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 O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O 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-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O O O O O O B-GENEPROD B-CELL_TYPE O O O B-TISSUE O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O O O B-GENEPROD B-CELL_TYPE O O O B-TISSUE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O 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-GENEPROD O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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 ( individual plots with mean ) of selected

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

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


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

O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE 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 . 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 ) .

O O O O O O O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE 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 . 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 .

O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O B-TISSUE O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 . 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 O O O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O B-TISSUE O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE 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 . 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 .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE 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 . 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 .

O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD 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 . 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 .

O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD 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 ( + 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 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 + 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 .

O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 . 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 .

O O O O O O O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 . 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 O O O O O O O O B-GENEPROD B-CELL_TYPE O O B-TISSUE O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 ( + 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-ORGANISM 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 ( individual plots with mean ) of selected mice .

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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 ) .

O O O O O O O O O O O B-CELL_TYPE O O O B-TISSUE O B-ORGANISM O O O O O O O

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 .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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 cells positive for IL - 1R1 , shown in ( D ) .

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


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 .

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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 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 .

O O O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

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 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 .

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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 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 .

O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 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 .

O O B-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE 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 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 .

O O B-EXP_ASSAY O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE 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 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 .

O O O B-EXP_ASSAY O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 .

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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 proteins in HeLa cells 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 proteins in 4KO or 4KO + GCN2 MEF cells 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 proteins in eIF2αS / S and eIF2αA / A MEF cells 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 proteins in eIF2αS / S and eIF2αA / A MEF cells treated with indicated concentrations of Tm for 2 . 5 or 5 h .

O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE 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 proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF .

O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O

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

O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation ( 10 mM ) .

O O B-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol ( His ) or borrelidin ( Bor ) .

O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 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 .

O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O

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 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 ) .

O O O B-GENEPROD O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 . 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 .

O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O 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) 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 analysis of LC3 - I / II expression in Panc02 cells under the conditions described in B . Actin was used as a loading control .

O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O

(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 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 .

O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

(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 , 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 ) .

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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 )

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 ) and immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 and IL - 1α ( b )

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

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


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 ) .

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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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O

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 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 ) .

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

O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY

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 , 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 .

O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O

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 and quantification of live and dead Salmonella at 4 h post - infection . Arrowheads , bacteria 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 , 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 .

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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 and 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 , 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 ) .

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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 and immunostained for LC3 and GBP2 . Arrowheads , bacteria 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 .

. c, Quantification of results from b.


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).


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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 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 ) .

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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 , Quantification 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 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 .

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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 , 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 ) .

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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 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 ) .

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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 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 .

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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 ) 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 .

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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 ) and NFAT1 ( deep red ) staining of healthy human skin and increasing melanoma stages ; P1 - P13 refer to the donor patient 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 the translocation of NFAT1 - GFP to the nucleus of WM3734 cells . ( A ) Ratio images ( F / F0 ) , ( B ) corresponding endpoint quantification ( 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 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 ) .

O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-DISEASE O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O O O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O

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 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 ) .

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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).


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 ) .

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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).


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 .

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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 cells were pre - incubated for 5 min with extracellular H2O2 ( 0 - 500 µM ) . Tg - stimulated NFAT1 - GFP nuclear 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.


The IC50 of H2O2 inhibition of NFAT1 nuclear 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 nuclear import in transient TMX1 silenced WM3734 cells and 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 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 ) .

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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).


Cytosolic calcineurin activity ( CaNAR2 ) in WM3734 cells 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.


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 ) .

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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 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 ) .

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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).


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 ) .

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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 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 ) .

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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).