averages of CD3 + cells were evaluated using unpaired t - test .

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

Figure 5(A-D) FACS analysis of spleen and muscle homogenates from 3m C57Bl (n=4), mdx (n=5) and mdx+ABX (n=7) mice demonstrates no significant alteration of CD45+CD11b+CD4-CD8- myeloid cells (A and C) and few differences in CD4+ or CD8+ naïve (CD62L+ CD44-), central memory (CD62L+ CD44+) and effector (CD62L- CD44+) T cells (B and D). Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.(E) FACS analysis of spleen of 3m C57Bl (n=7), mdx (n=9) and GFmdx (n=5) mice revealed similar proportions of CD4+ and CD8+ T cells but reduced activated CD44+ T cells in GFmdx mice. Representative plots are depicted. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.(F) Graphs show cumulative frequencies of CD4+ and CD8+ T cells on live cells of 3m C57Bl (n=7), mdx (n=4) and GFmdx (n=5) mice. Representative dot plots and cumulative frequencies of splenic CD4+GITR+CD25+ Treg. Frequencies of effector CD44+ T cells were significantly decreased in spleen of GFmdx mice. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.(G) Representative dot-plots showing the proportion of muscle-infiltrating CD45+ cells of 3m C57Bl (n=6), mdx (n=6) and GFmdx (n=5) mice. Cumulative frequencies of muscle-infiltrating CD45+ cells are shown. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.(H) Representative images of TA muscles from 3m mdx and GFmdx mice stained for CD45 (in green), isolectin (in red) and phalloidin (in purple). Nuclei were counterstained with DAPI (in blue). Scale bar: 10 μm.(I) Absolute number of CD3+ inflammatory cells (white arrows) were quantified in n=12 images of TA of 3m C57Bl, 3m mdx and 3m GFmdx mice (n=6 each). CD3 staining is shown in green and DAPI in blue. Scale bars: 50 μm. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 The comparisons among the averages of CD3+ cells were evaluated using unpaired t-test.


Figure 6 ( D ) RT - qPCR analysis of TA muscles of two independent experiments with 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx ( n = 5 ) mice determined the expression of myogenic markers . Data information : Data are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , ordinary one - way ANOVA and non - parametric test followed by Kruskal - Wallis test for RT - qPCR ) . ( E ) Representative Gomori modified staining and quantification of myofiber area and relative frequency of the myofiber cross - sectional area ( CSA ) expressed as the frequency distribution of the TA muscles of 3m C57Bl ( n = 4 ) , 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx mice ( n = 5 ) . Pooled samples for each group with n = 6240 for 3m C57Bl ; n = 6001 for 3m mdx ; n = 10556 for 3m mdx + ABX ; n = 23059 for GFmdx . For morphometric analysis , images were quantified with Image J software for each mouse . Scale bars : 50 μm . Data information : Data are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , ordinary one - way ANOVA ( G ) Representative images of skeletal muscle showed the distribution and composition of the myosin heavy chain ( MyHC ) isoforms ( Type IIa , Type IIx and Type IIb ) . ( H , I ) Graph portrays ( H ) the percentage of myofibers expressing different MyHC isoforms and ( I ) myofibers area per type of MyHC in TAs of 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx ( n = 5 ) mice ( n = 12 images per animal ) . Data information : Data are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , ordinary one - way ANOVA ( J ) Representative SDH staining and quantification of percentage of SDH + myofibers of TAs from 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx mice ( n = 5 ) ( n = 12 images per animal ) . Scale bars : 50 μm .

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

Figure 6(D) RT-qPCR analysis of TA muscles of two independent experiments with 3m mdx (n=4), mdx+ABX (n=4) and GFmdx (n=5) mice determined the expression of myogenic markers. Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA and non-parametric test followed by Kruskal-Wallis test for RT-qPCR).(E) Representative Gomori modified staining and quantification of myofiber area and relative frequency of the myofiber cross-sectional area (CSA) expressed as the frequency distribution of the TA muscles of 3m C57Bl (n=4), 3m mdx (n=4), mdx+ABX (n=4) and GFmdx mice (n=5). Pooled samples for each group with n=6240 for 3m C57Bl; n=6001 for 3m mdx; n=10556 for 3m mdx+ABX; n=23059 for GFmdx. For morphometric analysis, images were quantified with Image J software for each mouse. Scale bars: 50 μm. Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA(G) Representative images of skeletal muscle showed the distribution and composition of the myosin heavy chain (MyHC) isoforms (Type IIa, Type IIx and Type IIb). (H, I) Graph portrays (H) the percentage of myofibers expressing different MyHC isoforms and (I) myofibers area per type of MyHC in TAs of 3m mdx (n=4), mdx+ABX (n=4) and GFmdx (n=5) mice (n=12 images per animal). Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA(J) Representative SDH staining and quantification of percentage of SDH+ myofibers of TAs from 3m mdx (n=4), mdx+ABX (n=4) and GFmdx mice (n=5) (n=12 images per animal). Scale bars: 50 μm.


Figure 7 ( D , E ) Representative H & E staining ( D ) and quantification of myofiber area with Image J software ( E ) of TA muscles from 3m C57Bl ( n = 5 ) , mdx ( n = 5 ) and ABX - mdxFMT _ C57Bl ( n = 6 ) . Scale bars for H & E : 200 μm . Data information : Data for staining quantification are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison test ) . ( H ) Representative images of skeletal muscle showed the distribution and composition of MyHC isoforms ( Type IIa , Type IIx and Type IIb ) . Scale bar : 50 μm . ( I ) Graph portrays the percentage of myofibers expressing different MyHC isoforms . n = 12 images were analysed for each mouse . ( J - K ) Representative SDH staining and quantification of percentage of SDH + myofibers of TA muscles from mdx ( n = 5 ) and ABX - mdxFMT _ C57Bl ( n = 6 ) ( n = 12 images per mouse ) . Scale bar : 200 μm . Data information : Data for staining quantification are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison test ) . ( L - M ) Representative image of CD31 ( in cyan ) , α - SMA ( in green ) and isolectin ( in red ) staining and their quantification in TA muscles from mdx ( n = 5 ) and ABX - mdxFMT _ C57Bl ( n = 6 ) mice . Scale bar : 500 μm . Data information : Data for staining quantification are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison test ) .

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

Figure 7(D, E) Representative H&E staining (D) and quantification of myofiber area with Image J software (E) of TA muscles from 3m C57Bl (n=5), mdx (n=5) and ABX-mdxFMT_C57Bl (n=6). Scale bars for H&E: 200 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).(H) Representative images of skeletal muscle showed the distribution and composition of MyHC isoforms (Type IIa, Type IIx and Type IIb). Scale bar: 50 μm. (I) Graph portrays the percentage of myofibers expressing different MyHC isoforms. n=12 images were analysed for each mouse.(J-K) Representative SDH staining and quantification of percentage of SDH+ myofibers of TA muscles from mdx (n=5) and ABX-mdxFMT_C57Bl (n=6) (n=12 images per mouse). Scale bar: 200 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).(L-M) Representative image of CD31 (in cyan), α-SMA (in green) and isolectin (in red) staining and their quantification in TA muscles from mdx (n=5) and ABX-mdxFMT_C57Bl (n=6) mice. Scale bar: 500 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).


Figure 1 ( E ) Tadpoles at NF50 were amputated , fixed at the indicated time and then processed for whole mount in situ hybridisation using a probe specific for foxm1 . The two last panels show transverse section in the non - regenerating spinal cord ( nr ) and the regenerate at 3dpa . The red circle highlights the spinal cord and the asterisk the notochord . ( F ) Total RNA was isolated from regenerating tails at indicated time points post amputation , reversed transcribed into cDNA and analysed for foxm1 expression by qPCR , using ef1α as a reference gene . The graph represents the mean + / - SD of three independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test was used . * p = 0 .

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Figure 1(E) Tadpoles at NF50 were amputated, fixed at the indicated time and then processed for whole mount in situ hybridisation using a probe specific for foxm1. The two last panels show transverse section in the non-regenerating spinal cord (nr) and the regenerate at 3dpa. The red circle highlights the spinal cord and the asterisk the notochord.(F) Total RNA was isolated from regenerating tails at indicated time points post amputation, reversed transcribed into cDNA and analysed for foxm1 expression by qPCR, using ef1α as a reference gene. The graph represents the mean +/- SD of three independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was used. * p=0.0149


Figure 2 ( A ) NF40 tadpoles with the following genotypes foxm1 - / - ( mut ) , foxm1 + / - ( het ) and foxm1 + / + ( wt ) were amputated and left to regenerate for 7 days . The images show representative tails at 3 and 7 dpa . The white arrowheads indicate the amputation site . ( B ) Quantification of the rate of regeneration . The ratio of the length of the regenerate to the length that has originally be amputated was compared for the whole tail at 3 and 7 dpa . The graph represents the mean + / - SD of 5 independent experiments from 3 different clutches with at least 5 tadpoles in each experiment . ( D ) Representative images of EdU ( red ) and DAPI ( blue ) staining at 5 dpa . ( E ) The graph represents the mean + / - SD of EdU + cells over the total number of cells in the spinal cord of 5 to 12 tadpoles . Each data point represents a tadpole , with an average of 9 sections analysed per animal . ( G ) Representative images of EdU ( green ) , BrdU ( magenta ) , Sox3 ( white ) and DAPI ( blue ) staining at 3 dpa . ( H ) Quantification of images in ( G ) . The graph represents the mean + / - SD of 6 tadpoles with an average of 13 sections per tadpole analysed .

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Figure 2(A) NF40 tadpoles with the following genotypes foxm1-/- (mut), foxm1+/- (het) and foxm1+/+ (wt) were amputated and left to regenerate for 7 days. The images show representative tails at 3 and 7 dpa. The white arrowheads indicate the amputation site. (B) Quantification of the rate of regeneration. The ratio of the length of the regenerate to the length that has originally be amputated was compared for the whole tail at 3 and 7 dpa. The graph represents the mean+/-SD of 5 independent experiments from 3 different clutches with at least 5 tadpoles in each experiment. (D) Representative images of EdU (red) and DAPI (blue) staining at 5 dpa. (E) The graph represents the mean +/- SD of EdU+ cells over the total number of cells in the spinal cord of 5 to 12 tadpoles. Each data point represents a tadpole, with an average of 9 sections analysed per animal. (G) Representative images of EdU (green), BrdU (magenta), Sox3 (white) and DAPI (blue) staining at 3 dpa. (H) Quantification of images in (G). The graph represents the mean +/- SD of 6 tadpoles with an average of 13 sections per tadpole analysed.


Figure 4 ( C ) Representative sections labelled with an anti - PCNA antibody ( red ) and DAPI ( blue ) at the indicated day after amputation ( dpa ) . The white arrowheads point to cells in S phase in the spinal cord and the yellow arrowhead at cells in G1 , G2 and M phases . The right panels correspond to the inset indicated as a white box in the middle panels .

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Figure 4(C) Representative sections labelled with an anti-PCNA antibody (red) and DAPI (blue) at the indicated day after amputation (dpa). The white arrowheads point to cells in S phase in the spinal cord and the yellow arrowhead at cells in G1, G2 and M phases. The right panels correspond to the inset indicated as a white box in the middle panels.


Figure 5 ( A ) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa . After sectioning , the samples were labelled with antibodies against Sox3 ( green ) , EdU ( magenta ) and DAPI ( blue ) . White arrowheads show Sox3 positive extensions . ( B ) Sections of spinal cords at 5 dpa in the stump ( non - regen . ) or in the regenerate ( regen ) labelled with anti - Sox3 ( green ) , anti - Acetylated Tubulin ( AcTub , magenta ) and DAPI ( blue ) . White arrowheads show Sox3 positive extensions . ( E ) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa . After sectioning , the samples were labelled with antibodies against Myt1 ( green ) , EdU ( magenta ) and DAPI ( blue ) . ( F ) Quantification of images shown in ( E ) . The graph represents the mean + / - SD from 3 tadpoles with an average of 12 sections each .

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Figure 5(A) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Sox3 (green), EdU (magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.(B) Sections of spinal cords at 5 dpa in the stump (non-regen.) or in the regenerate (regen) labelled with anti-Sox3 (green), anti-Acetylated Tubulin (AcTub, magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.(E) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Myt1 (green), EdU (magenta) and DAPI (blue). (F) Quantification of images shown in (E). The graph represents the mean +/- SD from 3 tadpoles with an average of 12 sections each.


Figure 1Gene - trap - based genetic screen in haploid human cells for regulators of p38 kinase activation ( p38 Thr180 / Tyr182 phosphorylation ) in response to hyperosmotic shock ( 500 mM Sorbitol , 1 h ) . Per gene ( dots ) , the frequency of gene - trap insertions in the ' high ' phospho - p38 population divided by the frequency of insertions in the ' low ' population is plotted as mutation index ( y - axis ) against the total number of insertions assigned to the gene ( x - axis ) . Significant negative and positive regulators are colored in orange and blue , respectively ( two - sided Fisher ' s exact test , false discovery rate - corrected , p ≤ 0 . 05 ) . Human U2OS cells and cells deleted for ZAK ( ∆ ZAK ) were treated with anisomycin ( 1 h ) or 500 mM sorbitol ( 1 h ) . Lysates were analyzed by immunoblotting as indicated . Unique gene - trap insertions ( dots ) mapped to the genomic ZAK locus ( x - axis ) identified in the low ( blue ) and high ( orange ) channel of two individual haploid genetic screens for stress - induced p38 activation and Anisomycin ( Brockmann et al . , 2017 ) ) . The total number of identified insertions was similar for each channel within the individual screens . For visual purposes , insertion dots were spread on the y - axis and exons in the ZAK gene body schematic have been scaled up ( compared to introns ) . U2OS / S - HA - ZAKβ cells were induced for expression with doxycycline ( DOX ) and subjected to the indicated drugs and treatments ( 1 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . Sorb . , sorbitol ; Ani . , anisomycin ; Ars . , arsenite . U2OS cells stably expressing WT and kinase - dead ( KD ) versions of S - HA - ZAKβ were pre - treated with ZAK inhibitor ( ZAKi - 0 . 5 h ) and 500 mM sorbitol ( 1 h ) as indicated . Whole Cell Extracts ( WCE ) were analyzed by immunoblotting , and strep - pulldown material was used in a kinase assay , separated by SDS - PAGE and analyzed by autoradiography . U2OS and ∆ ZAK cells rescued with S - HA - ZAKβ , were DOX - induced and treated with 500 mM sorbitol ( 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies . U2OS and ∆ ZAK cells were transfected with the indicated siRNAs and treated with sorbitol ( 500 mM , 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies . T1 , TAK1 ; M2 , MEKK2 .

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Figure 1Gene-trap-based genetic screen in haploid human cells for regulators of p38 kinase activation (p38 Thr180/Tyr182 phosphorylation) in response to hyperosmotic shock (500 mM Sorbitol, 1 h). Per gene (dots), the frequency of gene-trap insertions in the 'high' phospho-p38 population divided by the frequency of insertions in the 'low' population is plotted as mutation index (y-axis) against the total number of insertions assigned to the gene (x-axis). Significant negative and positive regulators are colored in orange and blue, respectively (two-sided Fisher's exact test, false discovery rate-corrected, p≤0.05).Human U2OS cells and cells deleted for ZAK (∆ZAK) were treated with anisomycin (1 h) or 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting as indicated.Unique gene-trap insertions (dots) mapped to the genomic ZAK locus (x-axis) identified in the low (blue) and high (orange) channel of two individual haploid genetic screens for stress-induced p38 activation and Anisomycin(Brockmann et al., 2017)). The total number of identified insertions was similar for each channel within the individual screens. For visual purposes, insertion dots were spread on the y-axis and exons in the ZAK gene body schematic have been scaled up (compared to introns).U2OS/S-HA-ZAKβ cells were induced for expression with doxycycline (DOX) and subjected to the indicated drugs and treatments (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. Sorb., sorbitol; Ani., anisomycin; Ars., arsenite.U2OS cells stably expressing WT and kinase-dead (KD) versions of S-HA-ZAKβ were pre-treated with ZAK inhibitor (ZAKi - 0.5 h) and 500 mM sorbitol (1 h) as indicated. Whole Cell Extracts (WCE) were analyzed by immunoblotting, and strep-pulldown material was used in a kinase assay, separated by SDS-PAGE and analyzed by autoradiography.U2OS and ∆ZAK cells rescued with S-HA-ZAKβ, were DOX-induced and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.U2OS and ∆ZAK cells were transfected with the indicated siRNAs and treated with sorbitol (500 mM, 1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies. T1, TAK1; M2, MEKK2.


Figure 2U2OS cells stably expressing Strep - HA - ZAKβ or - ZAKα were treated with sorbitol ( 500 mM , 1 h ) as indicated . Cells were fixed , immunostained with HA antibody and counter - stained with DAPI . Data information : scale bars , 10 μm . U2OS cells stably expressing ZAKβ - GFP were treated with sorbitol ( 500 mM , 1 h ) , pre - extracted , fixed and immunostained with Lamin A / C antibody . Lower right : Intensity distribution graph showing fluorescence intensities along the magenta line . As in ( B ) except that Hela cells were transfected with ZAKbeta - GFP and immunostained with 53BP1 antibody . Data information : scale bars , 10 μm . Cells from ( B ) were imaged by live cell fluorescence microscopy . Sorbitol ( final concentration 500 mM ) was added after acquisition of the first frame . Data information : scale bars , 10 μm . As in ( D ) , except that cells were co - transfected with mCherry - FAK . Inserts show higher magnification of the yellow and red regions , white arrow indicates the direction of stress fibers . Data information : scale bars , 10 μm . U2OS cells were transfected with constructs from ( F ) and imaged by live cell fluorescence microscopy . Sorbitol ( final concentration 500 mM ) was added after acquisition of the first frame . Data information : scale bars , 10 μm . U2OS and ∆ ZAK cells stably expressing the indicated Strep - HA - tagged ZAKβ constructs were treated with sorbitol ( 500 mM , 5 min ) . Lysates were analyzed by immunoblotting with the indicated antibodies .

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Figure 2U2OS cells stably expressing Strep-HA-ZAKβ or -ZAKα were treated with sorbitol (500 mM, 1 h) as indicated. Cells were fixed, immunostained with HA antibody and counter-stained with DAPI. Data information: scale bars, 10 μm.U2OS cells stably expressing ZAKβ-GFP were treated with sorbitol (500 mM, 1 h), pre-extracted, fixed and immunostained with Lamin A/C antibody. Lower right: Intensity distribution graph showing fluorescence intensities along the magenta line. As in (B) except that Hela cells were transfected with ZAKbeta-GFP and immunostained with 53BP1 antibody. Data information: scale bars, 10 μm.Cells from (B) were imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.As in (D), except that cells were co-transfected with mCherry-FAK. Inserts show higher magnification of the yellow and red regions, white arrow indicates the direction of stress fibers. Data information: scale bars, 10 μm.U2OS cells were transfected with constructs from (F) and imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were treated with sorbitol (500 mM, 5 min). Lysates were analyzed by immunoblotting with the indicated antibodies.


Figure 3U2OS and ∆ ZAK cells were embedded in agarose and subjected to cyclic compression ( 0 . 5 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . U2OS and ∆ ZAK cells stably expressing the indicated Strep - HA - tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min . Lysates were analyzed as in ( B ) . U2OS and ∆ ZAK cells were pretreated with ZAK inhibitor ( ZAKi ) for 30 min and subjected to equibiaxial ( Equi ) or uniaxial ( Uni ) cyclic stretch ( 0 . 5 h ) as indicated . Lysates were analyzed as in ( B ) . U2OS and ∆ ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch ( 5 min ) as indicated . Lysates were analyzed as in ( B ) . Expression levels of MAP3 kinase genes in human skeletal muscle ( GTEx Portal ) . TPM , Transcripts Per Million . C2C12 cells were incubated in diff . medium for 3 or 6 days . Lysates were analyzed for ZAK isoform expression by immunoblotting . C2C12 myoblasts and myotubes ( 6 d differentiation ) were pre - treated with ZAK inhibitor ( ZAKi , 0 . 5 h ) and treated with 500 mM sorbitol ( 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies .

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Figure 3U2OS and ∆ZAK cells were embedded in agarose and subjected to cyclic compression (0.5 h). Lysates were analyzed by immunoblotting with the indicated antibodies.U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min. Lysates were analyzed as in (B).U2OS and ∆ZAK cells were pretreated with ZAK inhibitor (ZAKi) for 30 min and subjected to equibiaxial (Equi) or uniaxial (Uni) cyclic stretch (0.5 h) as indicated. Lysates were analyzed as in (B).U2OS and ∆ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch (5 min) as indicated. Lysates were analyzed as in (B).Expression levels of MAP3 kinase genes in human skeletal muscle (GTEx Portal). TPM, Transcripts Per Million.C2C12 cells were incubated in diff. medium for 3 or 6 days. Lysates were analyzed for ZAK isoform expression by immunoblotting.C2C12 myoblasts and myotubes (6 d differentiation) were pre-treated with ZAK inhibitor (ZAKi, 0.5 h) and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.


Figure 4Skeletal muscle ( tibialis anterior , TA ) and liver from WT and ZAK - / - mice were lysed and analyzed for ZAK isoform expression by immunoblotting . Ponceau staining of the membrane indicates equal loading . Mouse embryonic fibroblasts ( MEF ) isolated from WT and ZAK - / - mice were treated with 500 mM sorbitol ( 1 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . * , unspecific band . 16 - 18 - week - old WT and ZAK - / - male mice ( n = 3 biological replicates ) were subjected to the protocol in ( D ) . TA lysates were analyzed by immunoblotting with the indicated antibodies . ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from ( D ) . Values indicate absolute phospho - peptide abundances and error bars represent the standard deviation ( n = 3 biological replicates ) . TA muscle in an 8 - week old WT male mouse was electroporated with a ZAKβ - tdTomato construct . After 7 days the muscle was harvested , sectioned longitudinally and immunostained with an antibody against the Z - disc marker α - actinin1 . The right panel shows a higher magnification of the region highlighted in yellow . Data information : scale bars , 20 μm . C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP - ZAKβ constructs . Cells were fixed and immunostained with an antibody against α - actinin1 . Data information : scale bars , 20 μm . 16 - 18 - week - old WT and ZAK - / - male mice were subjected to the experimental protocol in ( I ) . TA muscle lysates were analyzed by immunoblotting with the indicated antibodies .

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Figure 4Skeletal muscle (tibialis anterior, TA) and liver from WT and ZAK-/- mice were lysed and analyzed for ZAK isoform expression by immunoblotting. Ponceau staining of the membrane indicates equal loading.Mouse embryonic fibroblasts (MEF) isolated from WT and ZAK-/- mice were treated with 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. *, unspecific band.16-18-week-old WT and ZAK-/- male mice (n=3 biological replicates) were subjected to the protocol in (D). TA lysates were analyzed by immunoblotting with the indicated antibodies.ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from (D). Values indicate absolute phospho-peptide abundances and error bars represent the standard deviation (n=3 biological replicates).TA muscle in an 8-week old WT male mouse was electroporated with a ZAKβ-tdTomato construct. After 7 days the muscle was harvested, sectioned longitudinally and immunostained with an antibody against the Z-disc marker α-actinin1. The right panel shows a higher magnification of the region highlighted in yellow. Data information: scale bars, 20 μm.C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP-ZAKβ constructs. Cells were fixed and immunostained with an antibody against α-actinin1. Data information: scale bars, 20 μm.16-18-week-old WT and ZAK-/- male mice were subjected to the experimental protocol in (I). TA muscle lysates were analyzed by immunoblotting with the indicated antibodies.


Figure 5H & E staining of soleus muscle cross sections from 8 - and 22 - week - old WT and ZAK - / - male mice . Arrows indicate the presence of centralized nuclei . Scale bars , 50 μm . Quantification of ( A ) . Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 and * * * , p < 0 . 001 in t - test with Bonferroni - Dunn correction for multiple comparison . Muscles from ( A ) were immunostained for type I ( red ) and type IIa ( green ) fibers using myosin isoform - specific antibodies . Scale bars , 500 μm . Quantification of ( C ) . Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 and ns , not significant in t - test with Bonferroni - Dunn correction for multiple comparison .

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Figure 5H&E staining of soleus muscle cross sections from 8- and 22-week-old WT and ZAK-/- male mice. Arrows indicate the presence of centralized nuclei. Scale bars, 50 μm. Quantification of (A). Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ***, p<0.001 in t-test with Bonferroni-Dunn correction for multiple comparison.Muscles from (A) were immunostained for type I (red) and type IIa (green) fibers using myosin isoform-specific antibodies. Scale bars, 500 μm. Quantification of (C). Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ns, not significant in t-test with Bonferroni-Dunn correction for multiple comparison.


Figure 1q - RT - PCR reveals that miR - 181a / b silencing leads to upregulation of miR - 181a / b predicted targets in SH - SY5Y cells . N = 3 independent experiments . miR - 181 - mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3 ' - UTR predicted target sequences . Point mutations ( mut ) in miR - 181a / b binding sites abolish Luciferase repression in all cases apart from PPARGC1A . Data are normalized to negative mimic transfection ( dashed line ) . N = 6 independent experiments . q - RT - PCR reveals upregulation of miR - 181a / b targets in the eyes of miR - 181a / b - 1 - / - vs . miR - 181a / b - 1 + / + animals . n ≥ 5 animals / genotype . miR - 181a / b - 1 - / - mice show increased mtDNA content vs . miR - 181a / b - 1 + / + mice as measured by q - PCR . N = 4 animals / genotype . WB analysis ( left panel ) reveals increased levels of mitochondrial proteins in the eyes of miR - 181a / b - 1 - / - ( - / - ) vs . miR - 181a / b - 1 + / + ( + / + ) mice ( quantified in the right panel ) . Data are normalized to either p115 or Gapdh . N ≥ 3 animals / genotype . Please note that all compared bands from + / + and - / - samples are from the same blots which were cropped and shown split for the sake of data presentation clarity . WB analysis on mitochondrial and cytosolic fractions ( left panel ) show increased levels of p62 and Parkin in mitochondrial fraction from the eye of - / - vs . + / + mice ( quantified in the right panel ) . Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions , respectively . N = 3 animals / genotype . Cell death analysis shows that miR - 181a / b silencing protects SH - SY5Y cells from FCCP treatment . N ≥ 7 independent experiments .

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

Figure 1q-RT-PCR reveals that miR-181a/b silencing leads to upregulation of miR-181a/b predicted targets in SH-SY5Y cells. N=3 independent experiments.miR-181-mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3'-UTR predicted target sequences. Point mutations (mut) in miR-181a/b binding sites abolish Luciferase repression in all cases apart from PPARGC1A. Data are normalized to negative mimic transfection (dashed line). N=6 independent experiments.q-RT-PCR reveals upregulation of miR-181a/b targets in the eyes of miR-181a/b-1-/- vs. miR-181a/b-1+/+ animals. n≥5 animals/genotype.miR-181a/b-1-/- mice show increased mtDNA content vs. miR-181a/b-1+/+ mice as measured by q-PCR. N=4 animals/genotype.WB analysis (left panel) reveals increased levels of mitochondrial proteins in the eyes of miR-181a/b-1-/- (-/-) vs. miR-181a/b-1+/+ (+/+) mice (quantified in the right panel). Data are normalized to either p115 or Gapdh. N≥3 animals/genotype. Please note that all compared bands from +/+ and -/- samples are from the same blots which were cropped and shown split for the sake of data presentation clarity.WB analysis on mitochondrial and cytosolic fractions (left panel) show increased levels of p62 and Parkin in mitochondrial fraction from the eye of -/- vs. +/+ mice (quantified in the right panel). Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions, respectively. N=3 animals/genotype.Cell death analysis shows that miR-181a/b silencing protects SH-SY5Y cells from FCCP treatment. N≥7 independent experiments.