figf2 ( a ) BCBL cells were used for immunoprecipitation ( IP ) with mouse IgG or anti - Atg3 , followed by immunoblotting ( IB ) with anti - cFLIP antibody ( left panel ) . NIH3T3 - KSHV - vFLIP cells were used for IP with mouse IgG or anti - Flag antibody , followed by IB with anti - Atg3 antibody ( right panel ) . ( b ) FLIP and Atg3 interaction after autophagy induction . HCT116 cells were treated with or without 2 μM rapamycin or Hank ' s solution ( HBSS ) for 4 h before IP with anti - Atg3 antibody , followed by IB with anti - cFLIP antibody . ( c ) Schematic diagram of human Atg3 ( top ) . N , amino terminus ; FR , flexible region ; C , carboxy terminus . At 48 h post - transfection with GST or GST - Atg3 along with Flag - vFLIP ( left ) or GST or GST - Atg3 along with GFP - LC3 ( right ) , HEK293T cells were used for GST pulldown , followed by IB with the indicated antibodies . ( d ) Schematic diagram of KSHV vFLIP ( top ) . The black boxes indicate the K - α2 and K - α4 peptides . At 48 h post - transfection with V5 - Atg3 and either GST - vFLIP DED1 mutants ( middle ) or GST - vFLIP DED2 mutants ( bottom ) , HEK293T cells were used for GST pulldown , followed by IB with anti - V5 antibody . ( e ) At 48 h post - transfection with GST , GST - Atg3 , and either Flag - vFLIP or Flag - vFLIP mAtg3 , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag antibody . ( f ) At 48 h post - transfection with GST - Atg3 and GFP - LC3 along with increasing amount of Flag - vFLIP , HEK293T cells were used for GST pulldown , followed by IB with anti - GFP or anti - Flag antibody . ( g ) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti - Atg3 antibody , followed by IB with anti - LC3 antibody . Finally , whole - cell lysates ( WCLs ) were used for IB with the indicated antibodies to show expression ( a - g ) .

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

figf2(a) BCBL cells were used for immunoprecipitation (IP) with mouse IgG or anti-Atg3, followed by immunoblotting (IB) with anti-cFLIP antibody (left panel). NIH3T3-KSHV-vFLIP cells were used for IP with mouse IgG or anti-Flag antibody, followed by IB with anti-Atg3 antibody (right panel).(b) FLIP and Atg3 interaction after autophagy induction. HCT116 cells were treated with or without 2 μM rapamycin or Hank's solution (HBSS) for 4 h before IP with anti-Atg3 antibody, followed by IB with anti-cFLIP antibody.(c) Schematic diagram of human Atg3 (top). N, amino terminus; FR, flexible region; C, carboxy terminus. At 48 h post-transfection with GST or GST-Atg3 along with Flag-vFLIP (left) or GST or GST-Atg3 along with GFP-LC3 (right), HEK293T cells were used for GST pulldown, followed by IB with the indicated antibodies.(d) Schematic diagram of KSHV vFLIP (top). The black boxes indicate the K-α2 and K-α4 peptides. At 48 h post-transfection with V5-Atg3 and either GST-vFLIP DED1 mutants (middle) or GST-vFLIP DED2 mutants (bottom), HEK293T cells were used for GST pulldown, followed by IB with anti-V5 antibody.(e) At 48 h post-transfection with GST, GST-Atg3, and either Flag-vFLIP or Flag-vFLIP mAtg3, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag antibody.(f) At 48 h post-transfection with GST-Atg3 and GFP-LC3 along with increasing amount of Flag-vFLIP, HEK293T cells were used for GST pulldown, followed by IB with anti-GFP or anti-Flag antibody.(g) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti-Atg3 antibody, followed by IB with anti-LC3 antibody. Finally, whole-cell lysates (WCLs) were used for IB with the indicated antibodies to show expression (a-g).


figf3 ( a ) TREX - BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy . The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis , autophagic death , apoptosis / autophagic death , and others forms of cell death . ( b ) At 12 - 16 h post - transfection with GFP - LC3 , doxycycline - treated TREX - BCBL vector , TREX - BCBL - vFLIP and TREX - BCBL - vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days ( top ) or 2 μM rapamycin for 12 h ( bottom ) . The percentage of cell death ( trypan blue staining ) and apoptosis level ( annexin V staining ) of rapamycin - treated cells was quantified as mean ± s . d . of three independent experiments . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope . Data are mean ± s . e . m . ; n = 200 - 300 cells ; three independent experiments ; * P < 0 . 01 . ( c ) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days . The percentage of cell death was determined by trypan blue staining . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 05 . ( d ) TREX - BCBL vector and TREX - BCBL - vFLIP cells were mock - treated or treated with 50 nM rapamycin for the indicated periods of time . The results were quantified as mean ± s . d . of the combined results from three independent experiments ; * P 0 . 001 . ( e ) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days . The results were quantified as mean ± s . d . of the combined results from three independent experiments ; * P 0 . 01 . Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer ( BC Z2 CS analyser ) were used to determine cell death and cell growth analysis ( b - e ) .

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

figf3(a) TREX-BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy. The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis, autophagic death, apoptosis/autophagic death, and others forms of cell death.(b) At 12-16 h post-transfection with GFP-LC3, doxycycline-treated TREX-BCBL vector, TREX-BCBL-vFLIP and TREX-BCBL-vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days (top) or 2 μM rapamycin for 12 h (bottom). The percentage of cell death (trypan blue staining) and apoptosis level (annexin V staining) of rapamycin-treated cells was quantified as mean ± s.d. of three independent experiments. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P<0.01.(c) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days. The percentage of cell death was determined by trypan blue staining. Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.05.(d) TREX-BCBL vector and TREX-BCBL-vFLIP cells were mock-treated or treated with 50 nM rapamycin for the indicated periods of time. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.001.(e) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.01. Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer (BC Z2 CS analyser) were used to determine cell death and cell growth analysis (b-e).


figf4 ( b ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 30 μM of the TAT , K - α2 or K - α4 peptide ( top left ) or the TAT , C - α2 , or C - α4 peptide ( bottom left ) for an additional 12 h . Subsequently , autophagy was quantified as means ± s . d . of the combined results from three independent experiments ( 2 μM rapamycin treatment was included as a control ) . TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 0 , 30 , or 50 μM of the TAT , K - α2 , or K - α4 peptide ( top right ) or the TAT , C - α2 , or C - α4 peptide ( bottom right ) for an additional 12 h . Trypan blue staining was then used to determine cell death ( as a percentage ) . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 01 ; * * P 0 . 05 . ( c ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 30 μM of the TAT , K - α2 or K - α4 peptide ( top left ) or the TAT , C - α2 , or C - α4 peptide ( bottom left ) for an additional 12 h . Subsequently , autophagy was quantified as means ± s . d . of the combined results from three independent experiments ( 2 μM rapamycin treatment was included as a control ) . TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 0 , 30 , or 50 μM of the TAT , K - α2 , or K - α4 peptide ( top right ) or the TAT , C - α2 , or C - α4 peptide ( bottom right ) for an additional 12 h and GFP - LC3 puncta were subsequently detected using an inverted fluorescence microscope . Scale bar , 5 μm . ( d ) KSHV - infected BCBL1 cells were treated with 30 μM of the K - α2 or the K - α4 peptide for 12 h and subjected to scanning electron microscopy . Scale bar , 50 nm . ( e ) TREX - BCBL - Vector and TREX - BCBL - vFLIP cells were treated with the K - α2 and the K - α4 ( 30 μM each ) peptide for 12 h , followed by immunoblotting ( IB ) with anti - LC3 and anti - actin antibody . ( f ) NOD / SCID mice received an injection of 5 × 106 BCBL1 - Luc cells , followed by intraperitoneal injections with 300 μg the TAT , K - α2 or Kα - 4 peptide for three weeks ( top three panels ) . After three weeks , a group of three mice treated with the TAT peptide was challenged with 300 μg of the K - α2 or Kα - 4 peptide for an additional three weeks ( bottom panel , see Supplementary Information , Fig . S15 ) . Tumours were measured by in vivo bioluminescence imaging .

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

figf4(b) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h. Trypan blue staining was then used to determine cell death (as a percentage). Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.01; **P 0.05.(c) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h and GFP-LC3 puncta were subsequently detected using an inverted fluorescence microscope. Scale bar, 5 μm.(d) KSHV-infected BCBL1 cells were treated with 30 μM of the K-α2 or the K-α4 peptide for 12 h and subjected to scanning electron microscopy. Scale bar, 50 nm.(e) TREX-BCBL-Vector and TREX-BCBL-vFLIP cells were treated with the K-α2 and the K-α4 (30 μM each) peptide for 12 h, followed by immunoblotting (IB) with anti-LC3 and anti-actin antibody.(f) NOD/SCID mice received an injection of 5 × 106 BCBL1-Luc cells, followed by intraperitoneal injections with 300 μg the TAT, K-α2 or Kα-4 peptide for three weeks (top three panels). After three weeks, a group of three mice treated with the TAT peptide was challenged with 300 μg of the K-α2 or Kα-4 peptide for an additional three weeks (bottom panel, see Supplementary Information, Fig. S15). Tumours were measured by in vivo bioluminescence imaging.


figf5 ( a ) At 16 h post - transfection with GST , GST - K - α2 or GST - K - α4 along with Flag - vFLIP ( top ) or Flag - cFLIPL ( bottom ) , HEK293T cells were used for GST pulldown , followed by immunoblotting ( IB ) with anti - Flag or anti - FLIP antibody . Whole - cell lysates ( WCLs ) were used for IB with anti - GST , anti - Flag or anti - FLIP antibody . ( b ) At 16 h post - transfection with V5 - Atg3 and Flag - vFLIP with increasing amounts of GST - K - α2 or GST - K - α4 , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag or anti - V5 antibody or for immunoprecipitation ( IP ) with anti - V5 antibody , followed by IB with anti - Flag antibody . WCLs were used for IB with anti - GST , anti - Flag or anti - V5 antibody . ( c ) At 12 - 16 h post - transfection with GST - Atg3 along with Flag - vFLIP ( first ) , Flag - cFLIPS ( second ) , or HA - LC3 ( third ) with increasing amounts of the K - α2 + K - α4 peptides ( top three panels ) or C - α2 + C - α4 peptides ( bottom panel ) , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag or anti - HA antibodies . WCLs were used for IB with anti - GST and anti - Flag antibody . ( d ) At 12 - 16 h post - transfection with Flag - vFLIP , HA - LC3 and V5 - Atg3 , HEK293T cells were treated with increasing amounts of the K - α2 + K - α4 peptides and used for IP anti - V5 antibody , followed by IB with anti - Flag or anti - HA antibody . WCLs were used for IB with anti - V5 , anti - HA and anti - Flag antibody .

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

figf5(a) At 16 h post-transfection with GST, GST-K-α2 or GST-K-α4 along with Flag-vFLIP (top) or Flag-cFLIPL (bottom), HEK293T cells were used for GST pulldown, followed by immunoblotting (IB) with anti-Flag or anti-FLIP antibody. Whole-cell lysates (WCLs) were used for IB with anti-GST, anti-Flag or anti-FLIP antibody.(b) At 16 h post-transfection with V5-Atg3 and Flag-vFLIP with increasing amounts of GST-K-α2 or GST-K-α4, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-V5 antibody or for immunoprecipitation (IP) with anti-V5 antibody, followed by IB with anti-Flag antibody. WCLs were used for IB with anti-GST, anti-Flag or anti-V5 antibody.(c) At 12-16 h post-transfection with GST-Atg3 along with Flag-vFLIP (first), Flag-cFLIPS (second), or HA-LC3 (third) with increasing amounts of the K-α2+K-α4 peptides (top three panels) or C-α2+C-α4 peptides (bottom panel), HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-HA antibodies. WCLs were used for IB with anti-GST and anti-Flag antibody.(d) At 12-16 h post-transfection with Flag-vFLIP, HA-LC3 and V5-Atg3, HEK293T cells were treated with increasing amounts of the K-α2+K-α4 peptides and used for IP anti-V5 antibody, followed by IB with anti-Flag or anti-HA antibody. WCLs were used for IB with anti-V5, anti-HA and anti-Flag antibody.


figf1 ( b ) IRGMC mRNA was significantly enriched in miR - 196B complexes . Extracts of cells expressing FLAG - tagged - AGO1 and transfected with biotinylated miR - 196B or control miR - 20 as indicated and then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification ( immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads ) . IRGM mRNA variants were quantified using quantitative RT - PCR ; results are presented as the ratio between miR - 196B and miR - 20 ( non - relevant miRNA ) pull - downs and the mean of three independent experiments ± standard deviation ( s . d . ) . IP strep , immunoprecipitation straptavidine . ( c ) HEK293 cells ( IRGMC / C ) were transfected with either FLAG - tagged IRGMC or FLAG - tagged IRGMT plasmids and co - transfected with miR - 196B . Immunoblotting with an IRGM antibody revealed the specificity of the downregulation effect mediated by the miRNA IRGM mRNA interaction . Quantification of the immunoblot signals are presented as IRGM expression relative to actin ( mean of at least three independent experiments ± s . d . ) .

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

figf1(b) IRGMC mRNA was significantly enriched in miR-196B complexes. Extracts of cells expressing FLAG-tagged-AGO1 and transfected with biotinylated miR-196B or control miR-20 as indicated and then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification (immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads). IRGM mRNA variants were quantified using quantitative RT-PCR; results are presented as the ratio between miR-196B and miR-20 (non-relevant miRNA) pull-downs and the mean of three independent experiments ± standard deviation (s.d.). IP strep, immunoprecipitation straptavidine.(c) HEK293 cells (IRGMC/C) were transfected with either FLAG-tagged IRGMC or FLAG-tagged IRGMT plasmids and co-transfected with miR-196B. Immunoblotting with an IRGM antibody revealed the specificity of the downregulation effect mediated by the miRNA IRGM mRNA interaction. Quantification of the immunoblot signals are presented as IRGM expression relative to actin (mean of at least three independent experiments ± s.d.).


figf2 ( a ) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls ( n = 40 ) or individuals with Crohn ' s disease ( CD ) ( n = 67 ) without or with active inflammation and labeled for miR - 196A . ( L , lumen ; LP , lamina propria ) . Scale bars in the upper panel , 25 μm ; scale bars in the lower panel , 10 μm . ( b ) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls ( n = 8 ) or individuals with Crohn ' s disease with no inflammation ( n = 16 ) , quiescent ( defined as low - grade inflammation ) ( n = 8 ) or acute inflammation ( n = 8 ) using laser capture microdissection . After RNA extraction , miR - 196A ( black bars ) and miR - 196B ( white bars ) relative expression was analyzed using RNU19 , 44 and U6 . To overcome possible inter - individual bias , the lamina propria fraction value was used for relative quantification . Due to high differences in expression between healthy and inflamed tissues , the results are presented as a log2 - fold ratio . Error bars indicate the s . d . of the ΔΔCt values . ( c ) Representative in situ staining for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn ' s disease with a defined genotype in a healthy non - inflamed or an acute inflamed phase . Scale bars , 32 μm . ( d ) Mean ( black line ) , s . e . m . ( white box ) and 95 % CI of the mean of the IRGM expression level for 40 healthy subjects ( 32 individuals with C / C and 8 individuals with C / T ) and 67 individuals with Crohn ' s disease ( 45 with C / C and 22 with C / T ) with quiescent or inflamed colon mucosa . We performed statistical analysis using ANOVA ( P = 0 . 0015 ) and an unpaired Student ' s t - test ( the one tail P value is indicated on the figure ) .

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

figf2(a) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls (n = 40) or individuals with Crohn's disease (CD) (n = 67) without or with active inflammation and labeled for miR-196A. (L, lumen; LP, lamina propria). Scale bars in the upper panel, 25 μm; scale bars in the lower panel, 10 μm.  (b) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls (n = 8) or individuals with Crohn's disease with no inflammation (n = 16), quiescent (defined as low-grade inflammation) (n = 8) or acute inflammation (n = 8) using laser capture microdissection. After RNA extraction, miR-196A (black bars) and miR-196B (white bars) relative expression was analyzed using RNU19, 44 and U6. To overcome possible inter-individual bias, the lamina propria fraction value was used for relative quantification. Due to high differences in expression between healthy and inflamed tissues, the results are presented as a log2-fold ratio. Error bars indicate the s.d. of the ΔΔCt values.(c) Representative in situ staining for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn's disease with a defined genotype in a healthy non-inflamed or an acute inflamed phase. Scale bars, 32 μm. (d) Mean (black line), s.e.m. (white box) and 95% CI of the mean of the IRGM expression level for 40 healthy subjects (32 individuals with C/C and 8 individuals with C/T) and 67 individuals with Crohn's disease (45 with C/C and 22 with C/T) with quiescent or inflamed colon mucosa. We performed statistical analysis using ANOVA (P = 0.0015) and an unpaired Student's t-test (the one tail P value is indicated on the figure).


figf3 ( a ) The basal flux of autophagy is affected by IRGM expression level . HEK293 cells transfected with an IRGM - expressing plasmid , miR - 196 or siIRGM were treated with bafilomycin A1 for 2 h and processed for immunoblotting with anti - LC3B . ( b ) Quantification of LC3 - II relative to actin ( mean of three independent experiments ± s . d . ) . ( c ) Downregulation of IRGM expression by miR - 196 abrogates AIEC - mediated autophagy in cells treated with autophagic inhibitors ( Inh ) or transfected with miR - 196B and infected for 4 h with AIEC LF82 ( mean of three independent experiments ± s . d . ) . ( d ) Confocal microscopic examination of LC3 revealed a significant decrease in the percentage of LC3 - associated ( red ) LF82 bacteria ( green ) in miR - 196 transfected cells compared to control cells ( mean ± s . d . ) . ( e ) miR - 196 transfection leads to increased intracellular LF82 replication . Results are expressed as a fold increase ± s . e . m . of intracellular bacteria . ( f ) IRGM overexpression did not inhibit autophagic flux and it increased LC3 - II accumulation slightly in response to AIEC infection . HEK293 cells were transfected with IRGM - expressing plasmid , treated with autophagic inhibitors and infected with AIEC bacteria for 4 h ( mean of three independent experiments ± s . d . ) . ( g ) Confocal microscopic examination showed an increased percentage of LC3 - associated AIEC bacteria in IRGM cells compared to control cells ( mean ± s . d . ) . ( h ) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria . ( i ) Most of the bacteria reside in non - acidic vacuoles , as shown with lysotracker at 8 h post infection ( means of three independent experiments ± s . d . ) .

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figf3(a) The basal flux of autophagy is affected by IRGM expression level. HEK293 cells transfected with an IRGM-expressing plasmid, miR-196 or siIRGM were treated with bafilomycin A1 for 2 h and processed for immunoblotting with anti-LC3B. (b) Quantification of LC3-II relative to actin (mean of three independent experiments ± s.d.).(c) Downregulation of IRGM expression by miR-196 abrogates AIEC-mediated autophagy in cells treated with autophagic inhibitors (Inh) or transfected with miR-196B and infected for 4 h with AIEC LF82 (mean of three independent experiments ± s.d.).(d) Confocal microscopic examination of LC3 revealed a significant decrease in the percentage of LC3-associated (red) LF82 bacteria (green) in miR-196 transfected cells compared to control cells (mean ± s.d.).(e) miR-196 transfection leads to increased intracellular LF82 replication. Results are expressed as a fold increase ± s.e.m. of intracellular bacteria.(f) IRGM overexpression did not inhibit autophagic flux and it increased LC3-II accumulation slightly in response to AIEC infection. HEK293 cells were transfected with IRGM-expressing plasmid, treated with autophagic inhibitors and infected with AIEC bacteria for 4 h (mean of three independent experiments ± s.d.).(g) Confocal microscopic examination showed an increased percentage of LC3-associated AIEC bacteria in IRGM cells compared to control cells (mean ± s.d.). (h) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria. (i) Most of the bacteria reside in non-acidic vacuoles, as shown with lysotracker at 8 h post infection (means of three independent experiments ± s.d.).


Figure 1Quantitative expression of Lif mRNA in the colon of DSS - challenged and control mice ( n = 6 per group ) . LIF measured by ELISA in mouse colon explant supernatants from DSS - challenged and control mice ( n = 4 per group ) . Quantitative expression of Lif mRNA in LPLs and IECs from DSS - challenged and control mice ( n = 4 per group ) . Quantitative expression of Lif mRNA in the colon of SPF mice treated with or without antibiotics ( Abx ) at the indicated time points during colitis induction ( n = 3 per group ) . Quantitative expression of Lif mRNA in LPLs and IECs from mice treated with or without Abx at the indicated time points during colitis induction ( n = 3 per group ) . mRNA expression of the indicated genes in IECs stimulated by LPS ( 2 . 5μg / ml ) , PGN ( 3μg / ml ) or LTA ( 2μg / ml ) for 2 hours . Serum concentrations of LIF in IEC supernatants untreated ( G ) followed by LPS ( 2 . 5μg / ml ) or LTA ( 2μg / ml ) for 12hours , as determined by ELISA . Serum concentrations of LIF in IEC supernatants pretreated with monensin ( H ) followed by LPS ( 2 . 5μg / ml ) or LTA ( 2μg / ml ) for 12hours , as determined by ELISA . Immunoblot analysis of the expression and modification of the indicated proteins in the total cell lysate of CD4 + T cells ( I ) and IECs ( J ) treated with LIF ( 20 ng / ml ) at different time points . The lower panel shows the intensity analysis of the bands from three independent experiments . Stat4 mRNA expression in LPLs and IECs ( n = 6 ) . The proliferation rate of CD4 + T cells ( L ) and DLD - 1 cells ( M ) was determined in the presence or absence of LIF .

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Figure 1Quantitative expression of Lif mRNA in the colon of DSS-challenged and control mice (n=6 per group).LIF measured by ELISA in mouse colon explant supernatants from DSS-challenged and control mice (n=4 per group).Quantitative expression of Lif mRNA in LPLs and IECs from DSS-challenged and control mice (n=4 per group).Quantitative expression of Lif mRNA in the colon of SPF mice treated with or without antibiotics (Abx) at the indicated time points during colitis induction (n=3 per group).Quantitative expression of Lif mRNA in LPLs and IECs from mice treated with or without Abx at the indicated time points during colitis induction (n=3 per group).mRNA expression of the indicated genes in IECs stimulated by LPS (2.5μg/ml), PGN (3μg/ml) or LTA (2μg/ml) for 2 hours.Serum concentrations of LIF in IEC supernatants untreated (G) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA.Serum concentrations of LIF in IEC supernatants pretreated with monensin (H) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA.Immunoblot analysis of the expression and modification of the indicated proteins in the total cell lysate of CD4+ T cells (I) and IECs (J) treated with LIF (20 ng/ml) at different time points. The lower panel shows the intensity analysis of the bands from three independent experiments.Stat4 mRNA expression in LPLs and IECs (n=6).The proliferation rate of CD4+ T cells (L) and DLD-1 cells (M) was determined in the presence or absence of LIF.


Figure 2Weight loss of wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . Macroscopic changes in the colon of the mice on day 10 of colitis induction Comparison of the colon length in the mice on day 10 of colitis inductionwild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . DAI ( stool consistency and bleeding score ) of colitis mice treatedwild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . H & E histology of representative colons from colitis mice . Scale bar , 20μm . Weight loss of Rag1 - / - ( n = 6 ) mice transferred with CD45RBhi CD4 + T cells and intraperitoneally injected with PBS or LIF for 8 weeks . Rag1 - / - ( n = 6 ) mice transferred with CD45RBhi CD4 + T cells and intraperitoneally injected with PBS or LIF for 8 weeks . Macroscopic changes in the colon of Rag1 - / - mice treated Comparison of colon lengths in the Rag1 - / - mice at week 8 ( n = 5 ) . Rag1 - / - ( n = 6 ) mice transferred with CD45RBhi CD4 + T cells and intraperitoneally injected with PBS or LIF for 8 weeks . H & E histology of representative mouse colons on day 54 of colitis

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Figure 2Weight loss of wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS.wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. Macroscopic changes in the colon of the mice on day 10 of colitis induction Comparison of the colon length in the mice on day 10 of colitis inductionwild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. DAI (stool consistency and bleeding score) of colitis mice treatedwild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. H&E histology of representative colons from colitis mice. Scale bar, 20μm.Weight loss of Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks.Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks. Macroscopic changes in the colon of Rag1-/- mice treated Comparison of colon lengths in the Rag1-/- mice at week 8 (n=5).Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks. H&E histology of representative mouse colons on day 54 of colitis induction


Figure 3Colons obtained from WT or Stat4 - KO mice on day10 of the colitis model induced as in Figure 2A were immunostained with an antibody against phosphorylated STAT4 . Scale bar , 50μm . Immunoblot analysis of STAT4 phosphorylation in spleen tissue from colitis mice . FACS staining of LPLs isolated on day10 from the colon of WT or Stat4 - KO colitis mice receiving PBS or LIF ( n = 4 per group ) . The percentage of IL - 17A + and / or IFNγ + CD4 + T cells in vivo was analyzed . Quantitative expression of Il17a mRNA in MLNs and LNs from colitis mice treatedFACS staining of WT and Stat4 - KO naïve CD4 + T cells treated with LIF ( 50 ng / ml ) on day 4 of induction into Th17 cell subsets . qPCR analysis of Th17 - related genes in WT or Stat4 - KO naïve CD4 + T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF ( n = 3 per group ) . FACS staining of WT or Stat4 - KO naïve CD4 + T cells treated with LIF ( 50 ng / ml ) on day 4 of induction into nonpathogenic or pathogenic Th17 cells .

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Figure 3Colons obtained from WT or Stat4-KO mice on day10 of the colitis model induced as in Figure 2A were immunostained with an antibody against phosphorylated STAT4. Scale bar, 50μm.Immunoblot analysis of STAT4 phosphorylation in spleen tissue from colitis mice.FACS staining of LPLs isolated on day10 from the colon of WT or Stat4-KO colitis mice receiving PBS or LIF (n=4 per group). The percentage of IL-17A+ and/or IFNγ+ CD4+ T cells in vivo was analyzed.Quantitative expression of Il17a mRNA in MLNs and LNs from colitis mice treatedFACS staining of WT and Stat4-KO naïve CD4+ T cells treated with LIF (50 ng/ml) on day 4 of induction into Th17 cell subsets.qPCR analysis of Th17-related genes in WT or Stat4-KO naïve CD4+ T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF (n=3 per group).FACS staining of WT or Stat4-KO naïve CD4+ T cells treated with LIF (50 ng/ml) on day 4 of induction into nonpathogenic or pathogenic Th17 cells.


Figure 4Immunoblot analysis of STAT4 phosphorylation in CD4 + T cells pretreated with PBS or LY2784544 ( 1μM ) for 12 hours followed by LIF for the indicated number of minutes . Immunoblot analysis of CD4 + T cells treated with LIF for the indicated number of minutes followed by immunoprecipitation with an anti - LIFR antibody . Coimmunoprecipitation and immunoblot analysis of LIF - treated HEK293T cells cotransfected with the indicated constructs . The red asterisks denote phosphorylated STAT bands . The mass spectrometry analysis of purified STAT4 protein revealed the phosphorylation of STAT4 at serine residues in the C terminus . Immunoblot analysis of the indicated protein modifications in the total lysate of CD4 + T cells treated with LIF ( 20 ng / ml ) at different time points . Coimmunoprecipitation and immunoblot analysis of LIF - treated HEK293T cells cotransfected with the indicated constructs . Coimmunoprecipitation and immunoblot analysis of LIF - treated HEK293T cells cotransfected with the indicated constructs . Coimmunoprecipitation and immunoblot analysis of LIF - treated HEK293T cells cotransfected with the indicated constructs . Immunoblot analysis of the distribution of the indicated proteins in the CD4 + T cell fraction . Cultured HeLa cells transfected with GFP - tagged STAT constructs were exposed to LIF and imaged 30 minutes later . Scale bar , 50μm .

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Figure 4Immunoblot analysis of STAT4 phosphorylation in CD4+ T cells pretreated with PBS or LY2784544 (1μM) for 12 hours followed by LIF for the indicated number of minutes.Immunoblot analysis of CD4+ T cells treated with LIF for the indicated number of minutes followed by immunoprecipitation with an anti-LIFR antibody.Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs. The red asterisks denote phosphorylated STAT bands.The mass spectrometry analysis of purified STAT4 protein revealed the phosphorylation of STAT4 at serine residues in the C terminus.Immunoblot analysis of the indicated protein modifications in the total lysate of CD4+ T cells treated with LIF (20 ng/ml) at different time points.Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs.Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs.Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs.Immunoblot analysis of the distribution of the indicated proteins in the CD4+ T cell fraction.Cultured HeLa cells transfected with GFP-tagged STAT constructs were exposed to LIF and imaged 30 minutes later. Scale bar, 50μm.


Figure 5Heatmap showing Th17 signature genes ( left ) or Th17 regulatory genes ( right ) up - or downregulated in WT or Stat4 - KO LPLs by LIF treatment ( n = 2 ) . Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice ( n = 3 per group ) . Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice ( n = 3 per group ) . Quantitative mRNA expression analysis of the indicated genes in WT or Stat4 - KO naïve CD4 + T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF . The GAS - luciferase reporter activity assay was performed in Stat3 - / - MEFs transfected with empty vector ( EV ) or STAT4 constructs and then treated with LIF for 8 hours . The GAS - luciferase reporter activity assay was performed in Stat3 - / - MEFs transfected with empty vector ( EV ) or STAT4 constructs and then treated with LIF for 8 hours . Relative SIE - ( I ) activities in Stat3 - / - MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours . Relative AGG - luciferase reporter activities in Stat3 - / - MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours . ChIP analysis of naïve CD4 + T cells obtained from IL - 17A - eGFP mice cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days . GFP + cells isolated by flow cytometry were restimulated with IL - 6 in the presence or absence of LIF , crosslinked with formaldehyde and immunoprecipitated with anti - STAT3 ( top ) or anti - STAT4 ( bottom ) antibodies , followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1 - p10 primer pairs . The results are presented relative to the amount of input DNA . ChIP analysis of naïve WT or Stat4 - KO CD4 + T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days . The cells were restimulated with IL - 6 or LIF for 30 minutes , crosslinked with formaldehyde and immunoprecipitated with the anti - STAT3 antibody , followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1 - p10 primer pairs . The results are presented relative to the amount of input DNA . Relative Il17a - SIE - ( left ) or AGG - luciferase reporter ( right ) activities in Stat3 - / - MEFs transfected with EV , STAT3 or STAT4 or cotransfected with STAT3 and STAT4 and then treated with IL - 6 or LIF for 8 hours . Relative Il17a promoter - luciferase reporter activities in Stat3 - / - MEFs transfected with the indicated constructs and then treated with LIF for 8 hours .

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Figure 5Heatmap showing Th17 signature genes (left) or Th17 regulatory genes (right) up- or downregulated in WT or Stat4-KO LPLs by LIF treatment (n=2).Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice (n=3 per group).Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice (n=3 per group).Quantitative mRNA expression analysis of the indicated genes in WT or Stat4-KO naïve CD4+ T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF.The GAS-luciferase reporter activity assay was performed in Stat3-/- MEFs transfected with empty vector (EV) or STAT4 constructs and then treated with LIF for 8 hours.The GAS-luciferase reporter activity assay was performed in Stat3-/- MEFs transfected with empty vector (EV) or STAT4 constructs and then treated with LIF for 8 hours.Relative SIE-(I) activities in Stat3-/- MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours.Relative AGG-luciferase reporter activities in Stat3-/- MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours.ChIP analysis of naïve CD4+ T cells obtained from IL-17A-eGFP mice cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. GFP+ cells isolated by flow cytometry were restimulated with IL-6 in the presence or absence of LIF, crosslinked with formaldehyde and immunoprecipitated with anti-STAT3 (top) or anti-STAT4 (bottom) antibodies, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.ChIP analysis of naïve WT or Stat4-KO CD4+ T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. The cells were restimulated with IL-6 or LIF for 30 minutes, crosslinked with formaldehyde and immunoprecipitated with the anti-STAT3 antibody, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.Relative Il17a-SIE- (left) or AGG-luciferase reporter (right) activities in Stat3-/- MEFs transfected with EV, STAT3 or STAT4 or cotransfected with STAT3 and STAT4 and then treated with IL-6 or LIF for 8 hours.Relative Il17a promoter-luciferase reporter activities in Stat3-/- MEFs transfected with the indicated constructs and then treated with LIF for 8 hours.


Figure 6Ki67 and cleaved caspase3 staining of representative colons obtained from mice on day10 of colitis induction as described in Figure 2A . The number of proliferating cells per crypt in ( A ) ( upper panel ) or apoptotic cells per field in ( B ) was determined ( n = 5 per group , 5 crypts were counted per mouse ) . Scale bar , 50μm . Quantitative mRNA expression analysis of the indicated genes in colon tissues obtained from WT or Stat4 - KO colitis mice treatedImmunoblot analysis of the expression and modification of the indicated proteins in DLD - 1 cells treated with LIF ( 20 ng / ml ) for different durations . Immunoblot analysis of YAP expression in WT or Stat4 - KO IECs treated with LIF ( 20 ng / ml ) for different durations ; β - actin was used as a loading control ( top ) . The statistical analysis of YAP protein intensity is shown in the bottom panel . Relative YAP promoter - luciferase reporter activities in HEK293T cells transfected with EV , STAT3 or STAT4 and then treated with IL - 6 or LIF for 8 hours . Heatmap showing the expression profile of cell proliferation - related genes in LIF - treated or control IECs ( Ctrl ) ( K ) .

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Figure 6Ki67 and cleaved caspase3 staining of representative colons obtained from mice on day10 of colitis induction as described in Figure 2A. The number of proliferating cells per crypt in (A) (upper panel) or apoptotic cells per field in (B) was determined (n=5 per group, 5 crypts were counted per mouse). Scale bar, 50μm.Quantitative mRNA expression analysis of the indicated genes in colon tissues obtained from WT or Stat4-KO colitis mice treatedImmunoblot analysis of the expression and modification of the indicated proteins in DLD-1 cells treated with LIF (20 ng/ml) for different durations.Immunoblot analysis of YAP expression in WT or Stat4-KO IECs treated with LIF (20 ng/ml) for different durations; β-actin was used as a loading control (top). The statistical analysis of YAP protein intensity is shown in the bottom panel.Relative YAP promoter-luciferase reporter activities in HEK293T cells transfected with EV, STAT3 or STAT4 and then treated with IL-6 or LIF for 8 hours.Heatmap showing the expression profile of cell proliferation-related genes in LIF-treated or control IECs (Ctrl) (K).


Figure 7Principal coordinate analysis ( PCoA ) of the microbiota composition determined by 16S rRNA gene sequencing of day 0 and day 9 fecal specimens from mice treated Each symbol represents an individual mouse . PC1 and PC2 represent principal components 1 and 2 , respectively . The bars depict the average relative abundance of the bacterial phyla of the microbiota in fecal specimens , as determined by taxon - based analyses as described in ( A ) ( n = 5 per group ) . 16S rRNA sequencing analyses of the Proteobacteria distribution in feces from WT or Stat4 - KO colitis mice . ( n = 5 per group ) . Heatmap depicting the relative abundance of microbes at the family level ( top ) and the genus level ( bottom ) in the fecal specimens described in ( A ) ( n = 3 per group ) . qPCR of the 16S rRNA genes of microbes in the phylum Proteobacteria , family Enterobacteriaceae , and genera Escherichia - Shigella in the feces of the indicated mice on day0 and day10 ( n = 6 per group ) of colitis induction . Quantitative mRNA expression analysis of proinflammatory genes in the colon of mice ( n = 3 per group )

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Figure 7Principal coordinate analysis (PCoA) of the microbiota composition determined by 16S rRNA gene sequencing of day 0 and day 9 fecal specimens from mice treated Each symbol represents an individual mouse. PC1 and PC2 represent principal components 1 and 2, respectively.The bars depict the average relative abundance of the bacterial phyla of the microbiota in fecal specimens, as determined by taxon-based analyses as described in (A) (n=5 per group).16S rRNA sequencing analyses of the Proteobacteria distribution in feces from WT or Stat4-KO colitis mice. (n=5 per group).Heatmap depicting the relative abundance of microbes at the family level (top) and the genus level (bottom) in the fecal specimens described in (A) (n=3 per group).qPCR of the 16S rRNA genes of microbes in the phylum Proteobacteria, family Enterobacteriaceae, and genera Escherichia-Shigella in the feces of the indicated mice on day0 and day10 (n=6 per group) of colitis induction.Quantitative mRNA expression analysis of proinflammatory genes in the colon of mice (n=3 per group) treated


Figure 1E WT and C2βD1212A / D1212A mice were subjected to thromboembolic stroke model . Representatives T2 - weighted MRI of coronal section taken 24 hours after the onset of in situ clot formation by α - thrombin were shown . Graph quantification of infarct volume measurements . Data represent mean ± SEM ( n = 23 - 25 mice per group ; * * p < 0 . 01 , Unpaired t - test ) .

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Figure 1E WT and C2βD1212A/D1212A mice were subjected to thromboembolic stroke model. Representatives T2-weighted MRI of coronal section taken 24 hours after the onset of in situ clot formation by α-thrombin were shown. Graph quantification of infarct volume measurements. Data represent mean ± SEM (n=23-25 mice per group; **p<0.01, Unpaired t-test).


Figure 2WT and C2βD1212A / D1212A mice were subjected to tMCAO followed by 24h of reperfusion . ( B ) Protein expression of TNFα , IL - 1β and IL - 6 . Data represent mean ± SEM ( n = 4 mice per group ; * p = 0 . 05 ; Mann - Whitney test ) . Data represent mean ± SEM ( n = 5 - 7 mice per group ; * * p < 0 . 01 ; Mann - Whitney test ) .

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Figure 2WT and C2βD1212A/D1212A mice were subjected to tMCAO followed by 24h of reperfusion. (B) Protein expression of TNFα, IL-1β and IL-6. Data represent mean ± SEM (n=4 mice per group; *p=0.05; Mann-Whitney test). Data represent mean ± SEM (n=5-7 mice per group; **p<0.01; Mann-Whitney test).


Figure 3A Gene expression of TNFα ( n = 8 - 10 mice for WT and n = 6 mice for C2βD1212A / D1212A ) , IL - 1β ( n = 8 mice for WT and n = 10 - 12 mice for C2βD1212A / D1212A ) , IL - 6 ( n = 8 mice for WT and n = 10 - 12 mice for C2βD1212A / D1212A ) and VCAM - 1 ( n = 8 - 10 mice for WT and n = 10 mice for C2βD1212A / D1212A ) after 1 hour of ischemia without reperfusion ( right panel ) . Gene expression of IL - 6 ( n = 10 - 12 mice for WT and n = 8 mice for C2βD1212A / D1212A ) and VCAM - 1 ( n = 12 mice for WT and n = 10mice for C2βD1212A / D1212A ) after 1 hour of ischemia followed by 4 hours of reperfusion ( bottom , left panel ) on WT and C2βD1212A / D1212A mice . The mRNA levels are given as the fold increase normalized to PGK1 relative to the healthy contralateral hemisphere of WT mice . Data represent mean ± SEM ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , Mann - Whitney test or unpaired t - test , as accordance ) .

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Figure 3A Gene expression of TNFα (n=8-10 mice for WT and n=6 mice for C2βD1212A/D1212A), IL-1β (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A), IL-6 (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A) and VCAM-1 (n=8-10 mice for WT and n=10 mice for C2βD1212A/D1212A) after 1 hour of ischemia without reperfusion (right panel). Gene expression of IL-6 (n=10-12 mice for WT and n=8 mice for C2βD1212A/D1212A) and VCAM-1 (n=12 mice for WT and n=10mice for C2βD1212A/D1212A) after 1 hour of ischemia followed by 4 hours of reperfusion (bottom, left panel) on WT and C2βD1212A/D1212A mice. The mRNA levels are given as the fold increase normalized to PGK1 relative to the healthy contralateral hemisphere of WT mice. Data represent mean ± SEM (***p<0.001, **p<0.01, *p<0.05, Mann-Whitney test or unpaired t-test, as accordance).


Figure 4Representative photographs of coronal brain sections and graph quantification of infarct volume ( dashed white line ) measurements in bone marrow ( BM ) chimeric mice 24 hours after tMCAO . WT > WT : transplantation of WT BM into WT hosts ; C2βD1212A > WT : transplantation of C2βD1212A / D1212A BM into WT hosts ; C2βD1212A > C2βD1212A : transplantation of C2βD1212A / D1212A BM into C2βD1212A / D1212A hosts ; WT > C2βD1212A transplantation of WT BM into kinase - dead hosts . Data represent mean ± SEM ; n = 13 - 16 mice per group ; * p < 0 . 05 * * p < 0 . 01 ; * * * p < 0 . 001 vs WT > WT , Mann - Whitney test or unpaired t - test , as accordance .

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Figure 4Representative photographs of coronal brain sections and graph quantification of infarct volume (dashed white line) measurements in bone marrow (BM) chimeric mice 24 hours after tMCAO. WT>WT: transplantation of WT BM into WT hosts; C2βD1212A>WT: transplantation of C2βD1212A/D1212A BM into WT hosts; C2βD1212A>C2βD1212A: transplantation of C2βD1212A/D1212A BM into C2βD1212A/D1212A hosts; WT> C2βD1212A transplantation of WT BM into kinase-dead hosts. Data represent mean ± SEM; n=13-16 mice per group; *p<0.05 **p<0.01; ***p<0.001 vs WT>WT, Mann-Whitney test or unpaired t-test, as accordance.


Figure 5B Analysis of APPL1 , EEA1 , LAMP1 and Rab11a staining in sh - Control and sh - PI3KC2β hCMEC / D3 cells . White dotted lines delineate cells ; Scale bar , 10µm ; nuclei are shown in blue ( DAPI ) . Quantification was performed to determine number of positive vesicles per cell ( ImageJ software ) . Nuclei are shown in blue ( DAPI ) . Data represent mean ± SEM of cell fields randomly selected ( n = 13 - 20 for ( C ) and n = 11 - 20 for ( D ) ) . Data are collected from at least 3 - 4 independent experiments . For violin plots ( D ) , thick lines represent median and thin lines interquartile ; * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 , unpaired t - test or Mann - Whitney , as accordance .

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Figure 5B Analysis of APPL1, EEA1, LAMP1 and Rab11a staining in sh-Control and sh-PI3KC2β hCMEC/D3 cells. White dotted lines delineate cells; Scale bar, 10µm; nuclei are shown in blue (DAPI).Quantification was performed to determine number of positive vesicles per cell (ImageJ software). Nuclei are shown in blue (DAPI). Data represent mean ± SEM of cell fields randomly selected (n=13-20 for (C) and n=11-20 for (D)). Data are collected from at least 3-4 independent experiments. For violin plots (D), thick lines represent median and thin lines interquartile; ***p<0.001; **p<0.01; *p<0.05, unpaired t-test or Mann-Whitney, as accordance.


Figure 6 ( A ) Representative VE - cadherin staining and graph quantification of fluorescence intensity normalized to total number of cells per field ( DAPI labelling ) in sh - Control and sh - PI3KC2β hCMEC / D3 in resting condition and after 24 hours activation with 25 ng / ml of TNFα . Control normalized to 100 % . Scale bar , 15 µm . Data represent mean ± SEM of cell fields randomly selected ( dots n = 14 - 19 ) . Data are collected from 4 - 5 independent experiments ; * * * p < 0 . 001 , unpaired t - test . ( B ) Graph quantification of fluorescence intensity of VE - Cadherin normalized to total cell number per field ( DAPI labelling ) in control condition and after 5 hours in oxygen - glucose deprivation ( OGD ) condition . Control normalized to 100 % . Data represent mean ± SEM of cell fields randomly selected ( dots n = 16 ) . Data were collected from 5 independent experiments ) ; * * * p < 0 . 001 ; * * p < 0 . 01 , Mann - Whitney test .

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Figure 6(A) Representative VE-cadherin staining and graph quantification of fluorescence intensity normalized to total number of cells per field (DAPI labelling) in sh-Control and sh-PI3KC2β hCMEC/D3 in resting condition and after 24 hours activation with 25 ng/ml of TNFα. Control normalized to 100%. Scale bar, 15 µm. Data represent mean ± SEM of cell fields randomly selected (dots n=14-19). Data are collected from 4-5 independent experiments; ***p<0.001, unpaired t-test.(B) Graph quantification of fluorescence intensity of VE-Cadherin normalized to total cell number per field (DAPI labelling) in control condition and after 5 hours in oxygen-glucose deprivation (OGD) condition. Control normalized to 100%. Data represent mean ± SEM of cell fields randomly selected (dots n=16). Data were collected from 5 independent experiments);***p<0.001; **p<0.01, Mann-Whitney test.


Figure 7A Representative images of PI3P and PI ( 3 , 4 ) P2 labeling and quantification using mCherry - FYVEHRS and GFP - PH ( TAPP1 ) probes respectively in sh - Control or sh - PI3KC2β hCMEC / D3 . Scale bar , 10 µm . Nuclei are shown in blue ( DAPI ) . Staining intensity was quantified by ImageJ software and normalized to total number of cells ( DAPI labelling ) per field . Data represent mean ± SEM of cell fields randomly selected for PI3P ( dots n = 21 ) and for PI ( 3 , 4 ) P2 ( dots n = 14 - 16 ) . Data were collected from at least 5 independent experiments ; * * * p < 0 . 001 , unpaired t - test with Welch ' s correction . C Graph quantification of APPL1 - , EEA1 - and Rab11 - endosomes colocalized with mCitrin - VE - cadherin in sh - Control and sh - PI3KC2β hCMEC / D3 cells . Data are shown as percentage of total count endosomes per cell . Data are expressed as mean ± SEM ( n = 33 - 35 cells for APPL1 and EEA1 endosomes and n = 16 - 18 cells for Rab11 endosomes ; 3 independent experiments ) ; * * p < 0 . 001 , Mann - Whitney test or unpaired t - test , as accordance . D Representative confocal microscopic images of VE - cadherin immunostaining in sh - Control cells , sh - PI3KC2β cells or sh - PI3KC2β cells transduced with Rab11S25N ( Rab11 - DN ) . Graph quantification of VE - cadherin ; control normalized to 100 % . Scale bar , 10 µm . Data represent mean ± SEM of cell fields randomly selected ( dots n = 6 - 9 ) . Data are collected from at least 3 independent experiments ; * * p < 0 . 01 Mann - Whitney test .

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Figure 7A Representative images of PI3P and PI(3,4)P2 labeling and quantification using mCherry-FYVEHRS and GFP-PH(TAPP1) probes respectively in sh-Control or sh-PI3KC2β hCMEC/D3. Scale bar, 10 µm. Nuclei are shown in blue (DAPI). Staining intensity was quantified by ImageJ software and normalized to total number of cells (DAPI labelling) per field. Data represent mean ± SEM of cell fields randomly selected for PI3P (dots n=21) and for PI(3,4)P2 (dots n=14-16). Data were collected from at least 5 independent experiments; ***p<0.001, unpaired t-test with Welch's correction.C Graph quantification of APPL1-, EEA1- and Rab11- endosomes colocalized with mCitrin-VE-cadherin in sh-Control and sh-PI3KC2β hCMEC/D3 cells. Data are shown as percentage of total count endosomes per cell. Data are expressed as mean ± SEM (n=33-35 cells for APPL1 and EEA1 endosomes and n=16-18 cells for Rab11 endosomes; 3 independent experiments); **p<0.001, Mann-Whitney test or unpaired t-test, as accordance.D Representative confocal microscopic images of VE-cadherin immunostaining in sh-Control cells, sh-PI3KC2β cells or sh-PI3KC2β cells transduced with Rab11S25N (Rab11-DN). Graph quantification of VE-cadherin; control normalized to 100%. Scale bar, 10 µm. Data represent mean ± SEM of cell fields randomly selected (dots n=6-9). Data are collected from at least 3 independent experiments; **p<0.01 Mann-Whitney test.


Figure 1A , MC38 cells in tissue culture were treated with mock ( 0Gy ) and 10Gy irradiation . Conditioned medium ( CM ) was collected after 48 hours and assayed for the indicated cytokines . Fold - changes in the amounts of the cytokines are shown . B , CSF - 1 mRNA expression was measured in MC38 and KPC cells exposed to 10Gy IR compared with mock irradiated cells . Cells were harvested at 24 , 48 and 72 hours after irradiation and RNA expression was analysed by RT - qPCR . Data are presented as mean ± SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 3 ) . C , CSF - 1 protein ( pg / mg total protein ) in CM collected from MC38 and KPC cells 48 hours after exposure to 10Gy IR compared to mock - irradiated cells . Data are presented as mean ± SEM and analysed by Mann - Witney ( n = 3 ) . D , CSF - 1 protein ( pg / mg total protein ) measured by ELISA in the sera of naïve mice , mice bearing mock - irradiated tumours , and mice bearing irradiated tumours analysed at time points as indicated ( n = 4 / group ) . Data are presented as mean ± SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison . E - F , MC38 ( E ) and KPC ( F ) tumours were irradiated with 10Gy . Average tumour volume ( red line ) is shown with mean TAM infiltrate ( blue line ) for each time point . For TAM quantification , tumours were disaggregated and CD11b + / F480 + TAMs identified by flow cytometry . Data are presented as mean ± SEM for TAMs and SEM for tumour volume ( n = 6 ) . G - H , Immunofluorescent staining of MC38 ( G ) and KPC ( H ) tumour sections ; blue = DAPI , green = CD68 ( TAMs ) . I - J , Flow cytometric analysis of immune cell populations within MC38 ( I ) and KPC ( J ) tumours 5 days following 10Gy IR compared to mock - irradiated tumours . Macrophages ( CD11b + F480 + ) , neutrophils ( CD11b + Ly6G + ) , myeloid - derived suppressor cells ( CD11b + Gr1 + ) , CD8 T cells ( CD45 + CD3 + CD8 + ) , CD4 T cells ( CD45 + CD3 + CD4 + , Natural Killer cells ( CD45 + NK1 . 1 + ) were identified . Pie charts represent the proportion of CD45 + leukocytes out of the total cells .

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

Figure 1A, MC38 cells in tissue culture were treated with mock (0Gy) and 10Gy irradiation. Conditioned medium (CM) was collected after 48 hours and assayed for the indicated cytokines. Fold-changes in the amounts of the cytokines are shown.B, CSF-1 mRNA expression was measured in MC38 and KPC cells exposed to 10Gy IR compared with mock irradiated cells. Cells were harvested at 24, 48 and 72 hours after irradiation and RNA expression was analysed by RT-qPCR. Data are presented as mean ± SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=3).C, CSF-1 protein (pg/mg total protein) in CM collected from MC38 and KPC cells 48 hours after exposure to 10Gy IR compared to mock-irradiated cells. Data are presented as mean ± SEM and analysed by Mann-Witney (n=3).D, CSF-1 protein (pg/mg total protein) measured by ELISA in the sera of naïve mice, mice bearing mock-irradiated tumours, and mice bearing irradiated tumours analysed at time points as indicated (n=4/group). Data are presented as mean ± SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison.E-F, MC38 (E)and KPC (F) tumours were irradiated with 10Gy. Average tumour volume (red line) is shown with mean TAM infiltrate (blue line) for each time point. For TAM quantification, tumours were disaggregated and CD11b+/F480+ TAMs identified by flow cytometry. Data are presented as mean ± SEM for TAMs and SEM for tumour volume (n=6).G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections; blue=DAPI, green = CD68 (TAMs).I-J, Flow cytometric analysis of immune cell populations within MC38 (I) and KPC (J) tumours 5 days following 10Gy IR compared to mock-irradiated tumours. Macrophages (CD11b+ F480+), neutrophils (CD11b+ Ly6G+), myeloid-derived suppressor cells (CD11b+ Gr1+), CD8 T cells (CD45+ CD3+ CD8+), CD4 T cells (CD45+CD3+CD4+, Natural Killer cells (CD45+ NK1.1+) were identified. Pie charts represent the proportion of CD45+ leukocytes out of the total cells.


Figure 2A , Quantification of iNOS expression on gated macrophages ( CD11b + F480 + ) from MC38 and KPC tumours , with representative histograms . Data are presented as mean ± SEM and analysed by Mann - Witney ( n = 6 mice / group ) . B , Quantification of CD206 expression on macrophages ( CD11b + F480 + ) from MC38 and KPC tumours , with representative histograms . Data are presented as mean ± SEM and analysed by Mann - Witney ( n = 6 mice / group ) . C , Quantification of the percentage of TAMs ( CD11b + F480 + ) in MC38 control tumours compared with irradiated tumours ( n = 6 mice / group ) . D - E Quantification of iNOShi ( D ) macrophages as a percentage of total cells in MC38 tumours ( n = 6 ) . D - E , Quantification of CD206hi ( E ) macrophages as a percentage of total cells in MC38 tumours ( n = 6 ) . F , The total number of iNOShi TAMs were divided by CD206hi TAMs to derive a M1 / M2 ratio in MC38 and KPC tumours receiving mock or 10Gy irradiation . Data are presented as mean ± SEM and analysed by Mann - Witney ( n = 6 mice / group ) . G - H , TAMs ( CD11b + F480 + ) were isolated by FACS . Expression of selected immune stimulatory and immunosuppressive genes in TAMs was determined by RT - qPCR ( n = 3 ) . Data are presented as mean fold change ± SEM compared to TAMs from mock - irradiated tumours ( n = 3 ) . Statistical significance was determined by Mann - Witney . I - J , Assessment of TAM suppression of T cells was assayed by evaluation of inhibition of T cell proliferation . TAMs were isolated by magnetic bead separation using F480 microbeads and co - cultured at a 1 : 1 ratio with CFSE labelled CD8 + T cells . CFSE dilution was analysed by flow cytometry to measure proliferation . Percentages of CFSElo T cells were analysed by Kruskal - Wallis with Dunn ' s multiple comparisons test . Representative histograms are shown ( right panel ) . Experiments were repeated twice for each tumour cell line ( n = 3 mice / group ) * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

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

Figure 2A, Quantification of iNOS expression on gated macrophages (CD11b+ F480+) from MC38 and KPC tumours, with representative histograms. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group).B, Quantification of CD206 expression on macrophages (CD11b+ F480+) from MC38 and KPC tumours, with representative histograms. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group).C, Quantification of the percentage of TAMs (CD11b+ F480+) in MC38 control tumours compared with irradiated tumours (n=6 mice/group).D-E Quantification of iNOShi (D) macrophages as a percentage of total cells in MC38 tumours (n=6).D-E, Quantification of CD206hi (E) macrophages as a percentage of total cells in MC38 tumours (n=6).F, The total number of iNOShi TAMs were divided by CD206hi TAMs to derive a M1/M2 ratio in MC38 and KPC tumours receiving mock or 10Gy irradiation. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group).G-H, TAMs (CD11b+F480+) were isolated by FACS. Expression of selected immune stimulatory and immunosuppressive genes in TAMs was determined by RT-qPCR (n=3). Data are presented as mean fold change ± SEM compared to TAMs from mock-irradiated tumours (n=3). Statistical significance was determined by Mann-Witney.I-J, Assessment of TAM suppression of T cells was assayed by evaluation of inhibition of T cell proliferation. TAMs were isolated by magnetic bead separation using F480 microbeads and co-cultured at a 1:1 ratio with CFSE labelled CD8+ T cells. CFSE dilution was analysed by flow cytometry to measure proliferation. Percentages of CFSElo T cells were analysed by Kruskal-Wallis with Dunn's multiple comparisons test. Representative histograms are shown (right panel). Experiments were repeated twice for each tumour cell line (n=3 mice/group) *P <0.05 **P <0.01 ***P <0.001.


Figure 3B - C Flow cytometric analysis of TAMs ( CD11b + F480 + ) in MC38 ( B ) and KPC ( C ) tumours receiving indicated treatments . Tumours were harvested 5 days following IR and disaggregated for analysis by flow cytometry . The left panels in B and C show the data derived from the flow cytometry with representative plots shown in the right panels . Data are presented as mean SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment ( n = 6 mice / group , 3 independent experiments ) . D - E , Tumour growth kinetics of MC38 ( D ) and KPC ( E ) tumours receiving the indicated treatments . Data are presented as mean tumour volume ± SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment . F - G , shows flow cytometric analysis of CD206hi ( F ) TAMs in MC38 and KPC tumours 5 days following IR . Data are presented as mean ± SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment ( n = 6 mice / group ) . F - G , shows flow cytometric analysis of iNOShi ( G ) TAMs in MC38 and KPC tumours 5 days following IR . Data are presented as mean ± SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment ( n = 6 mice / group ) . H - I , CD11b + F480 + TAMs were isolated from MC38 ( H ) and KPC ( I ) tumours 5 days following irradiation ( + / - aCSF ) and expression of selected immune stimulatory and immunosuppressive genes was analysed by RT - qPCR ( n = 3 ) . Data are presented as mean ± SEM ( 10Gy vs 10Gy + aCSF n = 6 mice / group ) . Statistical significance was determined by Mann - Witney . * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

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

Figure 3B-C Flow cytometric analysis of TAMs (CD11b+ F480+) in MC38 (B) and KPC (C) tumours receiving indicated treatments. Tumours were harvested 5 days following IR and disaggregated for analysis by flow cytometry. The left panels in B and C show the data derived from the flow cytometry with representative plots shown in the right panels. Data are presented as mean SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group, 3 independent experiments).D-E, Tumour growth kinetics of MC38 (D) and KPC (E) tumours receiving the indicated treatments. Data are presented as mean tumour volume ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment.F-G, shows flow cytometric analysis of CD206hi (F) TAMs in MC38 and KPC tumours 5 days following IR. Data are presented as mean ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group).F-G, shows flow cytometric analysis of iNOShi (G) TAMs in MC38 and KPC tumours 5 days following IR. Data are presented as mean ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group).H-I, CD11b+ F480+ TAMs were isolated from MC38 (H) and KPC (I) tumours 5 days following irradiation (+/-aCSF) and expression of selected immune stimulatory and immunosuppressive genes was analysed by RT-qPCR (n=3). Data are presented as mean ± SEM (10Gy vs 10Gy + aCSF n=6 mice/group). Statistical significance was determined by Mann-Witney. *P <0.05 **P <0.01 ***P <0.001.


Figure 4A - B MC38 ( A ) and KPC ( B ) tumours were harvested 5 days following 10Gy IR ± aCSF as indicated . The left panels show the percentage of CD45 + CD3 + CD8 + T cells in these tumours after the indicated treatments . Representative flow cytometry plots from the irradiated groups are shown in the right panels . Tumours that did not receive irradiation were harvested when tumours reached 500mm3 . Data are presented as mean SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment ( A ) and Kruskal - Wallis test with Dunn ' s multiple comparison ( B ) ( n = 6 mice / group , 3 independent experiments ) . C , Flow cytometric analysis of Ki67 expression in the CD8 + T cells in MC38 tumours from A . Data are presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 6 / group ) . D , Flow cytometric analysis of PD - 1 expression on CD8 + T cells from MC38 in tumours as in A . Data are presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 6 / group ) . E - F Flow cytometric analysis of IFN - γ expression in CD8 + T cells isolated from MC38 tumours in A . Data presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 6 mice / group , 3 independent experiments ) . E - F , Flow cytometric analysis of Granzyme B expression in CD8 + T cells isolated from MC38 tumours in A . Data presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 6 mice / group , 3 independent experiments ) . G - H , Immunofluorescent staining of MC38 ( G ) and KPC ( H ) tumour sections , blue = DAPI , green = CD8 . I - J , Tumour growth in CD8 depleted C57BL6 wild - type mice bearing MC38 ( I ) and KPC ( J ) tumours receiving treatment as indicated ( n = 6 mice / group ) . K , Flow cytometric quantification of intra - tumoural CD8 T cells following anti - CD8 treatment . Data presented as mean SEM and analysed by unpaired t - test ( n = 5 mice / group ) . L - M , Tumour growth in athymic nude mice bearing MC38 ( I ) and KPC ( J ) tumours receiving treatments as indicated ( n = 6 mice / group ) . Data are presented as mean tumour volume ± SEM and analysed by one - way ANOVA with Tukey post - hoc adjustment . * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

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

Figure 4A-B MC38 (A) and KPC (B) tumours were harvested 5 days following 10Gy IR ± aCSF as indicated. The left panels show the percentage of CD45+ CD3+ CD8+ T cells in these tumours after the indicated treatments. Representative flow cytometry plots from the irradiated groups are shown in the right panels. Tumours that did not receive irradiation were harvested when tumours reached 500mm3. Data are presented as mean SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (A) and Kruskal-Wallis test with Dunn's multiple comparison (B) (n=6 mice/group, 3 independent experiments).C, Flow cytometric analysis of Ki67 expression in the CD8+ T cells in MC38 tumours from A. Data are presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6/group).D, Flow cytometric analysis of PD-1 expression on CD8+ T cells from MC38 in tumours as in A. Data are presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6/group).E-F Flow cytometric analysis of IFN-γ expression in CD8+ T cells isolated from MC38 tumours in A. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6 mice/group, 3 independent experiments).E-F, Flow cytometric analysis of Granzyme B expression in CD8+ T cells isolated from MC38 tumours in A. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6 mice/group, 3 independent experiments).G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections, blue=DAPI, green = CD8.I-J, Tumour growth in CD8 depleted C57BL6 wild-type mice bearing MC38 (I) and KPC (J) tumours receiving treatment as indicated (n=6 mice/group).K, Flow cytometric quantification of intra-tumoural CD8 T cells following anti-CD8 treatment. Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group).L-M, Tumour growth in athymic nude mice bearing MC38 (I) and KPC (J) tumours receiving treatments as indicated (n=6 mice/group). Data are presented as mean tumour volume ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment. *P <0.05 **P <0.01 ***P <0.001.


Figure 5A - B , CD8 + T cells were isolated from the spleens of MC38 tumour bearing mice . The tumours were radiated with 10Gy and cells were harvested 5 days later . Quantification ( A ) and representative images ( B ) of MC38 tumour cell specific tumour - derived CD8 T cell responses detected by IFN - γ ELISpot . The tumour specific CD8 + T cell response was evaluated after T cell incubation with naïve or irradiated MC38 cells for 24 hours . Data presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 3 mice / group ) . C - D , Flow cytometric detection of major histocompatibility complex I ( MHCI ) expressed on MC38 ( C ) and KPC ( D ) tumour cells 48 hours following exposure to 10Gy IR . The left graph shows the overall data , with representative flow cytometry plots on the right . Data presented as mean ± SEM and analysed by Mann - Witney ( n = 3 / group ) . E - F , Flow cytometric quantification of major histocompatibility complex I ( MHCI ) expression in vivo . Gated MC38 ( E ) and KPC ( F ) tumour cells were analysed 48 hours following exposure to 10Gy IR . Data presented as mean ± SEM and analysed by unpaired t - test ( n = 3 / group ) . G - H , Flow cytometric quantification of dendritic cells ( CD11b + CD11c + MHCII + ) in MC38 ( G ) and KPC ( H ) tumours receiving treatment as indicated and as in E - F . Data presented as mean SEM and analysed by unpaired t - test ( n = 5 mice / group ) . J - K , Tumour growth in mice bearing two MC38 tumours receiving 10Gy IR to the primary lesion ( J ) ± systemic aCSF therapy ( K ) . The differences in tumour volume 9 days following IR was analysed by unpaired t - test ( n = 5 mice / group ) . L - M , Tumour growth in mice bearing two KPC tumours receiving 10Gy IR to the primary lesion ( J ) ± systemic aCSF therapy ( M ) . The difference in mean tumour volume 10 days following IR was analysed by unpaired t - test ( n = 8 mice / group ) . N - O , Flow cytometric analysis of macrophages ( N ) and CD8 T cells ( O ) in primary and secondary MC38 tumours . Data presented as mean SEM and analysed by Kruskal - Wallis test with Dunn ' s multiple comparison ( n = 5 mice / group ) . * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

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Figure 5A-B, CD8+ T cells were isolated from the spleens of MC38 tumour bearing mice. The tumours were radiated with 10Gy and cells were harvested 5 days later. Quantification (A) and representative images (B) of MC38 tumour cell specific tumour-derived CD8 T cell responses detected by IFN-γ ELISpot. The tumour specific CD8+ T cell response was evaluated after T cell incubation with naïve or irradiated MC38 cells for 24 hours. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=3 mice/group).C-D, Flow cytometric detection of major histocompatibility complex I (MHCI) expressed on MC38 (C) and KPC (D) tumour cells 48 hours following exposure to 10Gy IR. The left graph shows the overall data, with representative flow cytometry plots on the right. Data presented as mean ± SEM and analysed by Mann-Witney (n=3/group).E-F, Flow cytometric quantification of major histocompatibility complex I (MHCI) expression in vivo. Gated MC38 (E) and KPC (F) tumour cells were analysed 48 hours following exposure to 10Gy IR. Data presented as mean ± SEM and analysed by unpaired t-test (n=3/group).G-H, Flow cytometric quantification of dendritic cells (CD11b+ CD11c+ MHCII+) in MC38 (G) and KPC (H) tumours receiving treatment as indicated and as in E-F. Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group).J-K, Tumour growth in mice bearing two MC38 tumours receiving 10Gy IR to the primary lesion (J) ± systemic aCSF therapy (K). The differences in tumour volume 9 days following IR was analysed by unpaired t-test (n=5 mice/group). L-M, Tumour growth in mice bearing two KPC tumours receiving 10Gy IR to the primary lesion (J) ± systemic aCSF therapy (M). The difference in mean tumour volume 10 days following IR was analysed by unpaired t-test (n=8 mice/group).N-O, Flow cytometric analysis of macrophages (N) and CD8 T cells (O) in primary and secondary MC38 tumours. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=5 mice/group). *P <0.05 **P <0.01 ***P <0.001.


Figure 6PD - L1 expression on MC38 and KPC cells 48 hours following 10Gy irradiation in tissue culture ( A , B ) or 10 Gy irradiation of tumours ( C , D ) analysed by flow cytometry . Data presented as mean SEM and analysed by Mann - Witney ( n = 3 , A , B ) . Data presented as mean SEM and analysed by unpaired t - test ( n = 5 mice / group , C , D ) . E - F , Flow cytometric analysis of PD - L1 ( E ) and PD - L2 ( F ) on TAMs in MC38 tumours receiving treatment as indicated above . Data presented as mean SEM and analysed by Mann - Witney ( n = 5 mice / group ) G - H , Tumour growth in mice bearing MC38 ( G ) and KPC ( H ) tumours receiving the indicated treatments . Data presented for individual mice . Pie charts indicate the number of regressions observed . I - J , Tumour growth in mice bearing KPC tumours mice receiving 10Gy IR + systemic aPD - L1 to the primary lesion ( I ) ± systemic aPD - L1 + aCSF therapy ( J ) . The difference in tumour volume 8 ( I ) or 10 ( J ) days following IR was analysed by unpaired t - test ( n = 8 mice / group ) . * P < 0 . 05 * * P < 0 . 01 * * * P < 0 . 001 .

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Figure 6PD-L1 expression on MC38 and KPC cells 48 hours following 10Gy irradiation in tissue culture (A,B) or 10 Gy irradiation of tumours (C,D) analysed by flow cytometry. Data presented as mean SEM and analysed by Mann-Witney (n=3, A,B). Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group, C,D).E-F, Flow cytometric analysis of PD-L1 (E) and PD-L2 (F) on TAMs in MC38 tumours receiving treatment as indicated above. Data presented as mean SEM and analysed by Mann-Witney (n=5 mice/group)G-H, Tumour growth in mice bearing MC38 (G) and KPC (H) tumours receiving the indicated treatments. Data presented for individual mice. Pie charts indicate the number of regressions observed.I-J, Tumour growth in mice bearing KPC tumours mice receiving 10Gy IR + systemic aPD-L1 to the primary lesion (I) ± systemic aPD-L1 + aCSF therapy (J). The difference in tumour volume 8 (I) or 10 (J) days following IR was analysed by unpaired t-test (n=8 mice/group). *P <0.05 **P <0.01 ***P <0.001.


Figure 1A . Characteristics of IGR39 / IGR37 and WM115 / WM266 . 4 melanoma cell lines ( Upper Panel ) . Pigmentation of melanoma cells pellets ( lower panel ) . B . Growth curve of primitive IGR39 and metastatic IGR37 cell line ( N = 3 biological replicates ) . Data are mean ± SD . C . Phase - contrast images of IGR39 and IGR37 cells . Scale bar is 100µm . D . Analysis of IGR39 and IGR37 speed of migration by time - lapse microscopy .

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Figure 1A. Characteristics of IGR39 /IGR37 and WM115/WM266.4 melanoma cell lines (Upper Panel). Pigmentation of melanoma cells pellets (lower panel).B. Growth curve of primitive IGR39 and metastatic IGR37 cell line (N=3 biological replicates). Data are mean ±SD.C. Phase-contrast images of IGR39 and IGR37 cells. Scale bar is 100µm.D. Analysis of IGR39 and IGR37 speed of migration by time-lapse microscopy.