B , C B6 FoxP3 ‐ GFP mice were treated and evaluated as in ( A ) . Regulatory cytokine production in FoxP3 ‐ GFP + ( green ‐ dotted bars ) versus FoxP3 ‐ GFP − ( white bars ) cells was measured by intracellular staining and FACS analyses ( C ) .

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B, C B6 FoxP3‐GFP mice were treated and evaluated as in (A). Regulatory cytokine production in FoxP3‐GFP+ (green‐dotted bars) versus FoxP3‐GFP− (white bars) cells was measured by intracellular staining and FACS analyses (C).


A B6 Rag1 − / − mice were injected with 106CD4 + CD45RBhiT cells and then orally gavaged with NCK56 ( red ) , NCK2187 ( green ) , or SlpA ( blue ) , 1 and 3 days after transfer , and subsequently once a week for four consecutive weeks , or left untreated ( magenta ) . A group of mice was co ‐ transferred with CD4 + CD25 + T cells as a positive control for protection ( Tregs ; gray ) . Colitis severity was determined in part by weight loss , diarrhea scores , and FOB . See Supplementary Tables S1 , S2 and S3 for statistical analyses results .

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A B6 Rag1−/−mice were injected with 106CD4+CD45RBhiT cells and then orally gavaged with NCK56 (red), NCK2187 (green), or SlpA (blue), 1 and 3 days after transfer, and subsequently once a week for four consecutive weeks, or left untreated (magenta). A group of mice was co‐transferred with CD4+CD25+T cells as a positive control for protection (Tregs; gray). Colitis severity was determined in part by weight loss, diarrhea scores, and FOB. See Supplementary Tables S1, S2 and S3 for statistical analyses results.


B , C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in ( A ) were also used as measures of disease . Scale bar = 50 μm .

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B, C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in (A) were also used as measures of disease. Scale bar = 50 μm.


D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in ( A ) , or sham adoptive transferred ( white bars ) .

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D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in (A), or sham adoptive transferred (white bars).


Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Colonic expression of tight junction ‐ associated genes Cldn3 and Ocln , determined by RT - PCR relative to 18S rRNA ( A ) , were used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( white bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three individual experiments and are shown as mean ± SEM . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , and red asterisks to NCK56 ‐ treated mice .

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Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Colonic expression of tight junction‐associated genes Cldn3 and Ocln, determined by RT-PCR relative to 18S rRNA (A), were used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.


Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Passive transepithelial absorption of FITC ‐ dextran ( B ) , was used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( white bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three individual experiments and are shown as mean ± SEM . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , and red asterisks to NCK56 ‐ treated mice .

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Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Passive transepithelial absorption of FITC‐dextran (B), was used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.


A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187 , and the colonic gene expression of C ‐ type lectin receptors were measured by RT - PCR . Each box represents an individual mouse ; n = 4 . Data represent three individual experiments .

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A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187, and the colonic gene expression of C‐type lectin receptors were measured by RT-PCR. Each box represents an individual mouse; n = 4. Data represent three individual experiments.


B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry . Gray tinted line = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion protein ; blue = SlpA ‐ coated beads + SIGNR1 ‐ hFc ; red = SlpA ‐ coated beads + SIGNR3 ‐ hFc . Binding assay results were confirmed five independent times .

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B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; blue = SlpA‐coated beads + SIGNR1‐hFc; red = SlpA‐coated beads + SIGNR3‐hFc. Binding assay results were confirmed five independent times.


C Binding of SlpA to SIGNR3 , but not SIGNR1 expressed by CHO ‐ S cells . Gray tinted line = untransfected CHO ‐ S cells ; blue = untransfected CHO ‐ S cells + labeled SlpA ; green = SIGNR1 ‐ transfected CHO ‐ S cells + labeled SlpA ; red = SIGNR3 ‐ transfected CHO ‐ S cells + labeled SlpA . Binding assays in CHO ‐ S cells were performed three individual times .

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C Binding of SlpA to SIGNR3, but not SIGNR1 expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; green = SIGNR1‐transfected CHO‐S cells + labeled SlpA; red = SIGNR3‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.


D Binding of SlpA to recombinant human DC ‐ SIGN / CD209 ‐ Fc chimera was analyzed by flow cytometry . Gray tinted line = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion protein ; red = SlpA ‐ coated beads + DC ‐ SIGN ‐ Fc . Binding assays were performed and confirmed three times .

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D Binding of SlpA to recombinant human DC‐SIGN/CD209‐Fc chimera was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; red = SlpA‐coated beads + DC‐SIGN‐Fc. Binding assays were performed and confirmed three times.


E Binding of SlpA to DC ‐ SIGN expressed by CHO ‐ S cells . Gray tinted line = untransfected CHO ‐ S cells ; blue = untransfected CHO ‐ S cells + labeled SlpA ; red = DC ‐ SIGN ‐ transfected CHO ‐ S cells + labeled SlpA . Binding assays in CHO ‐ S cells were performed three individual times .

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E Binding of SlpA to DC‐SIGN expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; red = DC‐SIGN‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.


F , G IL ‐ 1β production by colonic DCs of naïve WT B6 GF or B6 Signr3 − / − ( KO ) mice treated with NCK56 ( blue ) or NCK2187 ( green ) four times , or left untreated ( black ) , as determined by flow cytometry . n = 5 mice / group . Data represent four individual experiments and are shown as mean ± SEM . * P 0 . 05 . Black asterisks compare NCK2187 to untreated ( PBS ) mice , and red asterisks to NCK56 ‐ treated mice .

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F, G IL‐1β production by colonic DCs of naïve WT B6 GF or B6 Signr3−/− (KO) mice treated with NCK56 (blue) or NCK2187 (green) four times, or left untreated (black), as determined by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM. *P 0.05. Black asterisks compare NCK2187 to untreated (PBS) mice, and red asterisks to NCK56‐treated mice.


H Frequency of colonic FoxP3 + Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry . n = 5 mice / group . Data represent four individual experiments and are shown as mean ± SEM .

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H Frequency of colonic FoxP3+ Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . A Colitis severity was determined in part by weight loss . n = 5 mice / group .

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WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.A Colitis severity was determined in part by weight loss. n = 5 mice/group.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . B , C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease . Scale bar = 50 μm . n = 5 mice / group . Empty bars = WT ; lined bars = KO ; white bars = untreated ; purple bars = DSS ; red bars = DSS + NCK56 ; green bars = DSS + NCK2187 ; blue bars = DSS + SlpA .

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

WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.B, C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease. Scale bar = 50 μm. n = 5 mice/group. Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . D Colonoscopies were performed in the different groups with a Multi ‐ Purpose Rigid ™ Telescope attached to a TELE PACK X on day 10 .

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WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.D Colonoscopies were performed in the different groups with a Multi‐Purpose Rigid™ Telescope attached to a TELE PACK X on day 10.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . E Mean relative colonic expression of tight junction ‐ associated genes in WT mice . n = 5 mice / group .

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WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.E Mean relative colonic expression of tight junction‐associated genes in WT mice. n = 5 mice/group.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . F Fecalalbumin levels in WT mice as a measure of intestinal permeability . n = 5 mice / group .

O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O B-ORGANISM O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O

WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.F Fecalalbumin levels in WT mice as a measure of intestinal permeability. n = 5 mice/group.


Signr3 + / + ( WT ) or Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 1 and − 3 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times , and immunity was analyzed by flow cytometry at day 10 . A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS ‐ treated WT ( left ) and KO mice ( right ) . Empty bars = WT ; lined bars = KO ; white bars = untreated ; purple bars = DSS ; red bars = DSS + NCK56 ; green bars = DSS + NCK2187 ; blue bars = DSS + SlpA .

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

Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS‐treated WT (left) and KO mice (right). Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.


Signr3 + / + ( WT ) or Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 1 and − 3 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times , and immunity was analyzed by flow cytometry at day 10 . B , C Colonic DCs and macrophages ( MΦs ) were analyzed by flow cytometry for the production of IL ‐ 1β ( B ) , and colonic FoxP3 + Tregs were evaluated for co ‐ expression of RORγt + ( C ) . n = 5 mice / group . Gray tinted line = isotype control ; black = untreated ; purple = DSS ; red = DSS + NCK56 ; green = DSS + NCK2187 ; blue = DSS + SlpA .

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Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.B, C Colonic DCs and macrophages (MΦs) were analyzed by flow cytometry for the production of IL‐1β (B), and colonic FoxP3+ Tregs were evaluated for co‐expression of RORγt+ (C). n = 5 mice/group. Gray tinted line = isotype control; black = untreated; purple = DSS; red = DSS + NCK56; green = DSS + NCK2187; blue = DSS + SlpA.


CRTH2 subcellular localization in GFP - fused CRTH2 expressing plasmid - transfected cells . Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane . Green , GFP - CRTH2 ; blue , DAPI ; red , Calnexin ; Scale bar , 20 μm .

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CRTH2 subcellular localization in GFP-fused CRTH2 expressing plasmid-transfected cells. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.


Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells . Myc - tagged CRTH2 expressing plasmid was transfected into NIH 3T3 and HEK293T cells . Whole cell lysate , plasma , and ER fractions were obtained 48 h after transfection . Calnexin and ATP1B were used as markers for ER and cell membrane , respectively .

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Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells. Myc-tagged CRTH2 expressing plasmid was transfected into NIH 3T3 and HEK293T cells. Whole cell lysate, plasma, and ER fractions were obtained 48 h after transfection. Calnexin and ATP1B were used as markers for ER and cell membrane, respectively.


Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF - β1 ( 10 ng / mL ) treatment . Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane . Green , GFP - CRTH2 ; blue , DAPI ; red , Calnexin ; Scale bar , 20 μm .

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

Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF-β1 (10 ng/mL) treatment. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.


Co - immunoprecipitation analysis of association of CRTH2 with LARP6 . HEK293T cells were co - transfected with Myc - CRTH2 and Flag - LARP6 expressing plasmids . Whole cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies .

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

Co-immunoprecipitation analysis of association of CRTH2 with LARP6. HEK293T cells were co-transfected with Myc-CRTH2 and Flag-LARP6 expressing plasmids. Whole cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies.


Co - IP analysis of association of CRTH2 with GPR78 . HEK293T cells were co - transfected with Myc - GPR78 expressing plasmids .

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

Co-IP analysis of association of CRTH2 with GPR78. HEK293T cells were co-transfected with Myc-GPR78 expressing plasmids.


Effect of LARP6 knockdown on Col1a1 , Col1a2 and Col3a1 mRNA stability in WT and CRTH2 - / - fibroblasts . * P < 0 . 05 vs WT , ( two - way ANOVA ) ; n = 8 for all groups .

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

Effect of LARP6 knockdown on Col1a1, Col1a2 and Col3a1 mRNA stability in WT and CRTH2-/- fibroblasts. *P < 0.05 vs WT, (two-way ANOVA); n = 8 for all groups.


Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2 - / - fibroblasts .

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

Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2-/- fibroblasts.


, Representative images of Masson ′ s trichrome staining of lung sections from CRTH2flox / floxCol1a2 - CreERT ( F - CRTH2 KO ) and CRTH2flox / flox ( Control ) mice 14 days after bleomycin challenge . Blue indicates the collagen deposition regions . Scale bar , 100 μm . Quantification of collagen content in mouse lung sections after bleomycin challenge . * P < 0 . 05 vs control , # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each ; bleomycin groups , n = 8 each .

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

, Representative images of Masson′s trichrome staining of lung sections from CRTH2flox/floxCol1a2-CreERT (F-CRTH2 KO) and CRTH2flox/flox (Control) mice 14 days after bleomycin challenge. Blue indicates the collagen deposition regions. Scale bar, 100 μm. Quantification of collagen content in mouse lung sections after bleomycin challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each; bleomycin groups, n = 8 each.


Representative images of Masson ′ s trichrome staining of liver sections from control and F - CRTH2 KO mice 4 weeks after CCl4 challenge ; Scale bar , 100 μm .

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

Representative images of Masson′s trichrome staining of liver sections from control and F-CRTH2 KO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm.


Representative images of Masson ′ s trichrome staining of heart sections from control and F - CRTH2 KO mice 21 days after ISO challenge ; Scale bar , 50 μm . Quantification of collagen content in mouse heart sections after ISO challenge . * P < 0 . 05 vs control , # P < 0 . 05 vs saline group ( two - way ANOVA ) ; PBS groups , n = 3 each , ISO groups , n = 7 - 8 .

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

Representative images of Masson′s trichrome staining of heart sections from control and F-CRTH2 KO mice 21 days after ISO challenge; Scale bar, 50 μm. Quantification of collagen content in mouse heart sections after ISO challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); PBS groups, n = 3 each, ISO groups, n = 7-8.


Representative images of Masson ' s trichrome staining of lung sections from F - LARP6 KO and F - LARP6 / F - CRTH2 double KO ( F - DKO ) mice 14 days after bleomycin challenge ; Scale bar , 100 μm . , Quantification of collagen content in mouse lung sections after bleomycin challenge . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each , bleomycin groups , n = 7 - 8 . Hydroxyproline content in lungs from F - LARP6 KO and F - DKO mice treated with bleomycin . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each , bleomycin groups , n = 8 each .

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

Representative images of Masson's trichrome staining of lung sections from F-LARP6 KO and F-LARP6/F-CRTH2 double KO (F-DKO) mice 14 days after bleomycin challenge; Scale bar, 100 μm. , Quantification of collagen content in mouse lung sections after bleomycin challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 7-8. Hydroxyproline content in lungs from F-LARP6 KO and F-DKO mice treated with bleomycin. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 8 each.


Representative images of Masson ' s trichrome staining of liver sections from F - LARP6 KO and F - DKO mice 4 weeks after CCl4 challenge ; Scale bar , 100 μm . Quantification of collagen content in mouse liver sections after CCl4 challenge . # P < 0 . 05 vs mineral oil group ( two - way ANOVA ) ; mineral oil groups , n = 4 each , CCl4 groups , n = 8 each . Hydroxyproline content in livers from F - LARP6 KO and F - DKO mice treated with CCl4 . # P < 0 . 05 vs mineral oil group ( two - way ANOVA ) ; saline groups , n = 4 each , CCl4 groups , n = 8 each .

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

Representative images of Masson's trichrome staining of liver sections from F-LARP6 KO and F-DKO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm. Quantification of collagen content in mouse liver sections after CCl4 challenge. #P < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, n = 4 each, CCl4 groups, n = 8 each. Hydroxyproline content in livers from F-LARP6 KO and F-DKO mice treated with CCl4. #P < 0.05 vs mineral oil group (two-way ANOVA); saline groups, n = 4 each, CCl4 groups, n = 8 each.


Representative images of Masson ' s trichrome staining of heart sections from F - LARP6 KO and F - DKO mice 21 days after ISO challenge ; Scale bar , 50 μm . Quantification of collagen content in mouse heart sections after ISO challenge . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 3 each , ISO groups , n = 7 each . , Hydroxyproline content in hearts from F - LARP6 KO and F - DKO mice treated with ISO . # P < 0 . 05 vs saline group ( two - way ANOVA ) ; saline groups , n = 4 each , ISO groups , n = 8 each .

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

Representative images of Masson's trichrome staining of heart sections from F-LARP6 KO and F-DKO mice 21 days after ISO challenge; Scale bar, 50 μm. Quantification of collagen content in mouse heart sections after ISO challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 3 each, ISO groups, n = 7 each. , Hydroxyproline content in hearts from F-LARP6 KO and F-DKO mice treated with ISO. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, ISO groups, n = 8 each.


Microscale thermophoresis ( MST ) analysis of the binding affinity of Bumetanide with MBP -

Microscale thermophoresis (MST) analysis of the binding affinity of Bumetanide with MBP-LARP6


Quantification of collagen content in lung sections from Bumetanide - treated control ( CRTH2flox / flox ) and F - CRTH2 KO mice . * P < 0 . 05 vs control , # P < 0 . 05 vs Vehicle ( two - way ANOVA ) ; n = 7 for all groups . Effect of Bumetanide treatment on pulmonary hydroxyproline levels in control and F - CRTH2 KO mice . * P < 0 . 05 vs control ; # P < 0 . 05 vs Vehicle ( two - way ANOVA ) ; n = 7 for all groups .

O O B-SMALL_MOLECULE O O B-TISSUE O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-DISEASE B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of collagen content in lung sections from Bumetanide-treated control (CRTH2flox/flox) and F-CRTH2 KO mice. *P < 0.05 vs control, #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups. Effect of Bumetanide treatment on pulmonary hydroxyproline levels in control and F-CRTH2 KO mice. *P < 0.05 vs control; #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups.


Bumetanide interferes with the interaction of CRTH2 with LARP6 in MRC - 5 human lung fibroblasts MRC - 5 . MRC - 5 cells were co - transfected with myc - LARP6 and flag - CRTH2 expressing plasmids for 24 h , and then treated with Bumetanide ( 0 . 1 , 2 μM ) for an additional 24 h . Whole cell lysates from MRC - 5 cells were immunoprecipitated and immunoblotted with the indicated antibodies .

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

Bumetanide interferes with the interaction of CRTH2 with LARP6 in MRC-5 human lung fibroblasts MRC-5. MRC-5 cells were co-transfected with myc-LARP6 and flag-CRTH2 expressing plasmids for 24 h, and then treated with Bumetanide (0.1, 2 μM) for an additional 24 h. Whole cell lysates from MRC-5 cells were immunoprecipitated and immunoblotted with the indicated antibodies.


Effect of Bumetanide on Collagen ( Col ) I and III protein expression in MRC - 5 cells .

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

Effect of Bumetanide on Collagen (Col) I and III protein expression in MRC-5 cells.


( A ) qRT - PCR analysis for Islr , Chd1 and Vim in total intestinal tissues , intestinal epithelium and lamina propria . n = 4 .

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

(A) qRT-PCR analysis for Islr, Chd1 and Vim in total intestinal tissues, intestinal epithelium and lamina propria. n = 4.


( B ) Western blotting for Islr and E - cadherin in total intestinal tissues , intestinal epithelium and lamina propria . α - Tubulin was used as a loading control .

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

(B) Western blotting for Islr and E-cadherin in total intestinal tissues, intestinal epithelium and lamina propria. α-Tubulin was used as a loading control.


( C ) qRT - PCR analysis for Islr in CD45 + , CD45 - Epcam + , CD45 - Epcam - CD90 + and CD45 - Epcam - CD90 - cells sorted from intestinal tissues . n = 3 technical replicates .

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

(C) qRT-PCR analysis for Islr in CD45+, CD45-Epcam+, CD45-Epcam-CD90+ and CD45-Epcam-CD90- cells sorted from intestinal tissues. n = 3 technical replicates.


( D ) In situ hybridization for Islr with RNAscope probe in intestine and colon from 8 - week - old mice , showing that Islr is primarily expressed in stromal cells . Negative and positive controls were shown in Appendix Figure S1A . Scale bar : 25 μm .

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

(D) In situ hybridization for Islr with RNAscope probe in intestine and colon from 8-week-old mice, showing that Islr is primarily expressed in stromal cells. Negative and positive controls were shown in Appendix Figure S1A. Scale bar: 25 μm.


( E ) In situ hybridization for ISLR with RNAscope probe in human mucosa , inflamed mucosa from CD and UC patients . Scale bar : 25 μm .

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

(E) In situ hybridization for ISLR with RNAscope probe in human mucosa, inflamed mucosa from CD and UC patients. Scale bar: 25 μm.


( F ) qRT - PCR analysis showing the dynamic changes of Islr in colonic tissues from mice upon DSS treatment and after DSS removal . n = 4 at each time points .

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

(F) qRT-PCR analysis showing the dynamic changes of Islr in colonic tissues from mice upon DSS treatment and after DSS removal. n = 4 at each time points.


( G ) In situ hybridization for Islr with RNAscope probe in mouse colons without or with 3 - day or 5 - day DSS treatments . Scale bar : 50 μm .

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

(G) In situ hybridization for Islr with RNAscope probe in mouse colons without or with 3-day or 5-day DSS treatments. Scale bar: 50 μm.


( H ) The cancer genome atlas ( TCGA ) RNA - seq analysis showing that ISLR is upregulated in human rectal adenocarcinoma relative to normal rectal tissues . The horizontal lines between the box limits represent the median , the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively . n = 65 .

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

(H) The cancer genome atlas (TCGA) RNA-seq analysis showing that ISLR is upregulated in human rectal adenocarcinoma relative to normal rectal tissues. The horizontal lines between the box limits represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively. n = 65.


( I ) Kaplan - Meier survival curve of 270 CRC cases . P = 0 . 02 ( log - rank test ) .

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

(I) Kaplan-Meier survival curve of 270 CRC cases. P = 0.02 (log-rank test).


( J ) ISLR level increased with nodal metastasis in colorectal cancer patients . N0 , no regional lymph node metastasis ; N1 , metastases in 1 to 3 axillary lymph nodes ; N2 , metastases in 4 to 9 axillary lymph nodes ; N3 , metastases in 10 or more axillary lymph nodes . The solid horizontal lines represent the median , the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively .

O O O B-GENEPROD O O O O O O B-DISEASE I-DISEASE O O O O O O B-TISSUE I-TISSUE O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) ISLR level increased with nodal metastasis in colorectal cancer patients. N0, no regional lymph node metastasis; N1, metastases in 1 to 3 axillary lymph nodes; N2, metastases in 4 to 9 axillary lymph nodes; N3, metastases in 10 or more axillary lymph nodes. The solid horizontal lines represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively.


( K In situ hybridization for ISLR / Islr with RNAscope probes in normal human colon , human malignant adenocarcinoma , metastatic adenocarcinoma ( K ) , t indicating tumor ; a indicating adjacent tissues of tumor . Scale bar : 50 μm .

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

(K In situ hybridization for ISLR/Islr with RNAscope probes in normal human colon, human malignant adenocarcinoma, metastatic adenocarcinoma (K), t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm.


L ) In situ hybridization for ISLR / Islr with RNAscope probes in mouse colon tumors from AOM - DSS model ( L ) . t indicating tumor ; a indicating adjacent tissues of tumor . Scale bar : 50 μm .

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

L) In situ hybridization for ISLR/Islr with RNAscope probes in mouse colon tumors from AOM-DSS model (L). t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm.


( B ) qRT - PCR analysis for Ets1 in colonic epithelium and lamina propria . n = 3 .

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

(B) qRT-PCR analysis for Ets1 in colonic epithelium and lamina propria. n = 3.


( C ) Double immunofluorescence for ETS1 and β - catenin in intestinal biopsies of healthy controls and inflamed mucosa from UC patients . Scale bar : 50 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O B-TISSUE O B-DISEASE O O O O O O O O

(C) Double immunofluorescence for ETS1 and β-catenin in intestinal biopsies of healthy controls and inflamed mucosa from UC patients. Scale bar: 50 μm.


( D ) qRT - PCR for Ets1 in colons with or without 5 - day DSS treatment . n = 4 .

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

(D) qRT-PCR for Ets1 in colons with or without 5-day DSS treatment. n = 4.


( E ) Double immunofluorescence for Ets1 and β - catenin in normal mouse colons without DSS treatment , mouse colons with 5 - day DSS treatments , and AOM - DSS mouse colon tumors . Scale bar : 50 μm .

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

(E) Double immunofluorescence for Ets1 and β-catenin in normal mouse colons without DSS treatment, mouse colons with 5-day DSS treatments, and AOM-DSS mouse colon tumors. Scale bar: 50 μm.


( F ) Pearson correlation analysis on ETS1 and ISLR in colorectal adenocarcinoma TCGA RNA - seq ( P < 0 . 001 ; R = 0 . 68 ) .

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

(F) Pearson correlation analysis on ETS1 and ISLR in colorectal adenocarcinoma TCGA RNA-seq (P < 0.001; R = 0.68).


( G ) Western blotting for ISLR and HA in lysates of UC - MSCs transfected with pCMV5 - HA or pCMV5 - ETS1 . α - Tubulin was used as a loading control .

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

(G) Western blotting for ISLR and HA in lysates of UC-MSCs transfected with pCMV5-HA or pCMV5-ETS1. α-Tubulin was used as a loading control.


( H ) Luciferase activity in lysates of UC - MSCs transfected with luciferase reporter plasmids of pGL3 - basic empty vector ( basic ) , wild type ISLR promoter or mutant promoter with mutation of ETS1 binding sites under normal and ETS1 overexpression conditions . n = 3 .

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

(H) Luciferase activity in lysates of UC-MSCs transfected with luciferase reporter plasmids of pGL3-basic empty vector (basic), wild type ISLR promoter or mutant promoter with mutation of ETS1 binding sites under normal and ETS1 overexpression conditions. n = 3.


( I ) Chromatin immunoprecipitation assay was carried out on UC - MSCs cells using antibodies against ETS1 and Histone H3 . The antibody against Histone H3 was used as a positive control . IgG was used as a negative control . The enrichment of ETS1 binding to ISLR promoter was quantified using qPCR . n = 3 technical replicates .

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

(I) Chromatin immunoprecipitation assay was carried out on UC-MSCs cells using antibodies against ETS1 and Histone H3. The antibody against Histone H3 was used as a positive control. IgG was used as a negative control. The enrichment of ETS1 binding to ISLR promoter was quantified using qPCR. n = 3 technical replicates.


( J ) Chromatin immunoprecipitation assay was carried out on primary intestinal stromal cells isolated from WT mice without DSS treatment or WT mice after 5 - day DSS treatment using antibodies against ETS1 and Histone H3 . The antibody against Histone H3 was used as a positive control . IgG was used as a negative control . The enrichment of Ets1 binding to Islr promoter was quantified using qPCR . n = 3 technical replicates .

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

(J) Chromatin immunoprecipitation assay was carried out on primary intestinal stromal cells isolated from WT mice without DSS treatment or WT mice after 5-day DSS treatment using antibodies against ETS1 and Histone H3. The antibody against Histone H3 was used as a positive control. IgG was used as a negative control. The enrichment of Ets1 binding to Islr promoter was quantified using qPCR. n = 3 technical replicates.


( A ) Quantification of body weight change in control ( n = 12 ) and cKO ( n = 13 ) mice after DSS treatment and DSS removal .

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(A) Quantification of body weight change in control (n=12) and cKO (n=13) mice after DSS treatment and DSS removal.


( B ) Fecal occult blood test in control and cKO mice at indicated timepoints . n = 3 at each timepoint .

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(B) Fecal occult blood test in control and cKO mice at indicated timepoints. n=3 at each timepoint.


( C ) Histological images of colonic tissues from control ( n = 10 ) and cKO ( n = 10 ) mice at indicated timepoints after DSS treatment . The Panels with non DSS are identical to Panels in Fig EV1F . Scale bar : 100 μm .

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(C) Histological images of colonic tissues from control (n=10) and cKO (n=10) mice at indicated timepoints after DSS treatment. The Panels with non DSS are identical to Panels in Fig EV1F. Scale bar: 100 μm.


( D ) Quantification of the clinical scores in control ( n = 12 ) and cKO ( n = 13 ) mice at indicated timepoints .

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

(D) Quantification of the clinical scores in control (n=12) and cKO (n=13) mice at indicated timepoints.


( E ) Gross images of colons and quantification of colon length from control ( n = 6 ) and cKO ( n = 6 ) mice two days after 5 - day DSS treatment .

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(E) Gross images of colons and quantification of colon length from control (n=6) and cKO (n=6) mice two days after 5-day DSS treatment.


( F ) Double immunofluorescence for Ki67 and β - catenin in colons from control ( n = 4 ) and cKO ( n = 4 ) mice two days after 5 - day DSS treatment . Quantification of percentage of Ki67 + epithelial cells . Scale bar : 100 m .

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

(F) Double immunofluorescence for Ki67 and β-catenin in colons from control (n=4) and cKO (n=4) mice two days after 5-day DSS treatment. Quantification of percentage of Ki67+ epithelial cells. Scale bar: 100 m.


( B ) Quantification of body weight changes in control ( n = 9 ) and cKO ( n = 10 ) mice during tumor development .

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(B) Quantification of body weight changes in control (n=9) and cKO (n=10) mice during tumor development.


( C ) Gross images of AOM - DSS tumors in control ( n = 9 ) and cKO ( n = 10 ) mice .

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(C) Gross images of AOM-DSS tumors in control (n=9) and cKO (n=10) mice.


( D ) Quantification of tumor number per mouse and tumor volume in control ( n = 9 ) and cKO ( n = 10 ) mice .

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(D) Quantification of tumor number per mouse and tumor volume in control (n=9) and cKO (n=10) mice.


( E ) Histological images of colon tumors from control and cKO mice . Scale bar : 2 mm .

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(E) Histological images of colon tumors from control and cKO mice. Scale bar: 2 mm.


( F Double immunofluorescence for Cyclin D1 and β - catenin ( F ) , in colon tumors from control ( n = 4 ) and cKO ( n = 4 ) mice . Scale bar : 100 μm .

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(F Double immunofluorescence for Cyclin D1 and β-catenin (F), in colon tumors from control (n=4) and cKO (n=4) mice. Scale bar: 100 μm.


G ) Double immunofluorescence for Ki67 and β - catenin ( G ) in colon tumors from control ( n = 4 ) and cKO ( n = 4 ) mice . Scale bar : 100 μm .

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G) Double immunofluorescence for Ki67 and β-catenin (G) in colon tumors from control (n=4) and cKO (n=4) mice. Scale bar: 100 μm.


( H ) Quantification of percentage of cyclin D1 + and Ki67 + cells in ( F and G ) . n = 4 .

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(H) Quantification of percentage of cyclin D1+ and Ki67+ cells in (F and G). n = 4.


( I ) The growth curve of HT29 CRC cells cultured in the supernatant with or without ISLR - HA . The supernatant were collected for HT29 CRC cells 24 hours after transfected with pcDNA3 . 1 empty vector or pcDNA3 . 1 - ISLR - HA vector . n = 5 at each timepoint .

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

(I) The growth curve of HT29 CRC cells cultured in the supernatant with or without ISLR-HA. The supernatant were collected for HT29 CRC cells 24 hours after transfected with pcDNA3.1 empty vector or pcDNA3.1-ISLR-HA vector. n=5 at each timepoint.


( A ) Heatmap of the altered Hippo related genes in colons from control and cKO mice one day after 5 - day DSS treatment . The parameter of the color key indicated the fold changes converted to log2 of signal value normalized .

O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(A) Heatmap of the altered Hippo related genes in colons from control and cKO mice one day after 5-day DSS treatment. The parameter of the color key indicated the fold changes converted to log2 of signal value normalized.


( B ) qRT - PCR analysis validates the altered Hippo - related genes in control and cKO mice . n = 4 .

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

(B) qRT-PCR analysis validates the altered Hippo-related genes in control and cKO mice. n=4.


( C ) Western blotting for pMst1 / 2 , Mst1 , pMob1 and Mob1 in colon epithelium from control and cKO mice two days after 5 - day DSS treatment . β - actin was used as a loading control . The quantification of pMst1 / 2 versus Mst1 and pMob1 versus Mob1 were shown under the corresponding protein bands .

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

(C) Western blotting for pMst1/2, Mst1, pMob1 and Mob1 in colon epithelium from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The quantification of pMst1/2 versus Mst1 and pMob1 versus Mob1 were shown under the corresponding protein bands.


( D ) Western blotting for Yap1 in nuclear and cytoplasmic proteins isolated from intestinal epithelial cells from control and cKO mice two days after 5 - day DSS treatment . Histone H3 , a positive control for nuclear proteins . α - Tubulin , a positive control for cytoplasmic proteins . The quantification of Yap1 versus Histone H3 in nuclear proteins was shown under the corresponding band .

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

(D) Western blotting for Yap1 in nuclear and cytoplasmic proteins isolated from intestinal epithelial cells from control and cKO mice two days after 5-day DSS treatment. Histone H3, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins. The quantification of Yap1 versus Histone H3 in nuclear proteins was shown under the corresponding band.


( E and F ) Double immunofluorescence for Yap1 and β - catenin in the colons from control ( n = 4 ) and cKO ( n = 4 ) mice two days after DSS removal ( E ) , and in AOM / DSS tumors from control ( n = 4 ) and cKO ( n = 4 ) mice ( F ) . The percentage of nuclear Yap1 + cells versus epithelial cells was quantified . Scale bar : 50 μm .

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

(E and F) Double immunofluorescence for Yap1 and β-catenin in the colons from control (n=4) and cKO (n=4) mice two days after DSS removal (E), and in AOM/DSS tumors from control (n=4) and cKO (n=4) mice (F). The percentage of nuclear Yap1+ cells versus epithelial cells was quantified. Scale bar: 50 μm.


( G ) Western blotting for Yap1 and pYap1 in colon epithelial cells from control and cKO mice two days after 5 - day DSS treatment . β - actin was used as a loading control . The ratio of Yap1 / pYap1 were quantified . n = 3 .

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

(G) Western blotting for Yap1 and pYap1 in colon epithelial cells from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The ratio of Yap1/pYap1 were quantified. n=3.


( H ) The growth curve of HCT116 colorectal cancer cells cultured in the supernatant from WT or cKO IMCs , concomitantly transfected with PCDH empty vector or PCDH - YAP1 - 5SA vector . n = 4 technical replicates .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE B-DISEASE I-DISEASE O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(H) The growth curve of HCT116 colorectal cancer cells cultured in the supernatant from WT or cKO IMCs, concomitantly transfected with PCDH empty vector or PCDH-YAP1-5SA vector. n = 4 technical replicates.


( I ) Pearson correlation analysis of ISLR and CTGF ( P < 0 . 001 ; R = 0 . 69 ) , ISLR and FSTL1 ( P < 0 . 001 ; R = 0 . 81 ) , as well as ISLR and CYR61 ( P < 0 . 001 ; R = 0 . 65 ) in human colorectal cancer based on TCGA RNA - seq database .

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(I) Pearson correlation analysis of ISLR and CTGF (P < 0.001; R = 0.69), ISLR and FSTL1 (P < 0.001; R = 0.81), as well as ISLR and CYR61 (P < 0.001; R = 0.65) in human colorectal cancer based on TCGA RNA-seq database.


( A ) Schematics of culture epithelial cells with supernatant from HEK293FT cells transfected with pcDNA3 . 1 empty vector or pcDNA3 . 1 - hISLR - HA plasmids . Western blotting for HA in the supernatant from HEK293FT cells transfected with the indicated plasmids .

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

(A) Schematics of culture epithelial cells with supernatant from HEK293FT cells transfected with pcDNA3.1 empty vector or pcDNA3.1-hISLR-HA plasmids. Western blotting for HA in the supernatant from HEK293FT cells transfected with the indicated plasmids.


( B ) Gene set enrichment for YAP / TAZ / TEAD direct target genes in transcriptome profiles for HEK293FT cells treated by hISLR - HA - enriched supernatant .

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(B) Gene set enrichment for YAP/TAZ/TEAD direct target genes in transcriptome profiles for HEK293FT cells treated by hISLR-HA-enriched supernatant.


( C ) Heatmap showing the altered Hippo related genes in the transcriptome profiles . The parameter of the color key indicated the fold changes converted to log2 of gene fragments per kilobase per million mapped reads ( FPKM ) values .

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

(C) Heatmap showing the altered Hippo related genes in the transcriptome profiles. The parameter of the color key indicated the fold changes converted to log2 of gene fragments per kilobase per million mapped reads (FPKM) values.


( D ) Western blotting for pMST1 / 2 , MST1 , pMOB1 and MOB1 , pYAP1 and YAP1 in lysates from HEK293FT cells cultured in the supernatants with or without hISLR - HA . β - actin was used as a loading control .

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

(D) Western blotting for pMST1/2, MST1, pMOB1 and MOB1, pYAP1 and YAP1 in lysates from HEK293FT cells cultured in the supernatants with or without hISLR-HA. β-actin was used as a loading control.


( E ) Western blotting for HA and ISLR in the supernatant from NCM460 colonic epithelial cells with pcDNA3 . 1 empty vector or pcDNA3 . 1 - hISLR - HA plasmids . Western blotting for pMST1 / 2 , MST1 , pMOB1 and MOB1 , pYAP1 and YAP1 in lysates from NCM460 CRC cells cultured in the supernatants with or without hISLR - HA . β - actin was used as a loading control .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE B-DISEASE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(E) Western blotting for HA and ISLR in the supernatant from NCM460 colonic epithelial cells with pcDNA3.1 empty vector or pcDNA3.1-hISLR-HA plasmids. Western blotting for pMST1/2, MST1, pMOB1 and MOB1, pYAP1 and YAP1 in lysates from NCM460 CRC cells cultured in the supernatants with or without hISLR-HA. β-actin was used as a loading control.


( F ) Immunofluorescence for YAP1 in HEK293FT cells cultured in the supernatant with or without hISLR - HA . Scale bar : 10 μm .

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(F) Immunofluorescence for YAP1 in HEK293FT cells cultured in the supernatant with or without hISLR-HA. Scale bar: 10 μm.


( G ) Western blotting for YAP1 in cytoplasmic or nuclear proteins from HEK293FT cells cultured in the supernatant with or without hISLR - HA . Lamin a / c , a positive control for nuclear proteins . α - Tubulin , a positive control for cytoplasmic proteins .

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

(G) Western blotting for YAP1 in cytoplasmic or nuclear proteins from HEK293FT cells cultured in the supernatant with or without hISLR-HA. Lamin a/c, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins.


( H ) qRT - PCR for CTGF and CYR61 in HEK293FT cells cultured in the supernatant with or without hISLR - HA . n = 3 .

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

(H) qRT-PCR for CTGF and CYR61 in HEK293FT cells cultured in the supernatant with or without hISLR-HA. n=3.


( A ) Western blotting for Islr in the supernatant from primary cultured intestinal epithelial cells ( IECs ) transfected with pcDNA3 . 1 or pcDNA3 . 1 - mIslr - HA plasmids , and western blotting for pMst1 / 2 , Mst1 , pMob1 and Mob1 , pYap1 and Yap1 in lysates from primary IECs cultured in the supernatants with or without mIslr - HA . β - actin was used as a loading control .

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

(A) Western blotting for Islr in the supernatant from primary cultured intestinal epithelial cells (IECs) transfected with pcDNA3.1 or pcDNA3.1-mIslr-HA plasmids, and western blotting for pMst1/2, Mst1, pMob1 and Mob1, pYap1 and Yap1 in lysates from primary IECs cultured in the supernatants with or without mIslr-HA. β-actin was used as a loading control.


( B ) Western blotting for Yap1 in nuclear or cytoplasmic proteins from primary intestinal epithelial cells cultured in the supernatant with or without mIslr . Histone H3 , a positive control for nuclear proteins . α - Tubulin , a positive control for cytoplasmic proteins .

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

(B) Western blotting for Yap1 in nuclear or cytoplasmic proteins from primary intestinal epithelial cells cultured in the supernatant with or without mIslr. Histone H3, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins.


( C ) qRT - PCR for Ctgf , Cry61 and Fstl1 in primary intestinal epithelial cells cultured in the supernatant with or without mIslr for 24 hours . n = 4 .

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

(C) qRT-PCR for Ctgf, Cry61 and Fstl1 in primary intestinal epithelial cells cultured in the supernatant with or without mIslr for 24 hours. n=4.


( D ) Activity of TEAD - Luciferase reporter in primary intestinal epithelial cells upon mIslr , Yap1 or mIslr and Yap1 . n = 4 .

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(D) Activity of TEAD-Luciferase reporter in primary intestinal epithelial cells upon mIslr, Yap1 or mIslr and Yap1. n=4.


( E ) The growth curve of primary intestinal epithelial cells upon mIslr or / and Yap1 siRNA over time . n = 5 .

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

(E) The growth curve of primary intestinal epithelial cells upon mIslr or/and Yap1 siRNA over time. n=5.


( F ) qRT - PCR for Ctgf and Cry61 in primary intestinal epithelial cells 48 hours after treatments of mIslr or / and Yap1 siRNA ( siYap1 ) . n = 4 .

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(F) qRT-PCR for Ctgf and Cry61 in primary intestinal epithelial cells 48 hours after treatments of mIslr or/and Yap1 siRNA (siYap1). n=4.


( G ) Representative images of primary intestinal organoids cultured at indicated conditions and timepoints . Scale bar : 100 μm . The organoids were co - cultured in a mixed medium of the supernatant with / without mIslr from CT26 cells and regular organoid culture solution at the ratio of 1 : 4 . Quantification of organoids diameter at indicated timepoints . n > 150 organoids at each timepoint .

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(G) Representative images of primary intestinal organoids cultured at indicated conditions and timepoints. Scale bar: 100 μm. The organoids were co-cultured in a mixed medium of the supernatant with/without mIslr from CT26 cells and regular organoid culture solution at the ratio of 1:4. Quantification of organoids diameter at indicated timepoints. n>150 organoids at each timepoint.


( H ) Western blotting for pMst1 / 2 , Mst1 , pMob1 and Mob1 , pYap1 and Yap1 24 hours after the treatment . β - actin was used as a loading control .

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(H) Western blotting for pMst1/2, Mst1, pMob1 and Mob1, pYap1 and Yap1 24 hours after the treatment. β-actin was used as a loading control.


( I ) qRT - PCR for Ctgf , Cry61 and Fstl1 in primary intestinal organoids 24 hours after the treatments . n = 3 .

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(I) qRT-PCR for Ctgf, Cry61 and Fstl1 in primary intestinal organoids 24 hours after the treatments. n=3.


( J ) Immunofluorescence for Yap1 and β - catenin in intestinal organoids 24 hours after the indicated treatments . The treatments are identical to ( G ) . The percentage of nuclear Yap1 + cells versus epithelial cells was quantified . Scale bar : 50 μm . n = 12 .

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(J) Immunofluorescence for Yap1 and β-catenin in intestinal organoids 24 hours after the indicated treatments. The treatments are identical to (G). The percentage of nuclear Yap1+ cells versus epithelial cells was quantified. Scale bar: 50 μm. n=12.


A PHIP - 1 interaction with GPB - 1 by yeast two - hybrid assay . The reporter strain PJ69 - 4A was co - transformed with expression vectors encoding GAL4 DBD - PHIP - 1 ( H45A ) and GAL4 AD - GPB - 1 , as indicated . Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5 - aminotriazole for 4 days .

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A PHIP-1 interaction with GPB-1 by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-PHIP-1(H45A) and GAL4 AD-GPB-1, as indicated. Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.


A His - phosphorylation of GPB - 1 in animals . The phip - 1 ( km96 ) mutant animals carrying the 3XFLAG : : gpb - 1 or 3XFLAG : : gpb - 1 ( H266F ) knock - in allele were lysed . The lysates were treated with or without heating ( 95 ° C ) and immunoblotted ( IB ) with anti - 3 - pHis and anti - FLAG antibodies .

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A His-phosphorylation of GPB-1 in animals. The phip-1(km96) mutant animals carrying the 3XFLAG::gpb-1 or 3XFLAG::gpb-1(H266F) knock-in allele were lysed. The lysates were treated with or without heating (95°C) and immunoblotted (IB) with anti-3-pHis and anti-FLAG antibodies.


B NDK - 1 phosphorylates GPB - 1 in vitro . COS - 7 cells were co - transfected with HA - GPB - 1 or HA - GPB - 1 ( H266F ) and T7 - GPC - 2 , and cell lysates were immunoprecipitated ( IP ) with anti - HA antibodies . Immunopurified GPB - 1 was subjected to the in vitro kinase assay with recombinant GST - NDK - 1 . Phosphorylated GPB - 1 was detected by immunoblotting ( IB ) with anti - 3 - pHis antibodies .

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B NDK-1 phosphorylates GPB-1 in vitro. COS-7 cells were co-transfected with HA-GPB-1 or HA-GPB-1(H266F) and T7-GPC-2, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies. Immunopurified GPB-1 was subjected to the in vitro kinase assay with recombinant GST-NDK-1. Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3-pHis antibodies.


C PHIP - 1 dephosphorylates GPB - 1 in vitro . COS - 7 cells were co - transfected with HA - GPB - 1 and T7 - GPC - 2 , and cell lysates were immunoprecipitated ( IP ) with anti - HA antibodies . The immunopurified HA - GPB - 1 was first subjected to the in vitro kinase assay with recombinant GST - NDK - 1 . Phosphorylated GPB - 1 was then equally aliquoted and subjected to the in vitro phosphatase assay with recombinant GST - PHIP - 1 or its variants . Phosphorylated GPB - 1 was detected by immunoblotting ( IB ) with anti - 3 - pHis antibodies .

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C PHIP-1 dephosphorylates GPB-1 in vitro. COS-7 cells were co-transfected with HA-GPB-1 and T7-GPC-2, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies. The immunopurified HA-GPB-1 was first subjected to the in vitro kinase assay with recombinant GST-NDK-1. Phosphorylated GPB-1 was then equally aliquoted and subjected to the in vitro phosphatase assay with recombinant GST-PHIP-1 or its variants. Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3-pHis antibodies.


A PHIP - 1 interaction with UNC - 51 by yeast two - hybrid assay . The reporter strain PJ69 - 4A was co - transformed with expression vectors encoding GAL4 DBD - PHIP - 1 ( H45A ) and GAL4 AD - UNC - 51 ( 274 - 856 ) , as indicated . Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5 - aminotriazole for 4 days .

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

A PHIP-1 interaction with UNC-51 by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-PHIP-1(H45A) and GAL4 AD-UNC-51(274-856), as indicated. Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.


C UNC - 51 phosphorylates PHIP - 1 at Ser - 112 . COS - 7 cells were co - transfected with Flag - PHIP - 1 ( WT or mutants ) and GFP - UNC - 51 [ WT or ∆ AIKAI ( KD ) ] , and cell lysates were analyzed using Phos - tag SDS - PAGE . Total lysates were immunoblotted ( IB ) with antibodies , as indicated . Filled and open arrowheads indicate unmodified and phosphorylated PHIP - 1 , respectively . Asterisk indicates non - specific band .

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

C UNC-51 phosphorylates PHIP-1 at Ser-112. COS-7 cells were co-transfected with Flag-PHIP-1 (WT or mutants) and GFP-UNC-51 [WT or ∆AIKAI (KD)], and cell lysates were analyzed using Phos-tag SDS-PAGE. Total lysates were immunoblotted (IB) with antibodies, as indicated. Filled and open arrowheads indicate unmodified and phosphorylated PHIP-1, respectively. Asterisk indicates non-specific band.


( A - B ) THP1 over - expressing IFITM3 , IFITM3 - Y20F , IFITM3 - ∆ 21 or control were pre - treated or not with Amphotericin B for 1 hour and then transduced with a VSV - gp ( A ) or Measles - gp ( B ) pseudotyped LV . Transduction efficiencies were evaluated at FACS five days later . Fold rescue of transduction over the DMSO control was calculated . Data are shown as mean ± SEM ( n = 5 - 8 biological replicates run in technical duplicate ) . P values are for one sample t test versus Oe - Luc DMSO = 1 , * * for p < 0 . 01 , * * * for p < 0 . 001 .

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

(A-B) THP1 over-expressing IFITM3, IFITM3-Y20F, IFITM3-∆21 or control were pre-treated or not with Amphotericin B for 1 hour and then transduced with a VSV-gp (A) or Measles-gp (B) pseudotyped LV. Transduction efficiencies were evaluated at FACS five days later. Fold rescue of transduction over the DMSO control was calculated. Data are shown as mean ± SEM (n=5-8 biological replicates run in technical duplicate). P values are for one sample t test versus Oe-Luc DMSO =1, ** for p<0.01, *** for p<0.001.


( E - F ) THP1 over - expressing IFITM3 , IFITM3 lysin - less mutant ( 4KR ) ( E ) , IFITM3 CIL - lysin mutant ( 3KR ) ( F ) or control were transduced with VSV - gp pseudotyped LV . Transduction efficiencies were evaluated by flow cytometry five days post - TD . Data represent the mean ± SEM ( n = 9 - 6 biological replicates run in technical duplicate ) . p values are for one sample t test verso Oe - Luc DMSO = 1 . * for p < 0 . 05 * * for p < 0 . 01 . * * * * for p < 0 . 0001 and ns for not significant .

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(E-F) THP1 over-expressing IFITM3, IFITM3 lysin-less mutant (4KR) (E), IFITM3 CIL-lysin mutant (3KR) (F) or control were transduced with VSV-gp pseudotyped LV. Transduction efficiencies were evaluated by flow cytometry five days post-TD. Data represent the mean ± SEM (n=9-6 biological replicates run in technical duplicate). p values are for one sample t test verso Oe-Luc DMSO= 1. * for p<0.05 ** for p<0.01. **** for p<0.0001 and ns for not significant.


( G - H ) THP1 over - expressing IFITM1 , IFITM1 - 3KR ( G ) , IFITM3 , IFITM3 - Y20F , IFITM3 - Y20F - 3KR ( H ) or control were transduced with Measles - gp pseudotyped LV . Transduction efficiencies were evaluated by flow cytometry five days post - TD . Data represent the mean ± SEM ( n = 4 - 9 biological replicates run in technical duplicate ) . p values are for one sample t test verso Oe - Luc DMSO = 1 . * for p < 0 . 05 * * for p < 0 . 01 . * * * * for p < 0 . 0001 .

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(G-H) THP1 over-expressing IFITM1, IFITM1-3KR (G), IFITM3, IFITM3-Y20F, IFITM3-Y20F-3KR (H) or control were transduced with Measles-gp pseudotyped LV. Transduction efficiencies were evaluated by flow cytometry five days post-TD. Data represent the mean ± SEM (n=4-9 biological replicates run in technical duplicate). p values are for one sample t test verso Oe-Luc DMSO= 1. * for p<0.05 ** for p<0.01.**** for p<0.0001.


( A - B ) Immunofluorescence images were performed using TCS SP5 Leica confocal microscope , 60x with oil on THP1 over - expressing IFITM3 - WT , IFITM3 - 4KR , IFITM3 - 3KR or IFITM3 - Y20F ( A ) ( n = 22 images acquired from 3 biological replicates ) and primary monocytes derived macrophages ( MDM ) over - expressing IFITM3 - WT , IFITM3 - 4KR , IFITM3 - 3KR ( B ) ( n = 12 images acquired from 3 independent donors ) . Co - localization ( purple areas marked by white arrows ) of IFITM3 ( in red ) with the lysosome associated membrane protein 1 ( LAMP1 ) marker ( in blue ) was evaluated . Representative zoomed images are shown ,

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

(A-B) Immunofluorescence images were performed using TCS SP5 Leica confocal microscope, 60x with oil on THP1 over-expressing IFITM3-WT, IFITM3-4KR, IFITM3-3KR or IFITM3-Y20F (A) (n=22 images acquired from 3 biological replicates) and primary monocytes derived macrophages (MDM) over-expressing IFITM3-WT, IFITM3-4KR, IFITM3-3KR (B) (n=12 images acquired from 3 independent donors). Co-localization (purple areas marked by white arrows) of IFITM3 (in red) with the lysosome associated membrane protein 1 (LAMP1) marker (in blue) was evaluated. Representative zoomed images are shown,


( D ) IFITM3 - IFITM3 interactions ( red dots ) were evaluated by proximity ligation assay ( PLA ) in THP1 knock out for endogenous IFITM3 and expressing either HA - IFITM3 and His - IFITM3 , HA - 3KR and His - 3KR or His - dimer mutant IFITM3 ( D . M ) and HA - dimer mutant . Images were acquired on TCS SP5 Leica confocal microscope 60x with oil . Representative images are shown ( n = 8 images of 4 biological replicates ) , scale bar 40μm .

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(D) IFITM3-IFITM3 interactions (red dots) were evaluated by proximity ligation assay (PLA) in THP1 knock out for endogenous IFITM3 and expressing either HA-IFITM3 and His-IFITM3, HA-3KR and His-3KR or His-dimer mutant IFITM3 (D.M) and HA-dimer mutant. Images were acquired on TCS SP5 Leica confocal microscope 60x with oil. Representative images are shown (n=8 images of 4 biological replicates), scale bar 40μm.


( F ) IFITM3 dimerization was assessed by native PAGE followed by WB analysis in THP1 . IFITM3 higher order protein complex bands are highlighted by the asterisk . IFITM3 dimers and higher molecular weight complexes were quantified over the normalizer using ImageJ ( mean ± SEM , n = 7 biological replicates ) . p values are for Mann Whitney test . * for p < 0 . 05 , * * for p < 0 . 01 .

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(F) IFITM3 dimerization was assessed by native PAGE followed by WB analysis in THP1. IFITM3 higher order protein complex bands are highlighted by the asterisk. IFITM3 dimers and higher molecular weight complexes were quantified over the normalizer using ImageJ (mean ± SEM, n=7 biological replicates). p values are for Mann Whitney test. * for p<0.05, ** for p<0.01.


( B ) K562 were prestimulated with IFNα and then transduced with VSV - gp pseudotyped LV . Transduction was evaluated five days after TD ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . Data are represented as Folds over the Oe - Luc control . p values are for one sample t test versus - IFNα = 1 . ns for not significant .

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(B) K562 were prestimulated with IFNα and then transduced with VSV-gp pseudotyped LV. Transduction was evaluated five days after TD (mean ± SEM, n=4 biological replicates run in technical duplicate). Data are represented as Folds over the Oe-Luc control. p values are for one sample t test versus -IFNα = 1. ns for not significant.


( C ) Interferon stimulated genes ( ISGs ) mRNA expression were measured 24 hours post - IFNα stimulation by RT - qPCR in K562 ( mean ± SEM , n = 3 biological replicates run in technical duplicate ) .

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(C) Interferon stimulated genes (ISGs) mRNA expression were measured 24 hours post-IFNα stimulation by RT-qPCR in K562 (mean ± SEM, n=3 biological replicates run in technical duplicate).


( E ) K562 were pre - stimulated or not with IFNα for 24 hours and then inoculated with the infectious ZIKV ( mean ± SEM , n = 3 biological replicates run in technical triplicates ) . Viral supernatant was collected at different time points and titered on Vero E6 cells . p values are for Mann Whitney test . * * for p < 0 . 01 , * * * for p < 0 . 001 .

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(E) K562 were pre-stimulated or not with IFNα for 24 hours and then inoculated with the infectious ZIKV (mean ± SEM, n=3 biological replicates run in technical triplicates). Viral supernatant was collected at different time points and titered on Vero E6 cells. p values are for Mann Whitney test. ** for p<0.01, *** for p<0.001.


( F ) K562 over - expressing IFITM1 , IFITM1 - 3KR or control were transduced with Measles - gp pseudotyped LV . Transduction efficiency was evaluated five days post - TD ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for one sample t test versus Oe - Luc = 1 . * * * * for p < 0 . 0001 .

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

(F) K562 over-expressing IFITM1, IFITM1-3KR or control were transduced with Measles-gp pseudotyped LV. Transduction efficiency was evaluated five days post-TD (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc = 1. **** for p<0.0001.


K562 over - expressing IFITM3 , IFITM3 - Y20F , IFITM3 - Y20F - 3KR or control were pre - exposed or not to Ampho B and then transduced with Measles - gp ( G ) , BaEV - gp ( H ) pseudotyped LV . Transduction efficiencies were measured by flow cytometry five days after TD ( mean ± SEM , n = 5 - 4 - 4 biological replicates run in technical duplicate ) . p values are for one sample t test versus Oe - Luc DMSO = 1 or unpaired t test for Ampho B versus DMSO . * for p < 0 . 05 , * * for p < 0 . 01 , * * * for p < 0 . 001 , * * * * for p < 0 . 0001 .

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

K562 over-expressing IFITM3, IFITM3-Y20F, IFITM3-Y20F-3KR or control were pre-exposed or not to Ampho B and then transduced with Measles-gp (G), BaEV-gp (H) pseudotyped LV. Transduction efficiencies were measured by flow cytometry five days after TD (mean ± SEM, n=5-4-4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc DMSO=1 or unpaired t test for Ampho B versus DMSO. * for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001.


( A ) Interaction between IFITM3 wild - type or 3KR and Phosphatydinositol - 3 phosphate ( PIP3 ) ( red dots ) was evaluated by proximity ligation assay . Images were acquired using TCS SP5 Leica confocal microscope 60x with oil in THP1 knock out for endogenous IFITM3 and expressing either IFITM3 wild - type or IFITM3 - 3KR . Number of foci were counted on imageJ and normalized over the number of nuclei ( mean ± SEM , n = 5 biological replicates ) . White arrows indicate foci , scale bar 40μm .

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

(A) Interaction between IFITM3 wild-type or 3KR and Phosphatydinositol-3 phosphate (PIP3) (red dots) was evaluated by proximity ligation assay. Images were acquired using TCS SP5 Leica confocal microscope 60x with oil in THP1 knock out for endogenous IFITM3 and expressing either IFITM3 wild-type or IFITM3-3KR. Number of foci were counted on imageJ and normalized over the number of nuclei (mean ± SEM, n=5 biological replicates). White arrows indicate foci, scale bar 40μm.


( B ) Phosphatydinositol - 3 phosphate ( PIP3 ) levels were measured by immunofluorescence in THP1 over - expressing the His - IFITM3 / HA - IFITM3 and His - 3KR / HA - 3KR . PIP3 was quantified via Imagej ( mean ± SEM , n = 5 biological replicates ) .

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

(B) Phosphatydinositol-3 phosphate (PIP3) levels were measured by immunofluorescence in THP1 over-expressing the His-IFITM3/HA-IFITM3 and His-3KR/HA-3KR. PIP3 was quantified via Imagej (mean ± SEM, n=5 biological replicates).


( C ) THP1 over - expressing IFITM3 or control were transduced with VSV - gp pseudotyped LV in presence or absence of Rapamycin ( Rapa ) . Transduction efficiency was measured at FACS five days post - TD ( mean ± SEM , n = 6 biological replicates run in technical duplicate . p values are for one sample t test versus Oe - Luc DMSO = 1 and Mann Whitney test for Rapa vs DMSO . * * for p < 0 . 01 , * * * * for p < 0 . 0001 .

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

(C) THP1 over-expressing IFITM3 or control were transduced with VSV-gp pseudotyped LV in presence or absence of Rapamycin (Rapa). Transduction efficiency was measured at FACS five days post-TD (mean ± SEM, n=6 biological replicates run in technical duplicate. p values are for one sample t test versus Oe-Luc DMSO=1 and Mann Whitney test for Rapa vs DMSO. ** for p<0.01, **** for p<0.0001.


( D ) THP1 over - expressing IFITM3 - Y20F - 3KR or control were exposed or not to Rapamycin and transduced with Measles - gp pseudotyped LV . Transduction efficiency was evaluated by flow cytometry five days post - TD ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for one sample t test versus Oe - Luc DMSO = 1 . * * for p < 0 . 0 .

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

(D) THP1 over-expressing IFITM3-Y20F-3KR or control were exposed or not to Rapamycin and transduced with Measles-gp pseudotyped LV. Transduction efficiency was evaluated by flow cytometry five days post-TD (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc DMSO=1. ** for p<0.0.


( E ) Hematopoietic stem and progenitor cells ( HSPC ) were transduced with VSV - gp or Measles - gp pseudotyped LV in presence or absence of Rapamycin . Transduction was evaluated at FACS five days later measuring the % of BFP expression ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for Mann Whitney test . * for p < 0 . 05 .

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

(E) Hematopoietic stem and progenitor cells (HSPC) were transduced with VSV-gp or Measles-gp pseudotyped LV in presence or absence of Rapamycin. Transduction was evaluated at FACS five days later measuring the % of BFP expression (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for Mann Whitney test. * for p<0.05.


Levels of phosphatidylinositol ( PI ) precursors of PIP3 and PIP2 were measured through lipidomic analysis in HSPC and K562 . The results are presented as log plots of the PI ratio in K562 over HSPC ( D ) .

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

Levels of phosphatidylinositol (PI) precursors of PIP3 and PIP2 were measured through lipidomic analysis in HSPC and K562. The results are presented as log plots of the PI ratio in K562 over HSPC (D).


( E ) PIP3 levels were measured by immunofluorescence ( scale bar 40μm ) and quantified as integrated density with ImageJ software in THP1 and K562 over - expressing IFITM3 ( mean ± SEM , n = 3 biological replicates ) . p values are for Mann Whitney test , * for p < 0 . 05 .

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

(E) PIP3 levels were measured by immunofluorescence (scale bar 40μm) and quantified as integrated density with ImageJ software in THP1 and K562 over-expressing IFITM3 (mean ± SEM, n=3 biological replicates). p values are for Mann Whitney test, * for p<0.05.