( A - E ) Control Tg ( bactin : igfbp1a ) - overexpressing larvae were treated with MTZ from 3 - 4 dpf to ablate β , analyzed at 6 dpf , after 2 days of regeneration . ( A - B ) Representative confocal of control Tg ( bactin : igfbp1a ) - overexpressing Tg ( ins : dsRed ) ; Tg ( gcg : GFP ) ; Tg ( ins : Flag - NTR ) larvae . A bihormonal glucagon - insulin - expressing ( gcg + ; ins + ) is indicated by an arrow . Scale bars : 20 μm . ( C - D ) Representative confocal of control Tg ( bactin : igfbp1a ) - overexpressing Tg ( gcg : GFP ) ; Tg ( ins : Flag - NTR ) larvae stained for Pdx1 . Pdx1 - glucagon - expressing ( pdx1 + ; gcg + ) are indicated by arrows . Scale bars : 20 μm . ( E ) of gcg + ; ins + pdx1 + ; gcg + in control Tg ( bactin : igfbp1a ) group ; P < 0 . 001 , P = 0 . 0012 ; n = 18 18 in control groups , n = 15 10 in Tg ( bactin : igfbp1a ) groups , for gcg + ; ins + pdx1 + ; gcg + , respectively .

O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O 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 O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 B-GENEPROD O B-GENEPROD O O B-GENEPROD 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 B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O

(A-E) Control and Tg(bactin:igfbp1a)-overexpressing larvae were treated with MTZ from 3-4 dpf to ablate β cells, and analyzed at 6 dpf, after 2 days of regeneration. (A-B) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(ins:dsRed);Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae. A bihormonal glucagon- and insulin-expressing cell (gcg+;ins+) is indicated by an arrow. Scale bars: 20 μm. (C-D) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae stained for Pdx1. Pdx1- and glucagon-expressing cells (pdx1+;gcg+) are indicated by arrows. Scale bars: 20 μm. (E) Quantification of gcg+;ins+ and pdx1+;gcg+ cells in the control and Tg(bactin:igfbp1a) group; P<0.001, P=0.0012; n=18 and 18 in the control groups, n=15 and 10 in the Tg(bactin:igfbp1a) groups, for the gcg+;ins+ and pdx1+;gcg+ quantification, respectively.


( F - I ) Lineage - tracing evidence supports α - to β - transdifferentiation . ( G ) of insulin - expressing that originate from α - . Control larvae , n = 13 ; bactin : igfbp1a larvae , n = 16 . P = 0 . 0051 .

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

(F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (G) Quantification of insulin-expressing cells that originate from α-cells. Control larvae, n=13; bactin:igfbp1a larvae, n=16. P=0.0051.


( F - I ) Lineage - tracing evidence supports α - to β - transdifferentiation . ( H - I ) Representative confocal at 6 dpf ( after 2 days of regeneration ) of control bactin : igfbp1a - overexpressing larvae stained for insulin , glucagon , to - pro , H2B - GFP . ins + ; H2B - GFP + are indicated by arrowheads , H ' I ' show against only insulin H2B - GFP . Moreover , most insulin + ; H2B - GFP + no longer express glucagon . Scale bars : 15 μm .

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

(F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (H-I) Representative confocal images at 6 dpf (after 2 days of regeneration) of control and bactin:igfbp1a-overexpressing larvae stained for insulin, glucagon, to-pro, and H2B-GFP. ins+;H2B-GFP+ cells are indicated by the arrowheads, and H' and I' show staining against only insulin and H2B-GFP. Moreover, most insulin+;H2B-GFP+ cells no longer express glucagon. Scale bars: 15 μm.


( A - H ) PPP , an IGF1R inhibitor , promotes β - regeneration . Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β then treated with EdU DMSO or with EdU PPP during regeneration from 4 - 6 dpf . ( A - B ) Representative confocal at 6 dpf of DMSO - PPP - treated larvae displaying β in green β that had incorporated EdU as yellow overlap ( arrowheads ) . Scale bars : 20 μm . ( C ) of total number of β ( green bars ) at 6 dpf , β that had incorporated EdU ( bars ) from 4 - 6 dpf during β - regeneration . P = 0 . 0003 , P = 0 . 8607 respectively . ( D ) Rate of β - proliferation , displayed as of β that incorporated EdU . P = 0 . 1194 . n = 18 larvae for DMSO - treated group , n = 17 larvae for PPP - treated group . ( E - H ) To examine whether PPP affected β - proliferation during regular development , Tg ( ins : H2B - GFP ) larvae were treated with EdU DMSO or PPP from 4 - 6 dpf . ( E - F ) Representative confocal of 6 dpf DMSO - PPP - treated larvae displaying β in green β that had incorporated EdU as yellow overlap . Scale bars : 20 μm . ( G ) of total number of β ( green bars ) β that had incorporated EdU ( bars ) per larva from 4 - 6 dpf . P = 0 . 9098 0 . 9976 , respectively . ( H ) Rate of β proliferation , displayed as of β that incorporated EdU . P = 0 . 7822 . n = 16 larvae for DMSO - treated group , 18 larvae for PPP - treated group .

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

(A-H) PPP, an IGF1R inhibitor, promotes β-cell regeneration. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate the β cells and then treated with EdU and DMSO or with EdU and PPP during regeneration from 4-6 dpf. (A-B) Representative confocal images at 6 dpf of DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap (arrowheads). Scale bars: 20 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and β cells that had incorporated EdU (white bars) from 4-6 dpf during β-cell regeneration. P=0.0003, P=0.8607 respectively. (D) Rate of β-cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.1194. n=18 larvae for the DMSO-treated group, n=17 larvae for the PPP-treated group. (E-H) To examine whether PPP affected β-cell proliferation during regular development, Tg(ins:H2B-GFP) larvae were treated with EdU and DMSO or PPP from 4-6 dpf. (E-F) Representative confocal images of 6 dpf DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap. Scale bars: 20 μm. (G) Quantification of the total number of β cells (green bars) and β cells that had incorporated EdU (white bars) per larva from 4-6 dpf. P=0.9098 and 0.9976, respectively. (H) Rate of β cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.7822. n=16 larvae for DMSO-treated group, 18 larvae for PPP-treated group.


( I - K ) Activation of Igf pathway reduces β - regeneration . Control bactin : igf2a - overexpressing Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β subsequently let to regenerate from 4 - 6 dpf . ( I - J ) Representative confocal of 6 dpf control bactin : igf2a - overexpressing larva displaying β in green . Scale bars : 15 μm . ( K ) of total number of β per larva at 6 dpf following β regeneration from 4 - 6 dpf . P < 0 . 0001 . n = 28 larvae for control , n = 15 larvae for bactin : igf2a .

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

(I-K) Activation of the Igf pathway reduces β-cell regeneration. Control and bactin:igf2a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently let to regenerate from 4-6 dpf. (I-J) Representative confocal images of 6 dpf control and bactin:igf2a-overexpressing larva displaying β cells in green. Scale bars: 15 μm. (K) Quantification of the total number of β cells per larva at 6 dpf following β cell regeneration from 4-6 dpf. P<0.0001. n=28 larvae for control, n=15 larvae for bactin:igf2a.


( L - P ) No synergistic effect was observed for igfbp1a PPP . Control bactin : igfbp1a - overexpressing Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β subsequently treated with DMSO or PPP during regeneration from 4 - 6 dpf . ( L ) of total number of β per larva . n > 10 ( n = 23 , 20 , 14 , 13 ) . P = 0 . 9546 . ( M - P ) Representative confocal of 6 dpf control or bactin : igfbp1a overexpressing larvae treated with either DMSO or PPP , displaying β after 2 days regeneration . Scale bars : 10 μm . See also Figure EV2 EV3 .

O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE 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 B-CELL_TYPE 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 B-GENEPROD O B-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O

(L-P) No synergistic effect was observed for igfbp1a and PPP. Control and bactin:igfbp1a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently treated with DMSO or PPP during regeneration from 4-6 dpf. (L) Quantification of the total number of β cells per larva. n>10 (n=23, 20, 14, 13). P=0.9546. (M-P) Representative confocal images of 6 dpf control or bactin:igfbp1a overexpressing larvae treated with either DMSO or PPP, displaying β cells after 2 days regeneration. Scale bars: 10 μm. See also Figure EV2 EV3.


( A - B ) Representative confocal projections of whole pancreas ( dashed lines ) of 35 - day - old Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , that were subjected to β - ablation between day 30 - 31 then allowed to regenerate for 4 days . Scale bars represent 100 μm . ( C ) of control Tg ( bactin : igfbp1a ) zebrafish ; P < 0 . 0001 . ( D - H ) of β - regeneration was automated with an ImageJ script . ( D ) Insulin - positive per zebrafish . ( E ) Relative insulin - positive per ; P < 0 . 01 . ( F ) of pancreas that was insulin - positive was significantly larger in Tg ( bactin : igfbp1a ) than in controls ; P < 0 . 0001 . ( G ) total number of recorded units of adjacent ins : H2B - GFP + pixels ( units ranging from single β to β - clusters ) did not differ between control Tg ( bactin : igfbp1a ) zebrafish . ( H ) of recorded ins : H2B - GFP + units was on average larger in Tg ( bactin : igfbp1a ) than in controls ; P = 0 . 039 . n = 26 in control group , n = 27 in Tg ( bactin : igfbp1a ) group .

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

(A-B) Representative confocal projections of the whole pancreas (dashed lines) of 35-day-old Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), that were subjected to β-cell ablation between day 30-31 and then allowed to regenerate for 4 days. Scale bars represent 100 μm. (C) Body length of control and Tg(bactin:igfbp1a) zebrafish; P<0.0001. (D-H) Quantification of β-cell regeneration was automated with an ImageJ script. (D) Insulin-positive area per zebrafish. (E) Relative insulin-positive area per body length; P<0.01. (F) The percentage of the pancreas area that was insulin-positive was significantly larger in Tg(bactin:igfbp1a) than in controls; P<0.0001. (G) The total number of recorded units of adjacent ins:H2B-GFP+ pixels (units ranging from single β cells to β-cell clusters) did not differ between control and Tg(bactin:igfbp1a) zebrafish. (H) The size of recorded ins:H2B-GFP+ units was on average larger in Tg(bactin:igfbp1a) than in controls; P=0.039. n=26 in the control group, n=27 in the Tg(bactin:igfbp1a) group.


Isolated islets from mice were treated for 3 days with recombinant mouseIGFBP1 , respectively . ( A - B ) Representative confocal of control Igfbp1 - treated mouse islets . gcg + ins + are indicated by arrows . Scale bars : 25 μm . ( C ) of of gcg + ins + bihormonal per ins + in mouse islets after treatment with increasing concentrations of IGFBP1 . P = 0 . 7304 , 0 . 0023 , 0 . 0013 , 0 . 0410 , respectively . ( D ) of β - proliferation in mouse islets after treatment with Igfbp1 ; data displayed as relative values so that proliferation markers EdU Ki67 can be compared .

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

Isolated islets from mice were treated for 3 days with recombinant mouseIGFBP1 protein, respectively. (A-B) Representative confocal images of control and Igfbp1-treated mouse islets. gcg+ins+ cells are indicated by arrows. Scale bars: 25 μm. (C) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in mouse islets after treatment with increasing concentrations of IGFBP1. P=0.7304, 0.0023, 0.0013, 0.0410, respectively. (D) Quantification of β-cell proliferation in mouse islets after treatment with Igfbp1; data displayed as relative values so that the proliferation markers EdU and Ki67 can be compared.


Isolated islets from human donors were treated for 3 days with recombinant human IGFBP1 , respectively . ( E - F ) Representative confocal of control or IGFBP1 - treated human islets . A gcg + ins + is indicated by an arrow . For clarity , colors for denoting insulin ( green ) , glucagon ( red ) , DAPI ( blue ) are also shown separately . Scale bars : 15 μm . ( G ) of of gcg + ins + bihormonal per ins + in human islets from 5 donors . P = 0 . 0036 . Co - expression of insulin glucagon is indicative of transdifferentiation , which could reflect either α transdifferentiating to β or vice versa .

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

Isolated islets from human donors were treated for 3 days with recombinant human IGFBP1 protein, respectively. (E-F) Representative confocal images of control or IGFBP1-treated human islets. A gcg+ins+ cell is indicated by an arrow. For clarity, individual colors for denoting insulin (green), glucagon (red), and DAPI (blue) are also shown separately. Scale bars: 15 μm. (G) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in human islets from 5 donors. P=0.0036. Co-expression of insulin and glucagon is indicative of transdifferentiation, which could reflect either α cells transdifferentiating to β cells or vice versa.


Volcano plots of piliation , aggregation adhesion mutation scores for pilE mutations . Stop synonymous mutations are highlighted in blue orange respectively . Adhesion aggregation mutation scores relative to piliation . correlation appears as a red dotted .

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Volcano plots of piliation, aggregation and adhesion mutation scores for all pilE mutations. Stop and synonymous mutations are highlighted in blue and orange respectively. Adhesion and aggregation mutation scores relative to piliation. The correlation appears as a red dotted line.


Scanning electron microscopy of single on a cellulose filter . Scale bars : 500 nm .

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

Scanning electron microscopy of single bacteria on a cellulose filter. Scale bars: 500 nm.


Two representative immunofluorescence labelling of TFP . Scale bar : 1 µm .

O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O

Two representative immunofluorescence labelling of TFP. Scale bar: 1 µm.


Piliation per bacterium as a function of proportion of piliated normalized to pilESB values as measured by flow cytometry using 20D9 monoclonal antibody . Black dots indicate de novo generated PilE mutants that were characterized . Blue dots indicate mutants of piliation machinery green dots mutants in α1N region . Solid lines indicate 95 % confidence interval . of aggregation normalized to pilESB for a subset of outliers corresponding pilT double mutants . Mean values ± are indicated for each . N ≥ 3 independent experiments . Paired t - . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

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

Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody. Black dots indicate all the individual de novo generated PilE mutants that were characterized. Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region. Solid lines indicate 95% confidence interval. Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants. Mean values ± SEM are indicated for each strain. N≥3 independent experiments. Paired t-test. p<0.05 (*), p<0.01 (**).


Piliation per bacterium as measured by flow cytometry using 20D9 monoclonal antibody F10 nanobody .

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

Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody.


Visualization of type IV pili by immunofluorescence either using 20D9 antibody or F10 nanobody as indicated ( magenta ) . In case of K140Q mutant labeled with 20D9 antibody background was made visible to convincingly show absence of pilus labeling . Scale bar : 2 µm .

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

Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated (magenta). In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling. Scale bar: 2 µm.


Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E pilEK140Q for which F10 nanobody value is reported . Each dot represents values for a PilE point mutant relative to SB . Hypo - aggregative mutants are highlighted in orange while hyper - aggregative mutants are in blue . solid lines indicate 95 % confidence interval .

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

Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported. Each dot represents values for a PilE point mutant relative to the SB strain. Hypo-aggregative mutants are highlighted in orange while hyper-aggregative mutants are in light blue. solid lines indicate the 95% confidence interval.


of PilX present in purified pili preparations sheared from surface using western blot . PilX were normalized using PilE as a reference normalized to pilESB values . Mean ± is indicated . N = 3 independent experiments . Paired t - .

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Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot. PilX levels were normalized using PilE as a reference and normalized to pilESB values. Mean ratio ± SEM is indicated. N=3 independent experiments. Paired t-test.


Early adhesion of pilE mutants as a function of aggregation . Values were normalized to that of pilESB . Each dot represents a pilE mutant pilV , pilD , pilX pilC1 reference mutants . Mutants in region surrounding A131 are in green those surrounding Y50 in purple . A blown - up view of zone of interest ( lower left quarter ) is presented on right panel . shaded highlights absence of significant adhesion .

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Early adhesion of pilE mutants as a function of aggregation. Values were normalized to that of pilESB. Each dot represents a pilE mutant and the pilV, pilD, pilX and pilC1 reference mutants. Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple. A blown-up view of the zone of interest (lower left quarter) is presented on the right panel. The shaded area highlights the absence of significant adhesion.


Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB .

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Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB.


Piliation of a selection of mutants determined by immunofluorescence . Average pilus ± is indicated . N = 6 experiments with at least 50 analyzed in each experiment . Paired t - . Number of pili per ± determined as in ( E ) . Paired t - . p < 0 . 05 ( * ) .

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Piliation length of a selection of mutants determined by immunofluorescence. Average pilus length ± SEM is indicated. N=6 experiments with at least 50 bacteria analyzed in each experiment. Paired t-test. Number of pili per bacteria ± SEM determined as in (E). Paired t-test. p<0.05 (*).


Time - lapse of a - adhered pilus fluorescently labeled with F10 nanobody at three different focal planes . Initiation of flow corresponds to t = 0 . Scale bar : 1 µm .

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Time-lapse of a cell-adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes. Initiation of flow corresponds to t=0. Scale bar: 1 µm.


Spinning - disk confocal immunofluorescence of pili stained with both SM1 ( magenta ) F10 ( green ) . thick appearance of TFP is due to secondary reagents used to detect them as well as diffraction of . In each field of view , a substrate - a - bound pilus are shown . Upper panels : z - projection from stacks in xy plane ; bottom panels : side view in z plane . Scale bar : 2 µm . arrows correspond to SM1 at tip of pili . of adhesion after infection of HUVEC for 30 minutes , expressed as number of adherent over number of nuclei . Prior to infection , were incubated at 37 ° C with or without antibody or nanobody for 15 minutes . Mean values ± are indicated for each . N = 5 independent experiments . Paired t - . p < 0 . 05 ( * ) .

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Spinning-disk confocal immunofluorescence of pili stained with both SM1 (magenta) and F10 (green). The thick appearance of TFP is due to the primary and secondary reagents used to detect them as well as diffraction of light. In each field of view, a substrate- and a cell-bound pilus are shown. Upper panels: z-projection from the stacks in the xy plane; bottom panels: side view in the z plane. Scale bar: 2 µm. White arrows correspond to SM1 staining at the tip of pili. Quantification of adhesion after infection of HUVEC cells for 30 minutes, expressed as the number of adherent bacteria over the number of cell nuclei. Prior to infection, bacteria were incubated at 37°C with or without antibody or nanobody for 15 minutes. Mean values ± SEM are indicated for each strain. N=5 independent experiments. Paired t-test. p<0.05 (*).


( A ) miR - 515 - 5p expression increases of tubulin cytoskeleton . Tubulin ( green ) nucleus ( blue ) . Objective x20 . Bar : 200 µm .

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(A) miR-515-5p expression increases the area of the tubulin cytoskeleton. Tubulin (green) and cell nucleus (blue). Objective x20. Bar: 200 µm.


( B - C ) miR - 515 - 5p inhibits random ( B ) directed ( C ) migration . indicated lines were transfected with miR - 515 - 5p for 48 h before time - lapse imaging was performed for 18 h ( A ) or transwell migration assays were performed for 9 h ( C ) . ( B - left panel ) Plots show overlays of representative trajectories travelled . ( B - right panel ) of migration was quantified represented as mean ± of values normalised to respective control condition . ( C - left panel ) Representative field of view from bottom of transwell chambers . Scale bar is 20 µm . ( C - right panel ) Data represent of average number migratory per field ( n = 5 fields per condition ) . Data are mean of three experiments ± . ( B - C ) P values were calculated by t - between miRNA ( 515 - 5p ) values their respective non - targeting control miR ( NC ) values ( * * * , P < 0 . 001 ) . data shown are representative of experiments performed at least in triplicate .

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(B-C) miR-515-5p inhibits random (B) and directed (C) cell migration. The indicated cell lines were transfected with miR-515-5p for 48 h before time-lapse imaging was performed for 18 h (A) or transwell migration assays were performed for 9 h (C). (B-left panel) Plots show overlays of representative trajectories travelled. (B-right panel) The distance of migration was quantified and represented as the mean ± SEM of values normalised to the respective control condition. (C-left panel) Representative field of view from the bottom of the transwell chambers. Scale bar is 20 µm. (C-right panel) Data represent percentage of the average number migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (B-C) P values were calculated by t-test between miRNA (515-5p) values and their respective non-targeting control miR (NC) values (***, P<0.001). All data shown are representative of experiments performed at least in triplicate.


( A ) - seq of MCF7 MDA - MB - 231 transfected with miR - 515 - 5p revealed down - regulation of five transcripts , NRAS , FZD4 , CDC42BPA , PIK3C2B MARK4 .

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(A) RNA-seq of MCF7 and MDA-MB-231 transfected with miR-515-5p revealed the down-regulation of five transcripts, NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4.


( B - D ) effect of miR - 515 - 5p overexpression on NRAS , FZD4 , CDC42BPA , PIK3C2B MARK4 mRNA in MCF7 ( B ) , MDA - MB - 231 ( C ) , A549 H1299 ( D ) was determined by qPCR 48 h following transfection of indicated with non - targeting control miR ( NC ) or miR - 515 - 5p . Data is displayed as normalised mean ± . P values were calculated by t - between miRNA conditions their respective NC conditions ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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(B-D) The effect of miR-515-5p overexpression on NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNA levels in MCF7 (B), MDA-MB-231(C), A549 and H1299 (D) was determined by qPCR 48 h following transfection of the indicated cell line with non-targeting control miR (NC) or miR-515-5p. Data is displayed as the normalised mean ± SEM. P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001).


( E - F ) miR - 515 - 5p directly interacts with NRAS , MARK4 PI3KC2B ' s 3 ' UTR . Relative luciferase activity were measured 24 h after co - transfection of MCF - 7 ( E ) MDA - MB - 231 ( F ) with 3 ' UTR - luciferase reporter constructs either with miR - 515 - 5p or miR NC . Data shown are normalised mean of three independent experiments ± . ( B - F ) P values were calculated by t - between miRNA conditions their respective NC conditions ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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(E-F) miR-515-5p directly interacts with NRAS, MARK4 and PI3KC2B's 3'UTR. Relative luciferase activity levels were measured 24 h after co-transfection of MCF-7 (E) and MDA-MB-231 (F) with 3'UTR-luciferase reporter constructs and either with miR-515-5p or miR NC. Data shown are normalised mean of three independent experiments ± SEM. (B-F) P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001).


MARK4 siRNA - mediated silencing increases tubulin cytoskeletal ( A ) . ( A ) Data shown are representative of experiments performed in triplicate . Bar graph represents average ± of n ≥ 50 per technical replicate with 3 technical replicates per conditions .

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MARK4 siRNA-mediated silencing increases the tubulin cytoskeletal area (A). (A) Data shown are representative of experiments performed in triplicate. Bar graph represents the average ± SEM of n ≥ 50 per technical replicate with 3 technical replicates per conditions.


MARK4 siRNA - mediated silencing reduces random ( B ) migration in MDA - MB - 231 ( MDA ) , A549 H1299 . were transfected either with siRNA targeting MARK4 ( M4 ) or a non - targeting control ( NC ) for 48 h before time - lapse imaging was performed for 18 h ( B ) . ( B - top panel ) Plots show overlays of representative trajectories described . ( B - bottom panel ) migration was quantified represented as normalised mean ± . ( C - top panel ) Representative field of view from bottom side of transwell . ( C - bottom panel ) Graphs indicate migration expressed as a of average of migratory per field ( n = 5 fields per condition ) . ( B - C ) Data are mean of three experiments ± . ( A - C ) P values were calculated by t - between siRNA - transfected respective control condition ( * * * P < 0 . 001 ) .

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MARK4 siRNA-mediated silencing reduces random (B) migration in MDA-MB-231 (MDA), A549 and H1299 cells.Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before time-lapse imaging was performed for 18 h (B). (B-top panel) Plots show overlays of representative trajectories described. (B-bottom panel) The migration distance was quantified and represented as the normalised mean ± SEM. (C-top panel) Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001).


MARK4 siRNA - mediated silencing reduces directed ( C ) migration in MDA - MB - 231 ( MDA ) , A549 H1299 . ( C - top panel ) . were transfected either with siRNA targeting MARK4 ( M4 ) or a non - targeting control ( NC ) for 48 h before transwell for 9h ( C ) . Representative field of view from bottom side of transwell . ( C - bottom panel ) Graphs indicate migration expressed as a of average of migratory per field ( n = 5 fields per condition ) . ( B - C ) Data are mean of three experiments ± . ( A - C ) P values were calculated by t - between siRNA - transfected respective control condition ( * * * P < 0 . 001 ) .

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MARK4 siRNA-mediated silencing reduces directed (C) migration in MDA-MB-231 (MDA), A549 and H1299 cells.(C-top panel). Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before transwell assay for 9h (C). Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001).


( A - B ) indicated lines were transfected with either miR - 515 - 5p or a non - targeting miR ( miR NC ) together with a MARK4 expressing or an empty vector ( EV ) plasmid . MARK4 over - expression rescues inhibition by miR - 515 - 5p of random ( A ) directed ( B ) migration of indicated lines . ( A - left panel ) Plots show overlays of representative trajectories described for MDA - MB - 231 ( n = 30 ) . ( A - right panel ) migration was quantified represented as normalised mean ± . ( B - left panel ) Representative field of view from bottom side of transwell for MDA - MB - 231 . ( B - right panel ) Graphs indicate migration expressed as a of average of migratory per field ( n = 5 fields per condition ) . Data are mean of three experiments ± . ( A - C ) P values were calculated by t - between transfected respective control condition ( EV + miR - NC ) ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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(A-B) The indicated cell lines were transfected with either miR-515-5p or a non-targeting miR (miR NC) together with a MARK4 expressing or an empty vector (EV) plasmid DNA. MARK4 over-expression rescues the inhibition by miR-515-5p of random (A) and directed (B) migration of the indicated cell lines. (A-left panel) Plots show overlays of representative trajectories described for MDA-MB-231 cells (n=30). (A-right panel) The migration distance was quantified and represented as the normalised mean ± SEM. (B-left panel) Representative field of view from the bottom side of the transwell membrane for MDA-MB-231. (B-right panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the transfected and the respective control condition (EV+miR-NC) (*,P<0.05; **,P<0.01; ***,P<0.001).


( A - B ) Overall survival of ER - negative metastatic breast cancer ( n = 226 ) ( A ) lung adenocarcinoma ( n = 719 ) ( B ) according to MARK4 mRNA found in their tumors was represented using Kaplan Meier - plotter [ 25 ] .

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(A-B) Overall survival of ER-negative metastatic breast cancer (n=226) (A) and lung adenocarcinoma (n=719) (B) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier-plotter [25].


( C ) A cDNA array for 24 matched lung cancers surrounding normal tissue was analysed for MARK4 expression . Expression from tumors were normalised to their respective normal control of cases for indicated fold expression in represented as a pie - chart .

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(C) A cDNA array for 24 matched lung cancers and surrounding normal tissue was analysed for MARK4 expression. Expression levels from tumors were normalised to their respective normal control and the percentage of cases for the indicated fold expression in the tumor represented as a pie-chart.


( D - F ) MARK4 expression was determined by histoimmunochemistry in ( D - E ) a tissue microarray comprising 90 lung 10 normal lung samples or ( F ) a home - made tissue microarray comprising 100 matched cases of metastatic tumors . ( D - E ) of positivity for MARK4 in lung tumors versus normal lung samples ( D ) or in lung tumors according to their histological subtypes ( E ) is represented . ( F ) of cases with increased , decreased or unchanged of MARK4 in metastatic lesions as compared to corresponding tumors is represented .

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(D-F) MARK4 protein expression was determined by histoimmunochemistry in (D-E) a tissue microarray comprising 90 lung tumor and 10 normal lung samples or (F) a home-made tissue microarray comprising 100 matched cases of primary and metastatic tumors. (D-E) The percentage of positivity for MARK4 staining in lung tumors versus normal lung samples (D) or in lung tumors according to their histological subtypes (E) is represented. (F) The percentage of cases with increased, decreased or unchanged levels of MARK4 in metastatic lesions as compared to the corresponding primary tumors is represented.


( A - D ) Low expression of miR - 515 - 5p is linked to survival in both Lung Breast cancer . ( A ) Survival of miR - 515 - 5p from 195 lung adenocarcinoma samples from TCGA . ( B ) Survival of miR - 515 - p from 34 lung squamous carcinoma from TCGA . ( C ) Survival of miR - 515 - 5p from 60 Breast cancer samples from Lyng Ditzel Breast Study ( GSE37405 ) . ( D ) Survival of miR - 515 - 5p from 33 Breast cancer from GSE7405 Breast study . plot were obtained with SurvMicro http : / / bioinformatica . mty . itesm . mx : 8080 / Biomatec / Survmicro . jsp for ( A - C ) with MIRUMIR for ( D ) .

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(A-D) Low expression of miR-515-5p is linked to survival in both Lung and Breast cancer. (A) Survival analysis of miR-515-5p from 195 lung adenocarcinoma samples from the TCGA. (B) Survival analysis of miR-515-p from 34 lung squamous cell carcinoma from the TCGA. (C) Survival analysis of miR-515-5p from 60 Breast cancer samples from the Lyng Ditzel Breast Study (GSE37405). (D) Survival analysis of miR-515-5p from 33 Breast cancer from the GSE7405 Breast study. Analysis and plot were obtained with SurvMicro http://bioinformatica.mty.itesm.mx:8080/Biomatec/Survmicro.jsp for (A-C) and with MIRUMIR for (D).


( E ) Comparison of miR - 515 - 5p expression in 18 matched metastatic lesions from breast cancer . miR - 515 - 5p were quantified by TaqMan qRT - PCR . Absolute values were normalized to u6 snRNA values displayed as a dot - plot . Bar ; Mean , Whiskers ; .

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(E) Comparison of miR-515-5p expression in 18 primary and matched metastatic lesions from breast cancer patients. miR-515-5p levels were quantified by TaqMan qRT-PCR. Absolute values were normalized to u6 snRNA values and displayed as a dot-plot. Bar; Mean, Whiskers; SEM.


( F ) MDA - MB - 231 were inoculated into mammary fat pads of nude mice allowed to form metastatic deposits [ 22 ] . miR - 515 - 5p MARK4 were then determined in indicated secondary lesions . Data are average ± of technical triplicates .

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(F) MDA-MB-231 cells were inoculated into the mammary fat pads of nude mice and allowed to form metastatic deposits [22]. miR-515-5p and MARK4 levels were then determined in the primary and indicated secondary lesions. Data are average ± SEM of technical triplicates.


( G ) Luciferase - expressing A549 overexpressing miR - 515 - 5p or a non - targeting control ( miR - NC ) were injected in tail - vein of SCID mice development of burden monitored by whole - imaging in response to luciferin administration at week 1 4 following injection . ( G - left panel ) Representative imaging of same for indicated conditions at week 1 4 . ( G - Right panel ) emitted photons were quantified signals normalised to corresponding readings at week 1 . Data represented are representative of three independent experiments . ( E - G ) P values were calculated by t - ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

O O O B-GENEPROD O O B-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase-expressing A549 cells overexpressing miR-515-5p or a non-targeting control (miR-NC) were injected in the tail-vein of SCID mice and the development of tumor burden monitored by whole animal-imaging in response to luciferin administration at week 1 and 4 following injection. (G-left panel) Representative imaging of the same animals for the indicated conditions at week 1 and 4. (G-Right panel) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1. Data represented are representative of three independent experiments. (E-G) P values were calculated by t-test (*, P<0.05; **, P<0.01; ***, P<0.001).


A549 - Luc transfected either with miR - 515 - 5p precursor ( miR ) , siRNAs to MARK4 ( siMARK4 ) or corresponding control sequences ( C NT , respectively ) were injected in Scid mice through tail vein . 4 later , mice were sacrificed , their lungs extracted fixed prior to for Luciferase expression . ( A ) Representative pictures showing presence of A549 - Luc in lungs of NT mice either as single ( upper panel ) or as a tumour mass ( lower panel ) . ( B ) of covered by tumour on randomly observed fields of views . was performed using Photoshop CS4 . Each spot corresponds to of an 1 mm x 1 . 5 mm field . Transversal bar ; Median . Statistics : Student t - . * , p ≤ 0 . 05 .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 B-ORGANISM I-ORGANISM O B-TISSUE I-TISSUE O O O O B-ORGANISM O O O O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O O B-CELL_LINE O B-GENEPROD O B-TISSUE O O B-ORGANISM O O O O O O O O O O B-TISSUE O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O

A549-Luc cells transfected either with miR-515-5p precursor (miR), siRNAs to MARK4 (siMARK4) or the corresponding control sequences (C and NT, respectively) were injected in Scid mice through the tail vein. 4 weeks later, the mice were sacrificed, their lungs extracted and fixed prior to staining for Luciferase expression. (A) Representative pictures showing the presence of A549-Luc cells in the lungs of NT mice either as single cells (upper panel) or as a tumour mass (lower panel). (B) Percentage of the area covered by tumour cells on randomly observed fields of views. Quantification was performed using Photoshop CS4. Each spot corresponds to quantification of an individual 1 mm x 1.5 mm field. Transversal bar; Median. Statistics: Student t-test. *, p≤0.05.


( C ) Representative of CD3 + T Iba1 + macrophages / MG in isotype αCD4 + αCD8 group . Scale bar ; 100 μm . ( D ) of Iba1 + CD3 + in sections of BrM from isotype ( n = 9 ) αCD4 + αCD8 group ( n = 7 ) .

O O O O O B-GENEPROD O B-CELL_TYPE B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE 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 B-GENEPROD O B-GENEPROD O O O O O O O

(C) Representative IHC images of CD3+ T cells and Iba1+ macrophages/MG in the isotype and αCD4+αCD8 group. Scale bar; 100 μm. (D) Quantification of Iba1+ and CD3+ cells in IHC sections of BrM from the isotype (n=9) and αCD4+αCD8 group (n=7).


( F ) Kaplan - Meier curves depict BrM free survival of mice in isotype αCD4 + αCD8 group ( Isotype n = 19 , αCD4 + αCD8 n = 19 ) .

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

(F) Kaplan-Meier curves depict BrM free survival of mice in the isotype and αCD4+αCD8 group (Isotype n=19, αCD4+αCD8 n=19).


( H ) displays relative expression level of T effector exhaustion marker in TIL - CD4 TIL - CD8 compared to blood lymphocytes . Values display average per group on vst values ( BL - CD4 or BL - CD8 with n = 3 for each group ) vs . BrM - associated TIL - CD4 or TIL - CD8 ( n = 3 for each group ) .

O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O B-GENEPROD 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 O

(H) Heatmap displays the relative expression level of T cell effector and exhaustion marker in TIL-CD4 and TIL-CD8 compared to blood lymphocytes. Values display the average per group based on vst values (BL-CD4 or BL-CD8 with n=3 for each group) vs. BrM-associated TIL-CD4 or TIL-CD8 (n=3 for each group).


( A ) Surface expression of PD - 1 in vivo in 99LN - BrM quantified by flow cytometry ( n = 3 ) . ( B ) PD - L1 in 99LN BrM quantified by flow cytometry ( n = 3 ) . ( C ) Flow cytometric of PD - L1 in - free brain 99LN - BrM ( n = 3 ) . ( D ) Flow cytometric of PD - L1 expression on 99LN - BrM associated my myeloid types . ( n = 6 ) .

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

(A) Surface expression of PD-1 in vivo in 99LN-BrM quantified by flow cytometry (n=3). (B) PD-L1 in 99LN BrM quantified by flow cytometry (n=3). (C) Flow cytometric analysis of PD-L1 in tumor-free brain and 99LN-BrM (n=3). (D) Flow cytometric analysis of PD-L1 expression on 99LN-BrM associated my myeloid cell types. (n=6).


( 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY 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-GENEPROD O 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 B-EXP_ASSAY O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY 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-GENEPROD O B-SUBCELLULAR O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY 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-GENEPROD O O B-GENEPROD O B-GENEPROD O B-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 O O O O O 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 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

(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 .

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

(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 .

O O O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O

(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 .

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

(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-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-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

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

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

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 B-GENEPROD 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 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 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

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 B-GENEPROD O O B-CELL_TYPE O O B-TISSUE 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

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 B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O B-TISSUE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O O B-TISSUE 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 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

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 B-GENEPROD 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 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 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

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 B-GENEPROD O O B-CELL_TYPE 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 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

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

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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 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 B-GENEPROD I-GENEPROD I-GENEPROD 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 ( 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

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

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

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

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 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 B-CELL_TYPE O O B-TISSUE 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 B-GENEPROD I-GENEPROD I-GENEPROD 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

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

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-GENEPROD O O B-TISSUE O B-ORGANISM 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 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 B-GENEPROD I-GENEPROD I-GENEPROD 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

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 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 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 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 ( plots with mean ) of selected mice .

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

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 ) .

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

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

O O O O O O O O O B-CELL_LINE 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 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 .

O O O O O O O O O O O B-CELL_LINE 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 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 B-EXP_ASSAY O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE 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 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 B-CELL_LINE 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 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 B-EXP_ASSAY O O O O O B-CELL_LINE 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 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 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 B-EXP_ASSAY O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE 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 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 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 - 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-CELL_LINE O 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 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 ) .

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

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 .

O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O

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 .

O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O

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