( H ) Representative HALO spatial plot showing spatial distribution of types with indicated marker combination in 99LN - BrM representative IF of multiplexed histology . Scale bar ; 100 μm .

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

(H) Representative HALO spatial plot showing the spatial distribution of cell types with indicated marker combination in 99LN-BrM and representative IF images of multiplexed histology. Scale bar; 100 μm.


( C ) of Iba1 + macrophages in sections of 99LN - BrM at trial endpoint ( Ctrl n = 27 , WBRT n = 18 ) . ( D ) Representative IF of 99LN - BrM stained for macrophage marker Iba1 ( red ) MG marker Tmem119 ( ) . Higher magnification present areas in peri - region in . DAPI was used as counterstain . Scale bar ; 100 µm .

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

(C) Quantification of Iba1+ macrophages in IHC sections of 99LN-BrM at trial endpoint (Ctrl n=27, WBRT n=18). (D) Representative IF images of 99LN-BrM stained for the macrophage marker Iba1 (red) and the MG marker Tmem119 (white). Higher magnification images present areas in the peri-tumor region and in the tumor core. DAPI was used as nuclear counterstain. Scale bar; 100 µm.


( G ) of CD11c + MHCII + immune by flow cytometry in 99LN - BrM with without WBRT . ( H , I ) Relative proportion of cDC1 cDC2 in 99LN - BrM with without WBRT . ( J ) cDC1 : cDC2 in 99LN - BrM on flow cytometry data ( H + I ) .

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

(G) Quantification of CD11c+MHCII+ immune cells by flow cytometry in 99LN-BrM with and without WBRT. (H, I) Relative proportion of cDC1 and cDC2 in 99LN-BrM with and without WBRT. (J) cDC1:cDC2 ratio in 99LN-BrM based on flow cytometry data (H+I).


( K ) Representative of CD3 + , FoxP3 + CD8 + T in 99LN - BrM sections with without WBRT . Scale bar ; 100 µm . ( L ) of CD3 + in sections of 99LN - BrM . ( Ctrl n = 26 , WBRT n = 18 ) . ( M ) FoxP3 : CD3 in sections of 99LN - BrM . ( Ctrl n = 25 , WBRT n = 18 ) . ( N ) CD8 : CD3 in sections of 99LN - BrM . ( Ctrl n = 25 , WBRT n = 18 ) .

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

(K) Representative IHC images of CD3+, FoxP3+ and CD8+ T cells in 99LN-BrM sections with and without WBRT. Scale bar; 100 µm. (L) Quantification of CD3+ cells in IHC sections of 99LN-BrM. (Ctrl n=26, WBRT n=18). (M) FoxP3:CD3 ratio in IHC sections of 99LN-BrM. (Ctrl n=25, WBRT n=18). (N) CD8:CD3 ratio in IHC sections of 99LN-BrM. (Ctrl n=25, WBRT n=18).


( B ) T of estimated in BrM CLN of mice from control or WBRT group .

(B) Percent T cells of estimated cells in BrM and CLN of mice from control or WBRT group.


( B ) Representative MRI of 99LN - BrM from mice at different time points after treatment start .

(B) Representative MRI images of 99LN-BrM from mice at different time points after treatment start.


( G ) Kaplan - Meier curves show of symptom - free mice .

(G) Kaplan-Meier curves show the percentage of symptom-free mice.


( K ) Relative Gzmb IFNγ level on CD4 + CD8 + T cultivated with different types , with or without aPD - 1 unstimulated or stimulated with conditioned media ( Cond . ) ( n = 4 for conditions including BMDM , n = 5 for other conditions ) .

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

(K) Relative Gzmb and IFNγ protein level on CD4+ and CD8+ T cells cultivated with different cell types, with or without aPD-1 and unstimulated or stimulated with tumor conditioned media (Cond.) (n=4 for conditions including BMDM, n=5 for all other conditions).


A . Frequencies total numbers ( mean + ) of CD11b + myeloid isolated from spleen peripheral LN of naïve non - immunized mice .

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

A. 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 + ) of CD11b + expressing IL - 1β shown in ( A ) restimulated with indicated stimuli for 4 h .

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

B. Total numbers (mean +SEM) of CD11b+ cells expressing IL-1β shown in (A) and restimulated with indicated stimuli for 4 h.


C . ( mean + ) of myeloid populations expressing IL - 1β , isolated from LN restimulated with GM - CSF shown in ( B ) .

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

C. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β, isolated from LN and restimulated with GM-CSF shown in (B).


D . Frequencies total numbers ( mean + ) of CD11b + myeloid isolated from spleen of immunized mice .

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

D. Frequencies and total numbers (mean +SEM) of CD11b+myeloid cells isolated from the spleen of immunized mice.


E . of IL - 1R1 expression by CD11b + isolated from dLN of mice treated as in ( D ) . Data are representative FACS histogram overlays , gated on VD - CD11b + with mean frequencies per group .

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

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 . ( mean + ) of IL - 1β expression by CD11b + shown in ( E ) .

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

F. Quantification (mean +SEM) of IL-1β expression by CD11b+ cells shown in (E).


G . of IL - 1β expressing CD11b + shown in ( E ) . Data are representative FACS plots , gated on VD - CD11b + IL - 1β + with mean frequencies per group . H . ( mean + ) of myeloid populations expressing IL - 1β shown in ( G ) .

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

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).


A - D . of IL - 1R1 expression by different populations of murine ( A ) CD4 T ( B ) Th17 isolated from dLN 9 days after immunization ; ( C ) CD4T ( D ) Th17 isolated from CNS at peak of EAE . ( A - D ) were restimulated in 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 + gated on IL - 17A + , IL - 17A - IFNγ + ( IFNγ + ) , IL - 17A - GM - CSF + ( GM - CSF + ) or IL - 17A - FoxP3 + ( FoxP3 + ) ( also see Appendix Fig S1 ) quantifications shown as bar diagram ( mean + ) . Data ( B , D , F ) are FACS plots of Th17 histograms of IL - 1R1 of indicated populations with mean ( MFI ) ± or mean frequencies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - unpaired t - . Experiments were performed twice with similar results .

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

A-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 . of IL - 1R1 expression by different populations of human ( E ) CD4 T ( F ) Th17 isolated from peripheral blood . ( 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 gated on CD14 - CD3 + CD4 + with mean frequencies per group quantifications shown as bar diagram ( mean + ) . Data ( B , D , F ) are FACS plots of Th17 histograms of IL - 1R1 of indicated populations with mean ( MFI ) ± or mean frequencies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - unpaired t - . Experiments were performed twice with similar results .

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

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.


A . of cytokine expression by CD4T isolated from spleen . Data are representative FACS plots , gated on VD - TCRβ + CD4 + CD44 + ( upper row ) with mean frequencies among CD4T per group ± , ( lower row ) with mean frequencies per group . ( A - D ) were isolated at day 9 after immunization restimulated in 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 - unpaired t - . Experiments were performed at least twice with similar results .

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

A. 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 total numbers ( mean + ) of MOG - specific Th17 isolated from dLN . ( A - D ) were isolated at day 9 after immunization restimulated in 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 - unpaired t - . Experiments were performed at least twice with similar results .

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

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 total numbers ( mean + ) of MOG - specific GM - CSF + Th17 isolated from spleen ( C ) dLN ( D ) . ( A - D ) were isolated at day 9 after immunization restimulated in 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 - unpaired t - . Experiments were performed at least twice with similar results .

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

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.


A . of CD4T isolated from dLN restimulated with PMA / Ionomycin for 4 h . Data are representative FACS plots , gated on VD - CD4 + with mean frequencies per group . ( 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 - unpaired t - . Experiments were performed twice with similar results .

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

A. 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 . of cytokine expression by CD4T isolated from spleen restimulated with MOG for 6 h . Data are representative FACS plots , gated on VD - TCRβ + CD4 + CD44 + ( upper row ) with mean frequencies among CD4T per group ± , ( lower row ) with mean frequencies per group . ( 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 - unpaired t - . Experiments were performed twice with similar results .

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

B. Analysis of 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 numbers ( mean + ) of MOG - specific Th17 isolated from spleen , shown in ( B , upper row ) . ( 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 - unpaired t - . Experiments were performed twice with similar results .

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

C. 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 total numbers ( mean + ) of MOG - specific GM - CSF + IFNγ + / - Th17 isolated from spleen , shown in ( B , lower row ) . ( 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 - unpaired t - . Experiments were performed twice with similar results .

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

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.


A - C . ( A ) Mean , ( B ) maximal EAE clinical scores ( C ) under curve ( AUC ) of mice immunized with MOG / CFA / PTx . Data shown as ( A ) mean ( + ) , ( B , C ) plots with mean are combined from at least four independent experiments consist of n = 41 WT , n = 24 IL - 1R1ΔT , n = 18 IL - 1R1 - / - mice . * p < 0 . 05 , * * * p < 0 . 001 ; two - unpaired t - . Black ( * ) signs in ( A ) represent statistical of IL - 1R1ΔT vs WT blue ( * ) of IL - 1R1ΔT vs IL - 1R1 - / - groups .

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

A-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 ( plots with mean ) of selected

D-E. (D) Maximal EAE clinical scores (individual plots with mean) of selected mice


D - E . ( E ) total numbers ( mean + ) of infiltrates isolated from CNS used for further .

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

D-E. (E) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.


F - H . of cytokine expression by CD4 T isolated from CNS of mice shown in ( D ) . Data ( F ) are representative FACS plots , gated on VD - CD90 + CD4 + CD44 + ( upper row ) with mean frequencies among CD4 T per group ± , ( lower row ) with mean frequencies per group . ( G ) Mean ( mean + ) of IL - 17A by MOG - specific Th17 , shown in ( F , upper row ) . ( H ) of MOG - specific GM - CSF + Th17 ( mean + ) shown in ( F , lower row ) .

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

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 . of Treg isolated from mice shown in ( D ) . ( I ) Total Treg numbers ( mean + ) isolated from CNS dLN . ( J ) of Treg Th17cells isolated from CNS . Data are representative FACS plots , gated on VD - CD90 + CD4 + with mean frequencies per group . ( K ) of Treg / Th17 ( mean + ) within CNS dLN . were restimulated with MOG for 6 h ( I - N ) in presence of PMA / Ionomycin for 4 h .

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

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 . of CCR6 expression by CD4 T isolated from dLN CNS of EAE diseased mice . Data ( L ) are representative FACS histogram overlays of ( black ) control , ( blue ) IL - 1R1ΔTcells , gated on CD3 + CD4 + IL - 17A + ; of ( black filled ) control , ( blue filled ) IL - 1R1ΔTcells , gated on CD3 + CD4 + IL - 17A - . ( M ) Mean ( mean + ) of CCR6 by CD4 T , shown in ( L ) . ( N ) Mean ( mean + ) of CCR6 by GM - CSF + GM - CSF - Th17 isolated from CNS , shown in ( L ) .

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

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).


A - C . of ( A ) IL - 17A expression by wild type CD4T , followed with of ( B ) GM - CSF ( C ) GM - CSF IFNγ co - expression by Th17 .

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

A-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 . of cytokine expression by isolated from WT ( black ) , IL - 1R1ΔT ( blue ) IL - 1R1 - / - ( red ) mice cultured with or without IL - 23 . Data ( D ) are representative FACS plots , gated on VD - TCRβ + with mean frequencies per group . ( A - G ) were isolated from dLN spleen of MOG / CFA immunized mice , cultured under indicated conditions for four days restimulated with MOG for 6 h prior to . Data consist of n = 4 of each genotype .

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

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 . of cytokine expression by isolated from WT ( black ) , IL - 1R1ΔT ( blue ) IL - 1R1 - / - ( red ) mice cultured with or without IL - 23 . Data ( D ) are representative FACS plots , gated on VD - TCRβ + with mean frequencies per group . ( mean + ) of ( E ) MOG - specific Th17 , ( F ) GM - CSF + Th17 ( G ) GM - CSF + IFNγ + Th17 . ( A - G ) were isolated from dLN spleen of MOG / CFA immunized mice , cultured under indicated conditions for four days restimulated with MOG for 6 h prior to . Data consist of n = 4 of each genotype .

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

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 ( + ) of Rag1 - / - mice which received 1 x 105 IL - 17A + of indicated genotypes ( I ) total numbers ( mean + ) of infiltrates isolated from CNS used for further . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) IL - 1R1 - / - ( n = 4 ) polarized in presence of IL - 23 prior to transfer . experiments were performed at least twice with similar results .

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

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 + of indicated genotypes ( I ) total numbers ( mean + ) of infiltrates isolated from CNS used for further . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) IL - 1R1 - / - ( n = 4 ) polarized in presence of IL - 23 prior to transfer . experiments were performed at least twice with similar results .

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

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 . of cytokine expression by CD4 T isolated from CNS of mice shown in ( H ) restimulated with MOG for 6 h . ( C ) Data are representative FACS plots , gated on VD - TCRβ + ( upper row ) with mean frequencies ± . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) IL - 1R1 - / - ( n = 4 ) polarized in presence of IL - 23 prior to transfer . experiments were performed at least twice with similar results .

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

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 . of cytokine expression by CD4T isolated from CNS of mice shown in ( H ) restimulated with MOG for 6 h . ( C ) Data are representative FACS plots , gated on VD - TCRβ + ( upper row ) with mean frequencies ± . Data ( H - L ) consist of Rag1 - / - mice which received WT ( n = 6 ) , IL - 1R1ΔT ( n = 3 ) IL - 1R1 - / - ( n = 4 ) polarized in presence of IL - 23 prior to transfer . experiments were performed at least twice with similar results .

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

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.


A . Mean EAE clinical scores ( + ) . Data consist of ( A ) n = 10 WT , n = 4 IL - 1R1 - / - PBS , n = 4 WT , n = 4 IL - 1R1ΔT n = 5 IL - 1R1 - / - PC61 treated mice .

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

A. 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 ( plots with mean ) of selected mice .

B. Maximal EAE clinical scores (individual plots with mean) of selected mice.


C . Total numbers ( mean + ) of infiltrates isolated from CNS of mice , shown in ( B ) .

C. Total cell numbers (mean +SEM) of infiltrates isolated from the CNS of mice, shown in (B).


D . of IL - 1R1 expression by Th17 isolated from CNS of EAE diseased mice , shown in ( B ) . Data are representative FACS histogram overlays of isolated from ( grey ) wild type mice treated with PBS , ( black ) wild type , ( blue ) IL - 1R1ΔT ( red ) IL - 1R1 - / - mice treated with PC61 , gated on VD - TCRβ + CD4 + IL - 17A + .

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

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 positive for IL - 1R1 , shown in ( D ) .

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

(E) Frequencies of Th17 cells positive for IL-1R1, shown in (D).


A Representative immunoblots of indicated in lysates from HeLa treated with indicated concentrations of halofuginone ( HF ) for 5 h . B of ATF4 P - eIF2α from experiments as in ( A ) . Data are mean ± SD ( n = 3 biological replicates ) . * p ≤ 0 . 0414 , * * p ≤ 0 . 0019 , * * * * p < 0 . 0001 , as determined by one - way ANOVA with Dunnett ' s multiple comparison . Data information : Representative results of at least three independent experiments are shown in each panel .

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

A Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with indicated concentrations of halofuginone (HF) for 5 h. B Quantification of ATF4 and P-eIF2α from experiments as in (A). Data are mean ± SD (n = 3 biological replicates). *p ≤ 0.0414, **p ≤ 0.0019, ****p < 0.0001, as determined by one-way ANOVA with Dunnett's multiple comparison test. Data information: Representative results of at least three independent experiments are shown in each panel.


C Similar to ( A ) , but with HeLa treated with indicated concentrations of tunicamycin for 5 h . Data information : Representative results of at least three independent experiments are shown in each panel .

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C Similar to (A), but with HeLa cells treated with indicated concentrations of tunicamycin for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.


D Similar to ( A ) , but using lysates from HeLa treated with 62 . 5 nM 312 . 5 nM HF for indicated times . Data information : Representative results of at least three independent experiments are shown in each panel .

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D Similar to (A), but using lysates from HeLa cells treated with 62.5 nM and 312.5 nM HF for indicated times. Data information: Representative results of at least three independent experiments are shown in each panel.


A Representative immunoblots of indicated in lysates from mouse embryonic fibroblasts ( MEFs ) of indicated genotype after treatment with 12 . 5 nM or 200 nM HF for 5 h . Data information : Representative results of at least three independent experiments are shown in each panel .

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

A Representative immunoblots of indicated proteins in lysates from mouse embryonic fibroblasts (MEFs) of indicated genotype after treatment with 12.5 nM or 200 nM HF for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.


B Similar to ( A ) , but using HeLa untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h . C Similar to ( A ) , but with high HF concentrations . Data information : Representative results of at least three independent experiments are shown in each panel .

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

B Similar to (A), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h. C Similar to (A), but with high HF concentrations. Data information: Representative results of at least three independent experiments are shown in each panel.


B Representative immunoblots of indicated in lysates from HeLa after 5 h treatment with indicated concentrations of HF . C Similar to ( B ) , but using HeLa untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF . Data information : Representative results of at least three independent experiments are shown in each panel .

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

B Representative immunoblots of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF. C Similar to (B), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF. Data information: Representative results of at least three independent experiments are shown in each panel.


D Representative immunoblots of lysates from HeLa treated with indicated concentrations of HF for 5 h with or without Nedd8 - activating enzyme inhibitor MLN4924 ( 1 μM ) . Data information : Representative results of at least three independent experiments are shown in each panel .

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

D Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without the Nedd8-activating enzyme inhibitor MLN4924 (1 μM). Data information: Representative results of at least three independent experiments are shown in each panel.


E . Representative immunoblots of lysates from HeLa treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG - 132 ( 10 μM ) . Data information : Representative results of at least three independent experiments are shown in each panel .

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

E. Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG-132 (10 μM). Data information: Representative results of at least three independent experiments are shown in each panel.


A Newly synthesized - labeled with 35S - methionine for 10 min in HeLa pre - treated with indicated compounds for 2 . 5h , except ( E ) ( Bottom ) Coomassie - stained gel .

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

A Newly synthesized proteins pulse-labeled with 35S-methionine for 10 min in HeLa cells pre-treated with indicated compounds for 2.5h, except (E) (Bottom) Coomassie-stained gel.


B Newly synthesized in HeLa treated with 2 . 5 μg / ml Tm or 50 μg / ml cycloheximide ( CHX ) .

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B Newly synthesized proteins in HeLa cells treated with 2.5 μg/ml Tm or 50 μg/ml cycloheximide (CHX).


C Newly synthesized in 4KO or 4KO + GCN2 MEF treated with 12 . 5 or 200 nM HF .

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C Newly synthesized proteins in 4KO or 4KO+GCN2 MEF cells treated with 12.5 or 200 nM HF.


D Newly synthesized in eIF2αS / S eIF2αA / A MEF treated with 12 . 5 or 200 nM HF .

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D Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with 12.5 or 200 nM HF.


E Newly synthesized in eIF2αS / S eIF2αA / A MEF treated with indicated concentrations of Tm for 2 . 5 or 5 h .

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

E Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with indicated concentrations of Tm for 2.5 or 5 h.


F Newly synthesized in HeLa untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF .

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F Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF.


G Newly synthesized in HeLa treated with indicated concentrations of HF with or without proline supplementation ( 10 mM ) .

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

G Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation (10 mM).


H Representative immunoblots of indicated in lysates from HeLa after treatment with indicated concentrations of HF with or without proline supplementation ( 10 mM ) .

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

H Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation (10 mM).


I Newly synthesized in HeLa untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol ( His ) or borrelidin ( Bor ) .

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

I Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol (His) or borrelidin (Bor).


G , H of IC50 values of HF on confluency at 72h endpoint of H441 ( G ) H23 ( H ) normalised to control conditions determined by nonlinear regression curve fit for inhibitory dose - response ( IC50 ) . Each point represents mean ( n = 3 biological replicates ) ± SD .

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G, H Analysis of IC50 values of HF on confluency at 72h endpoint of H441 (G) and H23 (H) cells normalised to control conditions and determined by nonlinear regression curve fit for inhibitory dose-response (IC50). Each point represents mean (n = 3 biological replicates) ± SD.


( A ) HMGB1 translocates from nucleus to cytosol during autophagy , but not apoptosis . Mouse embryonic fibroblasts ( MEFs ) human Panc2 . 03 were treated with 1 µM rapamycin ( Rap ) for 12 h , starvation ( HBSS ) for 3 h , or UV irradiation at 50 mJ / cm2 for 5 min before a 12 - h recovery then were immunostained with HMGB1 - specific antibody ( green ) Hoechst 33342 ( blue ) . mean ( Nuc ) ( Cyt ) HMGB1 per was determined by imaging cytometric as described in Materials methods . * , P < 0 . 05 * * , P < 0 . 005 versus untreated ( UT ) group ; n = 3 . Representative are depicted ( right ) .

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(A) HMGB1 translocates from the nucleus to the cytosol during autophagy, but not apoptosis. Mouse embryonic fibroblasts (MEFs) and the human Panc2.03 tumor cell line were treated with 1 µM rapamycin (Rap) for 12 h, starvation (HBSS) for 3 h, or UV irradiation at 50 mJ/cm2 for 5 min before a 12-h recovery and then were immunostained with HMGB1-specific antibody (green) and Hoechst 33342 (blue). The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was determined by imaging cytometric analysis as described in Materials and methods. *, P < 0.05 and **, P < 0.005 versus untreated (UT) group; n = 3. Representative images are depicted (right).


( B ) Inhibition of autophagy blocks HMGB1 translocation . were pretreated as indicated with 100 nM wortmannin or 10 µM Ly294002 for 1 h or ATG5 - specific shRNA for 48 h were stimulated with starvation ( HBSS ) for 3 h immunostained with HMGB1 - or LC3 - specific antibody Hoechst 33342 . mean / HMGB1 LC3 per were determined by imaging cytometric . A representative Western blot of ATG5 level after shRNA HMGB1 is depicted ( right ) . In parallel , indicated were transfected with GFP - LC3 plasmid assayed for autophagy by quantifying of with GFP - LC3 . * , P < 0 . 05 , * * , P < 0 . 005 , * * * , P < 0 . 0005 versus HBSS group ; n = 3 . Ctrl , control .

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(B) Inhibition of autophagy blocks HMGB1 translocation. Cells were pretreated as indicated with 100 nM wortmannin or 10 µM Ly294002 for 1 h or ATG5-specific shRNA for 48 h and were stimulated with starvation (HBSS) for 3 h and immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. A representative Western blot of ATG5 level after shRNA and HMGB1 staining is depicted (right). In parallel, the indicated cells were transfected with GFP-LC3 plasmid and assayed for autophagy by quantifying the percentage of cells with GFP-LC3 punctae. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus HBSS group; n = 3. Ctrl, control.


( C ) Knockdown of ATG5 inhibits LC3 - II expression . Western blot of LC3 - I / II expression in Panc02 under conditions described in B . Actin was used as a loading control .

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(C) Knockdown of ATG5 inhibits LC3-II expression. Western blot analysis of LC3-I/II expression in Panc02 cells under the conditions described in B. Actin was used as a loading control.


( D ) Hmgb1 − / − does not influence ATG5 . Hmgb1 − / − Hmgb1 + / + MEFs were immunostained with HMGB1 - specific antibody ( green ) , ATG - 5 - specific antibody ( red ) , Hoechst 33342 ( blue ) . Representative are depicted in right panels .

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(D) Hmgb1−/− does not influence ATG5 staining. Hmgb1−/− and Hmgb1+/+ MEFs were immunostained with HMGB1-specific antibody (green), ATG-5-specific antibody (red), and Hoechst 33342 (dark blue). Representative images are depicted in the right panels.


( E ) mETC inhibitors promote ROS production , autophagy , HMGB1 translocation . Panc2 . 03 were stimulated with rotenone ( Rot ) , thenoyltrifluoroacetone ( TTFA ) , antimycin A ( AA ) at indicated doses for 12 h , ROS production was assessed by measuring of CM - H2DCFDA in a plate reader . In parallel experiments , were then immunostained with HMGB1 - or LC3 - specific antibody Hoechst 33342 . mean / HMGB1 LC3 per were determined by imaging cytometric . * , P < 0 . 05 , * * , P < 0 . 005 , * * * , P < 0 . 0005 versus untreated group ; n = 3 .

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(E) mETC inhibitors promote ROS production, autophagy, and HMGB1 translocation. Panc2.03 cells were stimulated with rotenone (Rot), thenoyltrifluoroacetone (TTFA), and antimycin A (AA) at indicated doses for 12 h, and ROS production was assessed by measuring the fluorescent intensity of CM-H2DCFDA in a fluorescent plate reader. In parallel experiments, cells were then immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus untreated group; n = 3.


( F ) mETC inhibitors increase LC3 - II expression promote HMGB1 translocation . Western blot of LC3 - I / II / HMGB1 expression as indicated in E . Fibrillarin is a fraction control , tubulin is a fraction control .

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(F) mETC inhibitors increase LC3-II expression and promote HMGB1 translocation. Western blot analysis of LC3-I/II and nuclear/cytosolic HMGB1 expression as indicated in E. Fibrillarin is a nuclear fraction control, and tubulin is a cytoplasmic fraction control.


( G ) Antioxidant SOD RNAi limit starvation - induced autophagy HMGB1 translocation . Panc2 . 03 were pretreated with antioxidant ( NAC ) at indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h . Then were starved ( HBSS ) for 3 h analyzed by imaging cytometry to determine mean / HMGB1 LC3 per . * , P < 0 . 05 ; * * , P < 0 . 005 ; * * * , P < 0 . 0005 versus HBSS group ; n = 3 . A representative Western blot for SOD1 SOD2 level after siRNA is depicted here .

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(G) Antioxidant and SOD RNAi limit starvation-induced autophagy and HMGB1 translocation. Panc2.03 cells were pretreated with the antioxidant (NAC) at the indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h. Then cells were starved (HBSS) for 3 h and analyzed by imaging cytometry to determine the mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus HBSS group; n = 3. A representative Western blot for SOD1 and SOD2 level after siRNA is depicted here.


( H ) Antioxidant SOD RNAi limit starvation - induced autophagy as measured by LC3 - II expression . Western blot of LC3 - I / II expression under conditions indicated in G . Actin was used as a loading control . Data are means ± .

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(H) Antioxidant and SOD RNAi limit starvation-induced autophagy as measured by LC3-II expression. Western blot analysis of LC3-I/II expression under the conditions indicated in G. Actin was used as a loading control. Data are means ± SEM.


( A ) HMGB1 knockout inhibits LC3 formation . HMGB1 − / − HMGB1 + / + MEFs were treated with autophagic stimuli as indicated , LC3 formation was detected by LC3 antibody or GFP - LC3 as described in Materials methods . * , P < 0 . 05 ; * * , P < 0 . 005 ; * * * , P < 0 . 0005 versus Hmgb1 + / + group ; n = 3 . UT , untreated . ( B ) Representative of LC3 in Hmgb1 − / − HMGB1 + / + MEFs with indicated treatments are depicted . of showing accumulation of LC3 was reported in A .

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(A) HMGB1 knockout inhibits LC3 punctae formation. HMGB1−/− and HMGB1+/+ MEFs were treated with autophagic stimuli as indicated, and LC3 punctae formation was detected by LC3 antibody or GFP-LC3 as described in Materials and methods. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. UT, untreated. (B) Representative images of LC3 punctae in Hmgb1−/− and HMGB1+/+ MEFs with the indicated treatments are depicted. The percentage of cells showing accumulation of LC3 punctae was reported in A.


( C ) of LC3 processing by autophagy in presence or absence of lysosomal protease inhibitors pepstatin A ( pepA ) at 10 µg / ml E64D at 10 µg / ml after starvation treatment for 3 h . * * , P < 0 . 05 versus Hmgb1 + / + group ; n = 3 . Actin was used as a loading control . AU , arbitrary units .

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(C) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (pepA) at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.05 versus Hmgb1+/+ group; n = 3. Actin was used as a loading control. AU, arbitrary units.


( D ) Up - regulation of HMGB1 expression restores starvation - induced autophagy . Hmgb1 − / − MEFs were transfected with HMGB1 plasmid or empty vector then were treated with starvation for 3 h . LC3 formation was assayed by imaging cytometric . * * , P < 0 . 05 versus vector group ; n = 3 . Non , nontransfected .

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(D) Up-regulation of HMGB1 protein expression restores starvation-induced autophagy. Hmgb1−/− MEFs were transfected with HMGB1 plasmid or empty vector and then were treated with starvation for 3 h. LC3 punctae formation was assayed by imaging cytometric analysis. **, P < 0.05 versus vector group; n = 3. Non, nontransfected.


( E ) of p62 processing by autophagy in presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg / ml E64D at 10 µg / ml after starvation treatment for 3 h . * * , P < 0 . 005 * * * , P < 0 . 0005 versus Hmgb1 + / + group ; n = 3 . Representative are depicted ( left ) .

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(E) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.005 and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. Representative images are depicted (left).


( F ) Ultrastructural features in Hmgb1 − / − Hmgb1 + / + MEFs with or without starvation ( HBSS for 3 h ) treatment ( a - e point to autophagosomes autolysosomes ) . Data are means ± .

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(F) Ultrastructural features in Hmgb1−/− and Hmgb1+/+ MEFs with or without starvation (HBSS for 3 h) treatment (a-e point to autophagosomes and autolysosomes). Data are means ± SEM.


Inhibition of autophagy by HMGB1 . Hmgb1 − / − Hmgb1 + / + MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials methods then were treated with HBSS for 3 h , LC3 formation was detected by a confocal microscope . ( A ) Representative of LC3 ( arrows ) HMGB1 ( red arrows ) in cytoplasts of Hmgb1 − / − Hmgb1 + / + MEFs are depicted . ( B ) of showing accumulation of LC3 was reported ( * , P < 0 . 05 ; n = 3 ) . Data are means ± .

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Inhibition of autophagy by cytoplasmic HMGB1. Hmgb1−/− and Hmgb1+/+ MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h, and LC3 punctae formation was detected by a confocal microscope. (A) Representative images of LC3 punctae (white arrows) and HMGB1 (red arrows) in cytoplasts of Hmgb1−/− and Hmgb1+/+ MEFs are depicted. (B) The percentage of cells showing accumulation of LC3 punctae was reported (*, P < 0.05; n = 3). Data are means ± SEM.


( A ) MEK inhibitors block starvation - induced p - ERK Bcl - 2 . HMGB1 − / − HMGB1 + / + MEFs were starved in presence or absence of 10 µM U0126 20 µM PD98059 for 6 h . expression were assessed as indicated by co - IP or Western blotting .

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(A) MEK inhibitors block starvation-induced p-ERK and Bcl-2. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h. Protein expression levels were assessed as indicated by co-IP or Western blotting.


( B ) Knockout of HMGB1 limits disassociation of Bcl - 2 - Beclin1 during treatment with autophagic stimuli . HMGB1 − / − HMGB1 + / + MEFs were starved in presence or absence of 10 µM U0126 for 3 h . expression were then assayed as indicated by co - IP or Western blotting .

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(B) Knockout of HMGB1 limits the disassociation of the Bcl-2-Beclin1 complex during treatment with autophagic stimuli. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 for 3 h. Protein expression levels were then assayed as indicated by co-IP or Western blotting.


( C ) HMGB1 interacts directly with Beclin1 during autophagy . HCT116 Panc02 were treated with HBSS for 3 h then assayed for expression as indicated by co - IP or Western blotting .

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(C) HMGB1 interacts directly with Beclin1 during autophagy. HCT116 and Panc02 cells were treated with HBSS for 3 h and then assayed for protein expression levels as indicated by co-IP or Western blotting.


( D ) HMGB1 direct interactions with Bcl - 2 are dependent on Beclin1 . Knockdown of Beclin1 Bcl - 2 by siRNA in HMGB1 wild - type MEFs was performed , expression were then assayed as indicated by co - IP or Western blotting . Ctrl , control .

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(D) HMGB1 direct interactions with Bcl-2 are dependent on Beclin1. Knockdown of Beclin1 and Bcl-2 by siRNA in HMGB1 wild-type MEFs was performed, and protein expression levels were then assayed as indicated by co-IP or Western blotting. Ctrl, control.


( E F ) Quantitative data demonstrating interaction between HMGB1 - Beclin1 Beclin1 - HMGB1 using densitometry software to relative band in co - IP experiments as shown in A - D . * * , P < 0 . 005 * * * , P < 0 . 0005 ; n = 3 . UT , untreated . Data are means ± .

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(E and F) Quantitative data demonstrating the interaction between HMGB1-Beclin1 and Beclin1-HMGB1 using densitometry software to assay the relative protein band intensity in co-IP experiments as shown in A-D. **, P < 0.005 and ***, P < 0.0005; n = 3. UT, untreated. Data are means ± SEM.


( A ) C106 mutation ( C106S ) of HMGB1 impairs its localization . Hmgb1 − / − MEFs were transfected with wild - type cysteine mutant HMGB1 - GFP plasmids as indicated then were treated with 1 µM rapamycin for 12 h or starved ( HBSS ) for 3 h . mean ( Nuc ) ( Cyt ) HMGB1 per was analyzed by imaging cytometric . * , P < 0 . 05 versus HMGB1 + / + group ; n = 3 . Representative of HMGB1 location are shown on left ( green , HMGB1 ; blue , nucleus ) . right panel is a schematic diagram of HMGB1 structure illustrating basic A box B box as well as acidic C - terminal domain , with cysteine mutation locations identified .

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(A) The C106 mutation (C106S) of HMGB1 impairs its nuclear localization. Hmgb1−/− MEFs were transfected with wild-type and cysteine mutant HMGB1-GFP plasmids as indicated and then were treated with 1 µM rapamycin for 12 h or starved (HBSS) for 3 h. The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was analyzed by imaging cytometric analysis. *, P < 0.05 versus HMGB1+/+ group; n = 3. Representative images of HMGB1 location are shown on the left (green, HMGB1; blue, nucleus). The right panel is a schematic diagram of HMGB1 structure illustrating the basic A box and B box as well as the acidic C-terminal domain, with the cysteine mutation locations identified.


( B C ) HMGB1 enhances autophagy limits apoptosis . HMGB1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated then were starved ( HBSS ) for indicated time . In a parallel experiment , Hmgb1 + / + MEFs were pretreated with 5 mM ethyl pyruvate ( EP ) for 2 h then starved as indicated . LC3 formation was assayed by imaging cytometric ( B )

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(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+ MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. LC3 punctae formation was assayed by imaging cytometric analysis (B)


( B C ) HMGB1 enhances autophagy limits apoptosis . HMGB1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated then were starved ( HBSS ) for indicated time . In a parallel experiment , Hmgb1 + / + MEFs were pretreated with 5 mM ethyl pyruvate ( EP ) for 2 h then starved as indicated . Apoptosis was assayed by FACS ( C ) ( C ) as described in Materials methods . * , P < 0 . 05 * * , P < 0 . 005 ; n = 3 . UT , untreated . Non , nontransfection .

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(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/−MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. Apoptosis was assayed by FACS (C) (C) as described in Materials and methods. *, P < 0.05 and **, P < 0.005; n = 3. UT, untreated. Non, nontransfection.


( D ) C23 / C45 is required for binding of HMGB1 to Beclin1 . Hmgb1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated stimulated with starvation ( HBSS ) for 3 h . These were then assayed for expression as indicated by IP or Western blotting . Blots are representative of three independent experiments with similar results .

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(D) C23/C45 is required for the binding of HMGB1 to Beclin1. Hmgb1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and stimulated with starvation (HBSS) for 3 h. These cells were then assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of three independent experiments with similar results.


( E ) Reducing reagents disrupt interaction between wild - type / C106 HMGB1 Beclin1 . As a control , before IP , samples were incubated with 50 mM DTT ( + DTT ) assayed for expression as indicated by IP or Western blotting . Blots are representative of two independent experiments with similar results .

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(E) Reducing reagents disrupt the interaction between wild-type/C106 HMGB1 and Beclin1. As a control, before IP, samples were incubated with 50 mM DTT (+DTT) and assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of two independent experiments with similar results.


( F ) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from nucleus to cytosol . were stimulated with HBSS , rapamycin ( Rap ) , rotenone ( Rot ) , or thenoyltrifluoroacetone ( TTFA ) for 3 h or 12 h , LC3 formation was assayed as indicated . * * , P < 0 . 005 versus Beclin1 shRNA group ; n = 3 . Ctrl , control . Data are means ± .

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(F) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from the nucleus to the cytosol. Cells were stimulated with HBSS, rapamycin (Rap), rotenone (Rot), or thenoyltrifluoroacetone (TTFA) for 3 h or 12 h, and LC3 punctae formation was assayed as indicated. **, P < 0.005 versus Beclin1 shRNA group; n = 3. Ctrl, control. Data are means ± SEM.


( A B ) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1 - induced autophagy . as indicated were stimulated with 1 µg / ml HMGB1 ( rHMGB1 ) for 24 h , LC3 expression was detected by Western blotting ( A ) .

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(A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, and LC3 expression was detected by Western blotting (A).


( A B ) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1 - induced autophagy . as indicated were stimulated with 1 µg / ml HMGB1 ( rHMGB1 ) for 24 h , LC3 formation ( arrows ) was assayed by immunofluorescence ( B ; n = 3 ; * , P < 0 . 05 ) .

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(A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, LC3 punctae formation (arrows) was assayed by immunofluorescence (B; n = 3; *, P < 0.05).


( C D ) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS - induced autophagy with without pUNO1 - HMGB1 transfection . as indicated were stimulated with Earle ' s balanced salt solution for 3 h , LC3 expression was detected by Western blotting ( C ) .

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(C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 expression was detected by Western blotting (C).


( C D ) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS - induced autophagy with without pUNO1 - HMGB1 transfection . as indicated were stimulated with Earle ' s balanced salt solution for 3 h , LC3 formation ( arrows ) was assayed by immunofluorescence ( D ; n = 3 ; * , P < 0 . 05 ) . Data are means ± .

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(C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 punctae formation (arrows) was assayed by immunofluorescence (D; n = 3; *, P < 0.05). Data are means ± SEM.


Expression of HMGB1 or C106S mutant , but not C23S C45S mutants , restore autophagic flux in Hmgb1 − / − MEFs . ( A ) HMGB1 − / − MEFs were transfected with wild - type ( WT ) or cysteine mutant HMGB1 - GFP plasmids as indicated , starved ( HBSS ) for 3 h in presence or absence of 100 nM bafilomycin A1 , then immunostained with LAMP2 - specific antibody / Alexa Fluor 594 secondary antibody ( shown in red ) , LC3 - specific antibody / Alexa Fluor 647 secondary antibody ( shown in green ) , Hoechst 33342 ( shown in blue ) . were acquired digitally from a randomly selected pool of 10 - 15 fields under each condition . ( B ) Quantitative of of LAMP2 / LC3 colocalization was detected by Image - Pro Plus 5 . 1 software . Data are means ± .

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Expression of HMGB1 or the C106S mutant, but not C23S and C45S mutants, restore autophagic flux in Hmgb1−/− MEFs. (A) HMGB1−/− MEFs were transfected with wild-type (WT) or the cysteine mutant HMGB1-GFP plasmids as indicated, starved (HBSS) for 3 h in the presence or absence of 100 nM bafilomycin A1, and then immunostained with LAMP2-specific antibody/Alexa Fluor 594 secondary antibody (shown in red), LC3-specific antibody/Alexa Fluor 647 secondary antibody (shown in green), and Hoechst 33342 (shown in blue). Images were acquired digitally from a randomly selected pool of 10-15 fields under each condition. (B) Quantitative analysis of the percentage of LAMP2/LC3 colocalization was detected by Image-Pro Plus 5.1 software. Data are means ± SEM.


a , b , LDH release , IL - 1β secretion ( a ) immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 IL - 1α ( b )

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a, b, LDH release, IL-1β secretion (a) and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α(b)


immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 IL - 1α ( b ) from unprimed BMDMs infected for 16h with indicated ( grown to stationary phase ) .

and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α (b) from unprimed BMDMs infected for 16h with the indicated bacteria (grown to stationary phase).


c , Time course measuring LDH release from unprimed or IFN - γ - primed BMDMs infected with S .

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c, Time course measuring LDH release from unprimed or IFN-γ-primed BMDMs infected with S. typhimurium


d , e , LDH release IL - 1β secretion from primed BMDMs infected with SPI - 1 - expressing logarithmic phase S . typhimurium , treated with monosodium urate , alum nigericin or transfected with poly ( dA : dT ) LPS .

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d, e, LDH release and IL-1β secretion from primed BMDMs infected with SPI-1-expressing logarithmic phase S. typhimurium, treated with monosodium urate, alum and nigericin or transfected with poly(dA:dT) and LPS.


LDH release IL - 1β secretion from unprimed wild - type Gbp2 − / − BMDMs infected for 16h with indicated ( grown to stationary phase ) . Graphs show mean s . d . of quadruplicate wells data are representative of two ( b ) three ( a , c - f ) independent experiments . * Crossreactive band ; * * P < 0 . 01 ; NS , not significant ( two - t - ) .

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LDH release and IL-1β secretion from unprimed wild-type and Gbp2−/− BMDMs infected for 16h with the indicated bacteria (grown to stationary phase). Graphs show mean and s.d. of quadruplicate wells and data are representative of two (b) and three (a, c-f) independent experiments. *Crossreactive band; **P<0.01; NS, not significant (two-tailed t-test).


a , b , of live ( mCherry - positive ) dead ( mCherry - negative ) S . typhimurium per by immunofluorescence ( a ) or as of total by flow -

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a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry


a , b , of live ( mCherry - positive ) dead ( mCherry - negative ) S . typhimurium per by immunofluorescence ( a ) or as of total by flow - cytometry ( b ) in unprimed BMDMs at 16 h post - infection .

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a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry (b) in unprimed BMDMs at 16 h post-infection.


c , Immunostaining for GBP2 of live dead Salmonella at 4 h post - infection . Arrowheads , shown in insets .

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c, Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post-infection. Arrowheads, bacteria shown in insets.


d , of GBP - positive , LDH release IL - 1β secretion at indicated time points from BMDMs infected with Salmonella , live or killed by different means .

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d, Quantification of GBP-positive bacteria, LDH release and IL-1β secretion at indicated time points from BMDMs infected with Salmonella, live or killed by different means.


e , Immunohistochemistry for GBP2 Salmonella on spleen tissue from Salmonella ( mCherry - positive ) - infected mice ( representative of n = 3 per group ) . S . tm . , S . typhimurium .

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e, Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella (mCherry-positive)-infected mice (representative of n = 3 per group). S. tm., S. typhimurium.


f , g , of GBP - positive Salmonella in anti - IFN - γ - treated or control ( f ) live dead among GBP2 - negative / - positive Salmonella ( g ) ( n = 3 per group ) . Scale bars , 10 µm ( c ) , 1 µm ( e ) . Graphs show mean 5 - 95 percentile ( box plots ) or s . d . of technical triplicates , data are representative of three independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - t - ) .

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f, g, Quantification of GBP-positive Salmonella in anti-IFN-γ-treated or control animals (f) and live and dead bacteria among GBP2-negative/-positive Salmonella (g) (n = 3 per group). Scale bars, 10 µm (c), 1 µm (e). Graphs show mean and 5-95 percentile (box plots) or s.d. of technical triplicates, and data are representative of three independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


a , b , Unprimed BMDMs infected with S . typhimurium for 4 h immunostained for LC3 GBP2 . Arrowheads , shown in insets . Scale bars , 10 & amp ; amp ; mgr ; m .

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a, b, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for LC3 and GBP2. Arrowheads, bacteria shown in insets. Scale bars, 10 &amp;mgr;m.


. c, Quantification of results from b.


b . d - g , LDH release immunoblots for caspase - 1 caspase - 11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3 - methyladenine ( 3 - MA ) .

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b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).


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b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).


h , i , LDH release IL - 1b secretion from BMDMs infected for 16 h or transfected with LPS . Graphs show mean s . d . of quadruplicate wells data are representative of two ( e , i ) three ( a - d , f - h ) independent experiments . * P 0 . 05 , * * P 0 . 01 ; NS , not significant ( two - t - ) .

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h, i, LDH release and IL-1b secretion from BMDMs infected for 16 h or transfected with LPS. Graphs show mean and s.d. of quadruplicate wells and data are representative of two (e, i) and three (a-d, f-h) independent experiments. *P  0.05, **P  0.01; NS, not significant (two-tailed t-test).


a , of galectin - 8 - positive Salmonella in unprimed BMDMs at 4 h post - infection .

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a, Quantification of galectin-8-positive Salmonella in unprimed BMDMs at 4 h post-infection. b


c , Unprimed BMDMs infected with S . typhimurium for 4 h immunostained for galectin - 8 , GBP2 LC3 . Arrowheads , shown in insets . Scale bars , 10 µm . d , of galectin - 8 / LC3 - double - positive Salmonella at indicated time points post - infection .

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c, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for galectin-8, GBP2 and LC3. Arrowheads, bacteria shown in insets. Scale bars, 10 µm. d, Quantification of galectin-8/LC3-double-positive Salmonella at indicated time points post-infection.


e - h , of vacuolar by flow cytometry in BMDMs infected with mCherry - positive S . typhimurium ( e - g ) or S . flexneri ( h , wild - type or ΔT3SS ) for 4 h . Graphs show mean s . d . or 5 - 95 percentile ( Box plots ) of technical triplicates . Data are representative of 2 ( g , h ) , 3 ( a - d ) 4 ( e , f ) independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - t - ) .

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e-h, Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry-positive S. typhimurium (e-g) or S. flexneri (h, wild-type or ΔT3SS) for 4 h. Graphs show mean and s.d. or 5-95 percentile (Box plots) of technical triplicates. Data are representative of 2 (g, h), 3 (a-d) and 4 (e, f) independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


mRNA of TMX1 ( A ) NFAT1 ( B ) in melanocytes , keratinocytes melanoma lines . data are are normalized to expression of TBP are presented as mean ± ( n ≥ 3 ) .

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mRNA levels of TMX1 (A) and NFAT1 (B) in primary melanocytes, keratinocytes and melanoma cell lines. data are are normalized to the expression of TBP and are presented as mean ± SEM (n≥3).


Representative immunoblot ( from 3 ) depicting abundance of TMX1 NFAT1 in melanocytes melanoma lines ; actin was used as a loading control . Indicated band intensities were normalized to actin background signal was subtracted .

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Representative immunoblot (from 3) depicting protein abundance of TMX1 and NFAT1 in melanocytes and melanoma cell lines; actin was used as a loading control. Indicated band intensities were normalized to actin and background signal was subtracted.


Melan - A ( brown ) , TMX1 ( red - brown ) NFAT1 ( deep red ) ( ) in a stage 4 high risk nodular melanoma I ( out of four ) , enlarged regions show healthy tissue ( panels 2 , 5 8 ) tissue ( panels 3 , 6 9 ) . Arrows indicate Melan - A positive melanocytes .

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Melan-A (brown), TMX1 (red-brown) and NFAT1 (deep red) staining (IHC) in a primary stage 4 high risk nodular melanoma patient I (out of four), the enlarged regions show healthy tissue (panels 2, 5 and 8) and tumor tissue (panels 3, 6 and 9). Arrows indicate Melan-A positive melanocytes.


TMX1 ( red - brown ) NFAT1 ( deep red ) of healthy human skin increasing melanoma stages ; P1 - P13 refer to donor number .

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TMX1 (red-brown) and NFAT1 (deep red) staining of healthy human skin and increasing melanoma stages; P1-P13 refer to the donor patient number.


Insulin ( 50 µg / mL ) induced translocation of NFAT1 - GFP to nucleus of WM3734 . ( A ) ( F / F0 ) , ( B ) corresponding endpoint ( n = 5 ) .

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Insulin (50 µg/mL) induced the translocation of NFAT1-GFP to the nucleus of WM3734 cells. (A) Ratio images (F/F0), (B) corresponding endpoint quantification (n=5).


Reduced translocation of NFAT1 - GFP in TMX1 or TMX3 silenced ( siRNA ) melanoma . ( C ) show NFAT1 - GFP before after stimulation with thapsigargin ( Tg ; 1 µM ) in WM3734 . ( D ) Corresponding time - dependent import of NFAT1 as a change of F / F0 . ( E ) Normalized endpoint . Same was performed with Mel Juso with ( F ) , ( G ) time - dependent import ( H ) normalized endpoint . data are presented as mean ± ( n values : WM3734 , control = 142 , TMX1 kd = 116 , TMX3 kd = 148 ; Mel Juso , control = 75 , TMX1 kd = 47 , TMX3 kd = 67 ) .

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Reduced nuclear translocation of NFAT1-GFP in TMX1 or TMX3 silenced (siRNA) melanoma cells. (C) Images show NFAT1-GFP fluorescence intensity before and after stimulation with thapsigargin (Tg; 1 µM) in WM3734 cells. (D) Corresponding time-dependent nuclear import of NFAT1 as a change of F/F0. (E) Normalized endpoint quantification. Same analysis was performed with Mel Juso cells with (F) images, (G) time-dependent nuclear import and (H) normalized endpoint quantification. data are presented as mean ± SEM (n values: WM3734, control=142, TMX1 kd=116, TMX3 kd=148; Mel Juso, control=75, TMX1 kd=47, TMX3 kd=67).


Thapsigargin ( Tg ) induced Fura - 2 Ca2 + imaging in Ringer ' s buffer containing 0 . 25 mM Ca2 + . ( B ) of basal calcium ( C ) SOCE ( plateau - basal ) for WM3734 after stable silencing of TMX1 ( two clones ) . : In ( B - C ) , data are presented as mean ± ( n values : WM3734 , control = 939 , TMX1 kds 1 = 988 , TMX1 kds 2 = 508 ) .

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Thapsigargin (Tg) induced Fura-2 based cytosolic Ca2+ imaging in Ringer's buffer containing 0.25 mM Ca2+. (B) Quantification of basal cytosolic calcium levels and (C) SOCE quantification (plateau-basal) for WM3734 after stable silencing of TMX1 (two clones). : In (B-C), data are presented as mean ± SEM (n values: WM3734, control=939, TMX1 kds 1=988, TMX1 kds 2=508).


Ca2 + imaging ( Fura - 2 ) ( E ) SOCE ( plateau - basal ) for WM3734 after transient silencing of TMX1 or TMX3 . In ( E ) , data are presented as mean ± ( n values : WM3734 , control = 30 , TMX1 kd = 49 , TMX3 kd = 52 ) .

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Cytosolic Ca2+ imaging (Fura-2) and (E) SOCE quantification (plateau-basal) for WM3734 after transient silencing of TMX1 or TMX3. In (E), data are presented as mean ± SEM (n values: WM3734, control=30, TMX1 kd=49, TMX3 kd=52).


H2O2 ( HyPer ) was evaluated in two melanoma lines upon TMX1 kd TMX3 kd . ( F ) Exemplary ratiometric ( F505 nm / F420 nm ) are shown for WM3734 Mel Juso . of basal H2O2 in WM3734 ( G ) Mel Juso ( H ) . In ( G - H ) , data are presented as mean ± ( n values : WM3734 : control = 168 , TMX1 kd = 209 , TMX3 kd = 192 ; Mel Juso : control = 297 , TMX1 kd = 343 , TMX3 kd = 440 ) . In ( F ) scale bars : 10 µm ; color code : WM3734 : color code : blue = 0 , red = 3 ; Mel Juso : color code : blue = 0 , red = 1 . 5 .

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Cellular H2O2 (HyPer) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd. (F) Exemplary ratiometric images (F505 nm / F420 nm) are shown for WM3734 and Mel Juso. Quantification of basal cellular H2O2 in WM3734 (G) and Mel Juso (H) cells. In (G-H), data are presented as mean ± SEM (n values: WM3734: control=168, TMX1 kd=209, TMX3 kd=192; Mel Juso: control=297, TMX1 kd=343, TMX3 kd=440). In (F) scale bars: 10 µm; color code: WM3734: color code: blue=0, red=3; Mel Juso: color code: blue=0, red=1.5.


WM3734 were pre - incubated for 5 min with extracellular H2O2 ( 0 - 500 µM ) . Tg - stimulated NFAT1 - GFP translocation was measured as F / F0 .

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WM3734 cells were pre-incubated for 5 min with extracellular H2O2 (0 - 500 µM). Tg-stimulated NFAT1-GFP nuclear translocation was measured as F/F0.


IC50 of H2O2 inhibition of NFAT1 import was determined to be 13 . 2 µM ( red mark ) .

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The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13.2 µM (red mark).


Tg - induced NFAT1 - GFP import in transient TMX1 silenced WM3734 after pre - incubation with 100 µM N - acetylcysteine ( NAC ) for 48 h .

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Tg-induced NFAT1-GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre-incubation with 100 µM N-acetylcysteine (NAC) for 48 h.


Endpoint of data from ( C ) upon treatment with NAC , PEG - catalase ( 50 U / mL ) , or DTT ( 1 mM ) . data are presented as mean ± ( n values : control = 73 , control + NAC = 19 , TMX1 kd = 57 , TMX1 kd + NAC = 63 , TMX1 kd + catalase = 58 , TMX1 kd + DTT = 22 ) .

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Endpoint quantification of the data from (C) and upon treatment with NAC, PEG-catalase (50 U/mL), or DTT (1 mM). data are presented as mean ± SEM (n values: control=73, control+NAC= 19, TMX1 kd=57, TMX1 kd+NAC=63, TMX1 kd+catalase=58, TMX1 kd+DTT=22).


calcineurin activity ( CaNAR2 ) in WM3734 with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation .

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Cytosolic calcineurin activity (CaNAR2) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation.


of basal calcineurin activity maximal calcineurin activity ( plateau - basal ) . data are presented as mean ± ( n values : control = 49 , TMX1 kd = 48 , TMX3 kd = 63 ) .

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Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal). data are presented as mean ± SEM (n values: control=49, TMX1 kd=48, TMX3 kd=63).


Normalized Tg - induced calcineurin activity in Mel Juso after 5 min pre - incubation with extracellular H2O2 ( 0 - 200 µM ) . of maximum calcineurin activity ( plateau - basal ) .

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Normalized Tg-induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre-incubation with extracellular H2O2 (0 - 200 µM). Quantification of the maximum cytosolic calcineurin activity (plateau-basal).


of basal calcineurin activity maximal calcineurin activity ( plateau - basal ) 8 h after treatment with antioxidant NAC ( 100 µM ) in WM3734 with transient knockdown of TMX1 . data are presented as mean ± ( n values : control = 15 , TMX1 kd = 24 , TMX1 kd + NAC = 19 ) .

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Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal) 8 h after treatment with the antioxidant NAC (100 µM) in WM3734 with transient knockdown of TMX1. data are presented as mean ± SEM (n values: control=15, TMX1 kd=24, TMX1 kd+NAC=19).


Mitochondrial H2O2 ( mito - HyPer ) was measured in two lines upon transient TMX knockdown . Exemplary ratiometric ( F505 nm / F420 nm ) are shown ( A ) ; corresponding for ( B ) WM3734 ( C ) Mel Juso . , data are presented as mean ± ( n values : WM3734 : control = 546 , TMX1 kd = 510 , TMX3 kd = 621 ; Mel Juso : control = 416 , TMX1 kd = 418 , TMX3 kd = 442 ) .

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Mitochondrial H2O2 (mito-HyPer) was measured in two cell lines upon transient TMX knockdown. Exemplary ratiometric images (F505 nm / F420 nm) are shown (A); corresponding quantification for (B) WM3734 and (C) Mel Juso. , data are presented as mean ± SEM (n values: WM3734: control=546, TMX1 kd=510, TMX3 kd=621; Mel Juso: control=416, TMX1 kd=418, TMX3 kd=442).


Mitochondrial Ca2 + uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria - targeted calcium sensor ( 4mt - D3cpV ) ( D ) . were exposed to 0 . 25 mM Ca2 + containing Ringer ' s buffer Tg . ( E ) of basal mitochondrial Ca2 + ( F ) mitochondrial Ca2 + uptake ( basal - plateau ) . , data are presented as mean ± ( n values : control = 62 , TMX1 kds = 45 ) .

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Mitochondrial Ca2+ uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria-targeted calcium sensor (4mt-D3cpV) (D). Cells were exposed to 0.25 mM Ca2+ containing Ringer's buffer and Tg. (E) Quantification of basal mitochondrial Ca2+ levels and (F) mitochondrial Ca2+ uptake (basal-plateau). , data are presented as mean ± SEM (n values: control=62, TMX1 kds=45).