( E ) HeLa GFP - LC3 cells were transfected with myc - SNX18 W154S / W158S mutant , starved for 2 h , immunostained against myc , and analyzed by confocal imaging . Bar , 10 µm . ( F ) Indicated myc - SNX18 constructs were transfected into HEK GFP - LC3 cells , and the number of GFP - LC3 spots per cell was quantified . The graph shows mean ± SEM ( error bars ) , n = 3 . * , P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa GFP-LC3 cells were transfected with myc-SNX18 W154S/W158S mutant, starved for 2 h, immunostained against myc, and analyzed by confocal imaging. Bar, 10 µm. (F) Indicated myc-SNX18 constructs were transfected into HEK GFP-LC3 cells, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 3. *, P < 0.05.


SNX18 tubules are positive for TfR . HeLa GFP - LC3 cells were transfected with myc - SNX18 or a control myc plasmid and then immunostained for myc and TfR ( A ) or Rab11 ( B ) . Bars : ( A ) 10 µm ; ( B ) 5 µm . Insets show enlarged views of the boxed regions .

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

SNX18 tubules are positive for TfR. HeLa GFP-LC3 cells were transfected with myc-SNX18 or a control myc plasmid and then immunostained for myc and TfR (A) or Rab11 (B). Bars: (A) 10 µm; (B) 5 µm. Insets show enlarged views of the boxed regions.


( C , i ) Clusters of tubulo - vesicular structures together with small multivesicular body - like vesicles were observed upon plastic embedding of HeLa GFP - LC3 cells overexpressing myc - SNX18 . ( ii - vi ) Immuno - EM showed that similar clusters labeled strongly for GFP - LC3 ( ii , and enlarged in iii from the boxed region ) , myc - SNX18 ( v ) , and TfR ( vi ) . Strong GFP - LC3 labeling in structures resembling more typical autophagosomal vesicles was also detected ( iv ) . See also Fig . S4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, i) Clusters of tubulo-vesicular structures together with small multivesicular body-like vesicles were observed upon plastic embedding of HeLa GFP-LC3 cells overexpressing myc-SNX18. (ii-vi) Immuno-EM showed that similar clusters labeled strongly for GFP-LC3 (ii, and enlarged in iii from the boxed region), myc-SNX18 (v), and TfR (vi). Strong GFP-LC3 labeling in structures resembling more typical autophagosomal vesicles was also detected (iv). See also Fig. S4.


Recycling endosomes provide membranes for SNX18 - mediated GFP - LC3 membrane trafficking and remodeling . ( A ) HeLa cells were starved or not starved for 2 h before fixation and immunostaining against endogenous SNX18 , Rab11 , and TfR . ( B ) Cells were transfected to express flag - Atg16L1 , starved , and immunostained against Rab11 or TfR and flag . ( C ) Cells were starved and immunostained against TfR , Atg16L1 , and SNX18 . ( D ) Cells were transfected with control or SNX18 siRNA , starved , and immunostained against SNX18 and Rab11 . Inset panels show enlarged views of the boxed regions . ( E ) Cells were transfected with control or Rab11 a + b siRNA , starved , and immunostained against SNX18 and Atg16L1 . Cells were imaged by confocal microscopy . Bars , 5 µm .

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

Recycling endosomes provide membranes for SNX18-mediated GFP-LC3 membrane trafficking and remodeling. (A) HeLa cells were starved or not starved for 2 h before fixation and immunostaining against endogenous SNX18, Rab11, and TfR. (B) Cells were transfected to express flag-Atg16L1, starved, and immunostained against Rab11 or TfR and flag. (C) Cells were starved and immunostained against TfR, Atg16L1, and SNX18. (D) Cells were transfected with control or SNX18 siRNA, starved, and immunostained against SNX18 and Rab11. Inset panels show enlarged views of the boxed regions. (E) Cells were transfected with control or Rab11 a+b siRNA, starved, and immunostained against SNX18 and Atg16L1. Cells were imaged by confocal microscopy. Bars, 5 µm.


( B and C ) RNAi against SH3PX1 ( GFP - positive clones ) decreases formation of LTR punctae in response to 4 h of starvation in 20 % sucrose ( B ) and inhibits formation of mCherry - Atg8a - positive autophagosomes in response to 2 h starvation ( C ) . Genotypes : ( B and D ) hs - flp ; UAS - dicer / + ; Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ; ( C and E ) hs - flp ; UAS - dicer / + ; r4 : mCherry - Atg8a Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ;

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

(B and C) RNAi against SH3PX1 (GFP-positive clones) decreases formation of LTR punctae in response to 4 h of starvation in 20% sucrose (B) and inhibits formation of mCherry-Atg8a-positive autophagosomes in response to 2 h starvation (C). Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi;


( D and E ) RNAi against SH3PX1 inhibits formation of LTR ( D ) and mCherry - Atg8a - positive structures ( E ) at early stages of developmental autophagy in fat bodies of 110 - h - old larvae . Genotypes : ( B and D ) hs - flp ; UAS - dicer / + ; Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ; ( C and E ) hs - flp ; UAS - dicer / + ; r4 : mCherry - Atg8a Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ;

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

(D and E) RNAi against SH3PX1 inhibits formation of LTR (D) and mCherry-Atg8a-positive structures (E) at early stages of developmental autophagy in fat bodies of 110-h-old larvae. Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi;


( F ) There was no change in mCherry - Atg8a in SH3PX1 mutant clones ( GFP negative , outlined ) in well - fed larvae . Genotypes : ( F and G ) hs - flp ; Cg - GAL4 UAS - mChAtg8a / + ; FRT80B UAS - 2XeGFP / FRT80B SH3PX1HK62b .

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

(F) There was no change in mCherry-Atg8a in SH3PX1 mutant clones (GFP negative, outlined) in well-fed larvae. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B UAS-2XeGFP/FRT80B SH3PX1HK62b.


( G ) Decreased formation of mCherry - Atg8a - positive autophagosomes in SH3PX1 mutant clones ( GFP negative , outlined ) in fat bodies from larvae starved in 20 % sucrose for 3 h . Bars , 20 µm . ( H ) The graph shows quantification of total mCherry - Atg8a spot intensity per cell for WT and SH3PX1 mutant cells . The graph shows mean ± SEM ( error bars ) ; * * * , P < 0 . 001 . Genotypes : ( F and G ) hs - flp ; Cg - GAL4 UAS - mChAtg8a / + ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Decreased formation of mCherry-Atg8a-positive autophagosomes in SH3PX1 mutant clones (GFP negative, outlined) in fat bodies from larvae starved in 20% sucrose for 3 h. Bars, 20 µm. (H) The graph shows quantification of total mCherry-Atg8a spot intensity per cell for WT and SH3PX1 mutant cells. The graph shows mean ± SEM (error bars); ***, P < 0.001. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B


( B ) Microarray heat map showing the upregulation in expression of the 11 candidate genes in β cell - ablated versus control islets . Igfbp1a and b were the genes whose expression increased the most after β - cell ablation .

O O O O O O O O O O O O O O O O O O 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) Microarray heat map showing the upregulation in expression of the 11 candidate genes in β cell-ablated versus control islets. Igfbp1a and b were the genes whose expression increased the most after β-cell ablation.


( D - E ) Representative images at 6 dpf of Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenic larvae that had been injected at the 1 - 2 - cell stage with transposase mRNA ( control ) or transposase mRNA + bactin : igfbp1a ( bactin : igfbp1a ) , subjected to β - cell ablation by metronidazole ( MTZ ) during 3 - 4 dpf , and subsequently allowed to regenerate for 2 days . The GFP + heart ( arrowhead ) visualizes successful integration of the construct . Islets are indicated by white arrows . Scale bars : 100 μm . ( F ) Quantification of β - cell regeneration at 6 dpf in control ( n = 23 ) , bactin : igfbp1a - overexpressing ( n = 13 ) , and bactin : igfbp1b - overexpressing ( n = 8 ) Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) larvae ; * * * P = 0 . 0002 , ns = nonsignificant ( P = 0 . 3106 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Representative images at 6 dpf of Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae that had been injected at the 1-2-cell stage with transposase mRNA (control) or transposase mRNA + bactin:igfbp1a (bactin:igfbp1a), subjected to β-cell ablation by metronidazole (MTZ) during 3-4 dpf, and subsequently allowed to regenerate for 2 days. The GFP+ heart (arrowhead) visualizes successful integration of the construct. Islets are indicated by white arrows. Scale bars: 100 μm. (F) Quantification of β-cell regeneration at 6 dpf in control (n=23), bactin:igfbp1a-overexpressing (n=13), and bactin:igfbp1b-overexpressing (n=8) Tg(ins:kaede);Tg(ins:CFP-NTR) larvae; ***P=0.0002, ns= nonsignificant (P=0.3106).


( G ) Immunohistochemistry showing Igfbp1 protein expression in 6 dpf Tg ( ins : GFP ) following β - cell ablation between 3 - 4 dpf . Scale bar : 50 μm .

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

(G) Immunohistochemistry showing Igfbp1 protein expression in 6 dpf Tg(ins:GFP) following β-cell ablation between 3-4 dpf. Scale bar: 50 μm.


( H ) Both liver - specific ( lfabp promoter ; n = 46 ) and widespread ( bactin promoter ; n = 18 ) overexpression of igfbp1a increase β - cell regeneration when compared to control ( n = 77 ) .

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

(H) Both liver-specific (lfabp promoter; n=46) and widespread (bactin promoter; n=18) overexpression of igfbp1a increase β-cell regeneration when compared to control (n=77).


( I - M ) Quantification of β cells with or without β - cell ablation and igfbp1a overexpression by confocal microscopy , which detects even weakly insulin - expressing β cells . Scale bars : 15 μm . See also Figure EV1 .

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

(I-M) Quantification of β cells with or without β-cell ablation and igfbp1a overexpression by confocal microscopy, which detects even weakly insulin-expressing β cells. Scale bars: 15 μm. See also Figure EV1.


( A - C ) Igfbp1a promotes β cell regeneration , rather than β cell survival . To trace β cells , we exposed Tg ( ins : kaede ) larvae to UV light ( which causes the existing Kaede protein to switch from emitting green fluorescence to emitting red fluorescence ) just before ablating the β cells by MTZ treatment from 3 - 4 dpf . After regeneration , the newly formed β cells are green , whereas the β cells that survived ablation are yellow ( overlap of green and red ) . ( A & amp ; amp ; B ) Representative confocal images at 6 dpf of control and bactin : igfbp1a - overexpressing Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenic larvae ; arrows indicate surviving ( yellow ) β cells . Scale bars : 10 μm . ( C ) Quantification of β cell regeneration ( green bars ) and β cell survival ( yellow bars ) per larva at 6 dpf . * * * P < 0 . 001 : n = 20 larvae in the control group ; n = 27 larvae in the bactin : igfbp1a - overexpressing group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Igfbp1a promotes β cell regeneration, rather than β cell survival. To trace β cells, we exposed Tg(ins:kaede) larvae to UV light (which causes the existing Kaede protein to switch from emitting green fluorescence to emitting red fluorescence) just before ablating the β cells by MTZ treatment from 3-4 dpf. After regeneration, the newly formed β cells are green, whereas the β cells that survived ablation are yellow (overlap of green and red). (A&amp;B) Representative confocal images at 6 dpf of control and bactin:igfbp1a-overexpressing Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae; arrows indicate surviving (yellow) β cells. Scale bars: 10 μm. (C) Quantification of β cell regeneration (green bars) and β cell survival (yellow bars) per larva at 6 dpf. ***P<0.001: n=20 larvae in the control group; n=27 larvae in the bactin:igfbp1a-overexpressing group.


( D - F ) Igfbp1a does not promote δ - cell regeneration . We treated control and bactin : igfbp1a - overexpressing Tg ( sst : flag - NTR ) ; Tg ( sst : dsRed ) larvae with MTZ from 3 - 4 dpf to ablate δ cells , and then allowed them to regenerate until 6 dpf . ( D & amp ; amp ; E ) Representative confocal images at 6 dpf of control and bactin : igfbp1a - overexpressing larvae showing comparable number of δ cells after 2 days of regeneration . Scale bars : 15 μm . ( F ) Quantification of the total number of δ cells per δ - cell ablated larva at 6 dpf compared to the baseline number of δ cells in non - ablated control larvae . P = 0 . 2325 . n = 13 in the control group , n = 7 in the bactin : igfbp1a group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Igfbp1a does not promote δ-cell regeneration. We treated control and bactin:igfbp1a-overexpressing Tg(sst:flag-NTR);Tg(sst:dsRed) larvae with MTZ from 3-4 dpf to ablate δ cells, and then allowed them to regenerate until 6 dpf. (D&amp;E) Representative confocal images at 6 dpf of control and bactin:igfbp1a-overexpressing larvae showing comparable number of δ cells after 2 days of regeneration. Scale bars: 15 μm. (F) Quantification of the total number of δ cells per δ-cell ablated larva at 6 dpf compared to the baseline number of δ cells in non-ablated control larvae. P=0.2325. n=13 in the control group, n=7 in the bactin:igfbp1a group.


( G ) Other Igfbps do not promote β - cell regeneration . We injected Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenics with either transposase mRNA ( control ; n = 31 ) or transposase mRNA + one of six different igfbps , igfbp2a ( n = 25 ) , igfbp2b ( n = 25 ) , igfbp3 ( n = 26 ) , igfbp5a ( n = 17 ) , igfbp6b ( n = 34 ) , and igfbp7 ( n = 17 ) , treated them with MTZ from 3 - 4 dpf to ablate their β cells , and then quantified their β cells after 2 days of regeneration , at 6 dpf ; ns = nonsignificant ; ( igfbp2aP = 0 . 986 , igfbp2bP = 0 . 037 , igfbp3P = 0 . 979 , igfbp5aP = 0 . 999 , igfbp6bP = 0 . 997 , and igfbp7P = 0 . 999 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Other Igfbps do not promote β-cell regeneration. We injected Tg(ins:kaede);Tg(ins:CFP-NTR) transgenics with either transposase mRNA (control; n=31) or transposase mRNA + one of six different igfbps, igfbp2a (n=25), igfbp2b (n=25), igfbp3 (n=26), igfbp5a (n=17), igfbp6b (n=34), and igfbp7 (n=17), treated them with MTZ from 3-4 dpf to ablate their β cells, and then quantified their β cells after 2 days of regeneration, at 6 dpf; ns=nonsignificant; (igfbp2aP=0.986, igfbp2bP=0.037, igfbp3P=0.979, igfbp5aP=0.999, igfbp6bP=0.997, and igfbp7P=0.999).


( H ) Tg ( pcsk1 : GFP ) is expressed in regenerating β cells within the islet ( arrowheads ) , but not outside the islet ( arrow ) , at 6 dpf in larvae overexpressing igfbp1a . Scale bar : 15 μm .

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

(H) Tg(pcsk1:GFP) is expressed in regenerating β cells within the islet (arrowheads), but not outside the islet (arrow), at 6 dpf in larvae overexpressing igfbp1a. Scale bar: 15 μm.


( I & amp ; amp ; J ) Free - glucose levels during β - cell regeneration in control ( bactin : mCherry ) and bactin : igfbp1a - overexpressing Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) larvae ( I ) , as well as in Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) larvae injected in the pericardial sac at 4 dpf with 1 ng of recombinant mouse Igfbp1 ( J ) . We treated larvae with MTZ from 3 - 4 dpf to ablate their β cells , and monitored their free - glucose levels at 3 - 7 dpf . Free - glucose levels were significantly lower after genetic igfbp1a overexpression or Igfbp1 - protein injection ( red lines ) than in controls ( black lines ) at 7 and 6 dpf , respectively . Baseline reference levels of free glucose throughout development are shown for a different set of larvae without β - cell ablation . n = 24 larvae ( four pools of six larvae ) per data point .

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

(I&amp;J) Free-glucose levels during β-cell regeneration in control (bactin:mCherry) and bactin:igfbp1a-overexpressing Tg(ins:kaede);Tg(ins:CFP-NTR) larvae (I), as well as in Tg(ins:kaede);Tg(ins:CFP-NTR) larvae injected in the pericardial sac at 4 dpf with 1 ng of recombinant mouse Igfbp1 (J). We treated larvae with MTZ from 3-4 dpf to ablate their β cells, and monitored their free-glucose levels at 3-7 dpf. Free-glucose levels were significantly lower after genetic igfbp1a overexpression or Igfbp1-protein injection (red lines) than in controls (black lines) at 7 and 6 dpf, respectively. Baseline reference levels of free glucose throughout development are shown for a different set of larvae without β-cell ablation. n=24 larvae (four pools of six larvae) per data point.


( A - C ) Tg ( ins : GFP ) ; Tg ( tp1 : H2B - mCherry ) ; Tg ( ins : Flag - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , were treated with MTZ from 3 - 4 dpf to ablate the β cells , and were then allowed to regenerate from 4 - 6 dpf . Representative confocal images at 6 dpf of control ( A ) and Tg ( bactin : igfbp1a ) ( B ) larvae showing a modest number of ins + tp1 + co - expressing cells , indicated by arrows , after 2 days of regeneration . Scale bars : 15 μm . ( C ) Quantification of the total number of β cells ( green bars ) at 6 dpf , and of β cells expressing Tg ( tp1 : H2B - mCherry ) , i . e . , of ductal origin ( yellow bars ) . * * * * P < 0 . 0001 , ns = nonsignificant . n = 23 larvae in the control group , n = 17 larvae in the Tg ( bactin : igfbp1a ) group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Tg(ins:GFP);Tg(tp1:H2B-mCherry);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3-4 dpf to ablate the β cells, and were then allowed to regenerate from 4-6 dpf. Representative confocal images at 6 dpf of control (A) and Tg(bactin:igfbp1a) (B) larvae showing a modest number of ins+ tp1+ co-expressing cells, indicated by arrows, after 2 days of regeneration. Scale bars: 15 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells expressing Tg(tp1:H2B-mCherry), i.e., of ductal origin (yellow bars). ****P<0.0001, ns= nonsignificant. n=23 larvae in the control group, n=17 larvae in the Tg(bactin:igfbp1a) group.


( D - F ) Tg ( ins : Venus - zGeminin ) was examined in control and Tg ( bactin : igfbp1a ) larvae at 6 dpf , after β - cell ablation 3 - 4 dpf by using Tg ( ins : Flag - NTR ) . ( D - E ) Representative confocal images at 6 dpf of control and Tg ( bactin : igfbp1a ) larvae showing cell cycle activation of β cells in green . Scale bars : 15 μm . ( F ) Quantification of the total number of β cells with activated cell cycle at 6 dpf , * * * P < 0 . 001 , n = 32 larvae in the control group , n = 41 larvae in the Tg ( bactin : igfbp1a ) group . The number of β cells with activated cell cycle are displayed together with the total number of β cells in experiments with the same setup , * * * * P < 0 . 0001 . n = 39 larvae in the control group , n = 33 larvae in the Tg ( bactin : igfbp1a ) group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Tg(ins:Venus-zGeminin) was examined in control and Tg(bactin:igfbp1a) larvae at 6 dpf, after β-cell ablation 3-4 dpf by using Tg(ins:Flag-NTR). (D-E) Representative confocal images at 6 dpf of control and Tg(bactin:igfbp1a) larvae showing cell cycle activation of β cells in green. Scale bars: 15 μm. (F) Quantification of the total number of β cells with activated cell cycle at 6 dpf, ***P<0.001, n=32 larvae in the control group, n=41 larvae in the Tg(bactin:igfbp1a) group. The number of β cells with activated cell cycle are displayed together with the total number of β cells in experiments with the same setup, ****P<0.0001. n=39 larvae in the control group, n=33 larvae in the Tg(bactin:igfbp1a) group.


( G - I ) EdU was used as a marker for cell cycle progression . Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , were treated with MTZ from 3 - 4 dpf to ablate their β cells , and subsequently incubated with EdU during regeneration from 4 - 6 dpf . ( G - H ) Representative confocal images at 6 dpf of control and Tg ( bactin : igfbp1a ) larvae showing β cells in green and the β cells that had incorporated EdU in yellow ( green and red overlap ; arrowheads ) . Scale bars : 20 μm . ( I ) Quantification of the total number of β cells ( green bars ) at 6 dpf , and of β cells that incorporated EdU ( yellow bars ) during β - cell regeneration from 4 - 6 dpf . * * P < 0 . 01 , ns = nonsignificant . n = 16 in both the control and the Tg ( bactin : igfbp1a ) group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) EdU was used as a marker for cell cycle progression. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3-4 dpf to ablate their β cells, and subsequently incubated with EdU during regeneration from 4-6 dpf. (G-H) Representative confocal images at 6 dpf of control and Tg(bactin:igfbp1a) larvae showing β cells in green and the β cells that had incorporated EdU in yellow (green and red overlap; arrowheads). Scale bars: 20 μm. (I) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells that incorporated EdU (yellow bars) during β-cell regeneration from 4-6 dpf. **P<0.01, ns= nonsignificant. n=16 in both the control and the Tg(bactin:igfbp1a) group.


( J - L ) Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , were injected with a control morpholino or a morpholino that knocked down arx , which is necessary for α - cell differentiation and thus glucagon expression ( J - K ) . Scale bars : 20 μm . ( L ) Quantification of the total number of regenerating β cells at 4 dpf ( after β - cell ablation at 2 - 3 dpf ) . * * * * P < 0 . 0001 , * P < 0 . 05 , ns = nonsignificant . n = 42 , 20 , 48 and 20 , respectively .

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

(J-L) Tg(ins:kaede);Tg(ins:CFP-NTR) transgenics, with or without Tg(bactin:igfbp1a), were injected with a control morpholino or a morpholino that knocked down arx, which is necessary for α-cell differentiation and thus glucagon expression (J-K). Scale bars: 20 μm. (L) Quantification of the total number of regenerating β cells at 4 dpf (after β-cell ablation at 2-3 dpf). ****P<0.0001, *P<0.05, ns=nonsignificant. n=42, 20, 48 and 20, respectively.


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 - 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 .

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

(F-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 - 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 .

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

(F-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 - 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 .

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

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

(I-K) 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 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 .

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

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

(A-B) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 bacteria on a cellulose filter . Scale bars : 500 nm .

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


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

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


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 ( * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the 20D9 monoclonal antibody and the F10 nanobody .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

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

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 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 .

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

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 .

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


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 ( * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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 ) 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 ( * ) .

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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 the area of the tubulin cytoskeleton . Tubulin ( green ) and cell nucleus ( blue ) . Objective x20 . Bar : 200 µm .

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) 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 .

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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 ) 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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

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 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 ) .

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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 ) 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 ) .

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

(A-B) 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 ) and lung adenocarcinoma ( n = 719 ) ( B ) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier - plotter [ 25 ] .

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

(A-B) 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 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 .

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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(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 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 .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 the isotype and αCD4 + αCD8 group ( Isotype n = 19 , αCD4 + αCD8 n = 19 ) .

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

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


( 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 ) .

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

(H) 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 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 ) .

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

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(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 ) 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 .

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

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


( G ) 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 ) .

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

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


( K ) Representative 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 ) .

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

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


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

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


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

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


( 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 ) .

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(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 and total numbers ( mean + SEM ) of CD11b + myeloid cells isolated from the spleen and peripheral LN of naïve non - immunized mice .

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

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


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

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


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

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


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

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


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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . Analysis of IL - 1R1 expression by different populations of human ( E ) CD4 T cells and ( F ) Th17 cells isolated from the peripheral blood . Cells ( E - F ) were restimulated with PMA / Ionomycin for 4 h . Data consist of PBMC isolated from n = 4 healthy individuals . Data ( E ) are FACS plots of cells gated on CD14 - CD3 + CD4 + cells with mean frequencies per group and quantifications shown as bar diagram ( mean + SEM ) . Data ( B , D , F ) are FACS plots of Th17 cells and histograms of IL - 1R1 staining of indicated cell populations with mean fluorescent intensity ( MFI ) ± SEM or mean frequencies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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

E-F. Analysis of IL-1R1 expression by different populations of human (E) CD4 T cells and (F) Th17 cells isolated from the peripheral blood.Cells (E-F) were restimulated with PMA/Ionomycin for 4 h. Data consist of PBMC isolated from n=4 healthy individuals.Data (E) are FACS plots of cells gated on CD14-CD3+CD4+cells with mean frequencies per group and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.


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

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

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


B . Frequencies and total numbers ( mean + SEM ) of MOG - specific Th17 cells isolated from the dLN . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results .

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

B. Frequencies and total numbers (mean +SEM) of MOG-specific Th17 cells isolated from the dLN.Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.


C - D . Frequencies and total numbers ( mean + SEM ) of MOG - specific GM - CSF + Th17 cells isolated from the spleen ( C ) and dLN ( D ) . Cells ( A - D ) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h . Data consist of n = 5 WT PBS , n = 4 WT PTx , n = 4 IL - 1R1 - / - PBS , n = 4 IL - 1R1 - / - PTx , MOG / CFA immunized mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed at least twice with similar results .

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

C-D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+ Th17 cells isolated from the spleen (C) and dLN (D).Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.


A . Analysis of CD4T cells isolated from the dLN and restimulated with PMA / Ionomycin for 4 h . Data are representative FACS plots , gated on VD - CD4 + cells with mean frequencies per group . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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

A. Analysis of CD4T cells isolated from the dLN and restimulated with PMA/Ionomycin for 4 h. Data are representative FACS plots, gated on VD-CD4+cells with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.


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

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

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


C . Total cell numbers ( mean + SEM ) of MOG - specific Th17 cells isolated from the spleen , shown in ( B , upper row ) . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

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

C. Total cell numbers (mean +SEM) of MOG-specific Th17 cells isolated from the spleen, shown in (B, upper row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.


D . Frequencies and total numbers ( mean + SEM ) of MOG - specific GM - CSF + IFNγ + / - Th17 cells isolated from the spleen , shown in ( B , lower row ) . Cells ( A - D ) were isolated at day 9 after MOG / CFA / PTx immunization . Data consist of n = 5 mice of each genotype . * p < 0 . 05 , * * p < 0 . 01 , N . S . - not significant ; two - tailed unpaired t - test . Experiments were performed twice with similar results .

D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+IFNγ+/- Th17 cells isolated from the spleen, shown in (B, lower row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.


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

A-C. (A) Mean, (B) maximal EAE clinical scores and (C) area under the curve (AUC) of mice immunized with MOG/CFA/PTx. Data shown as (A) mean (+SEM), (B, C) individual plots with mean and are combined from at least four independent experiments and consist of n=41 WT, n=24 IL-1R1ΔT, n=18 IL-1R1-/-mice. *p < 0.05, ***p < 0.001; two-tailed unpaired t-test. Black (*) signs in (A) represent statistical analysis of IL-1R1ΔT vs WT and blue (*) of IL-1R1ΔT vs IL-1R1-/- groups.


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

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


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

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