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

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

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-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O 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 O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O 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 O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O 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 O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

(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 O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

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

(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 O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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 O O O O O B-MEASURED_VAR O O O O O O 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 O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O 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 O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR 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 O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(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 O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(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 O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O 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 O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O 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 .

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

(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 .

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

(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 .

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

(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 O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O 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) 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 O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O 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 .

(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 .

O O 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) 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 .

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

(D) 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 .

(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 .

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

(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 .

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

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 .

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

(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 O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 O O O O O O O O O O O O O O O O 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 O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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 O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O 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 O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O 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 O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(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 .

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

(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 O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 O O O O O O 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 O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(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 O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(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 .

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

(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 O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O 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 O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O 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 O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(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 O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O 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 O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR 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 .

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

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

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

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 .

O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

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

(G-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 O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 .

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

(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 .

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

(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 .

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

(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 ) .

O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(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 O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( F ) Interaction between IFITM3 and Phosphatydinositol - 3 phosphate ( PIP3 ) ( red dots ) was evaluated by proximity ligation assay using TCS SP5 Leica confocal microscope 60x with oil in THP1 or K562 expressing same levels of IFITM3 . Number of foci and PLA foci intensity were measured on imageJ and normalized over the number of nuclei ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) , scale bar 40μm . p values are for Mann Whitney test , * * for p < 0 . 01 , * * * for p < 0 . 001 . ) .

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

(F) Interaction between IFITM3 and Phosphatydinositol-3 phosphate (PIP3) (red dots) was evaluated by proximity ligation assay using TCS SP5 Leica confocal microscope 60x with oil in THP1 or K562 expressing same levels of IFITM3. Number of foci and PLA foci intensity were measured on imageJ and normalized over the number of nuclei (mean ± SEM, n=4 biological replicates run in technical duplicate), scale bar 40μm. p values are for Mann Whitney test, ** for p<0.01, *** for p<0.001.).


PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in THP1 and K562 ( G ) pre - stimulated or not with IFN - α ( mean ± SEM , n = 3 biological replicates . p values are for Mann Whitney test , * * * for p < 0 . 001 .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in THP1 and K562 (G) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.


PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in HSPC ( H ) pre - stimulated or not with IFN - α ( mean ± SEM , n = 3 biological replicates . p values are for Mann Whitney test , * * * for p < 0 . 001 .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in HSPC (H) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.


PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in MDM ( I ) pre - stimulated or not with IFN - α ( mean ± SEM , n = 3 biological replicates . p values are for Mann Whitney test , * * * for p < 0 . 001 .

PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in MDM (I) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.


( A ) Calu3 over - expressing IFITM1 , IFITM2 , IFITM3 , PM - mutant IFITM3 Y20A or control were transduced with SARS - CoV2 Spike - pseudotyped LV ( mean ± SEM , n > 6 biological replicates run in technical duplicate ) . Transduction efficiency was measured by FACS five days after transduction , p values are for one sample paired T test versus Oe - Luc Ctrl = 1 , * * for p < 0 . 01 .

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

(A) Calu3 over-expressing IFITM1, IFITM2, IFITM3, PM-mutant IFITM3 Y20A or control were transduced with SARS-CoV2 Spike-pseudotyped LV (mean ± SEM, n>6 biological replicates run in technical duplicate). Transduction efficiency was measured by FACS five days after transduction, p values are for one sample paired T test versus Oe-Luc Ctrl=1, **for p<0.01.


( B - C ) Calu3 cells over - expressing IFITM1 , IFITM2 , IFITM3 , IFITM3 Y20A or control were infected with SARS - CoV2 . Viral titer was calculated by plaque assay in Vero E6 cells ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for Mann Whitney test , * for p < 0 . 05 , * * * for p < 0 . 001 .

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

(B-C) Calu3 cells over-expressing IFITM1, IFITM2, IFITM3, IFITM3 Y20A or control were infected with SARS-CoV2. Viral titer was calculated by plaque assay in Vero E6 cells (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for Mann Whitney test, * for p<0.05, *** for p<0.001.


( G ) Calu3 over - expressing IFITM1 , IFITM3 or control were challenged to SARS - CoV2 Omicron pseudotyped LV . Transduction efficiency was evaluated 5 days post - transduction ( mean ± SEM , n = 7 biological replicates run in technical duplicate ) . p values are for one sample T test versus Oe - Luc Ctrl = 1 , * * for p < 0 . 01 , * * * * for p < 0 . 0001 .

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

(G) Calu3 over-expressing IFITM1, IFITM3 or control were challenged to SARS-CoV2 Omicron pseudotyped LV. Transduction efficiency was evaluated 5 days post-transduction (mean ± SEM, n=7 biological replicates run in technical duplicate). p values are for one sample T test versus Oe-Luc Ctrl=1, **for p<0.01, ****for p<0.0001.


( H - I ) Calu3 cells over - expressing IFITM1 , IFITM2 , IFITM3 or control ( H ) or IFITM3 , Y20A or control ( I ) were infected with Omicron at MOI 1 . Viral supernatants were collected at different time points and tittered on Vero E6 cells . Data are presented as mean ± SEM ( n = 4 - 4 biological replicates run in technical triplicate ) . p values are for Mann Whitney test , * * for p < 0 . 01 , * * * for p < 0 . 001 , * * * * for p < 0 . 0001 .

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

(H-I) Calu3 cells over-expressing IFITM1, IFITM2, IFITM3 or control (H) or IFITM3, Y20A or control (I) were infected with Omicron at MOI 1. Viral supernatants were collected at different time points and tittered on Vero E6 cells. Data are presented as mean ± SEM (n=4-4 biological replicates run in technical triplicate). p values are for Mann Whitney test , **for p<0.01, ***for p<0.001, ****for p<0.0001.


( A - B ) VSV ( A ) or ZIKV ( B ) viral supernatants were collected one day post - infection in THP1 ( A ) or Calu3 ( B ) over - expressing IFITM3 , IFITM3 lysin - less mutant ( 4KR ) , IFITM3 CIL - lysin mutant ( 3KR ) or control and titered on VERO E6 cells . Data represent the mean ± SEM ( n = 5 - 3 biological replicates run in technical duplicate ) . p values are for Mann Whitney test . * for p < 0 . 05 , * * for p < 0 . 01 .

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

(A-B) VSV (A) or ZIKV (B) viral supernatants were collected one day post-infection in THP1 (A) or Calu3 (B) over-expressing IFITM3, IFITM3 lysin-less mutant (4KR), IFITM3 CIL-lysin mutant (3KR) or control and titered on VERO E6 cells. Data represent the mean ± SEM (n=5-3 biological replicates run in technical duplicate). p values are for Mann Whitney test. * for p<0.05, ** for p<0.01.


( C - D ) K562 were inoculated with the infectious VSV ( C ) or ZIKV ( D ) ( mean ± SEM , n = 3 biological replicates run in technical duplicate ) . Viral supernatant was collected at different time points and titered on Vero E6 cells .

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

(C-D) K562 were inoculated with the infectious VSV (C) or ZIKV (D) (mean ± SEM, n=3 biological replicates run in technical duplicate). Viral supernatant was collected at different time points and titered on Vero E6 cells.


( I ) IFITM3 and PIP3 level were measured by immunofluorescence ( scale bar 100µm ) and quantified as integrated density with ImageJ software in Calu3 infected or not with Omicron ( n = 3 biological replicates ) . p values are for Mann Whitney test , * * for p < 0 . 01 . ( J ) PIP3 coupled to a specific PIP carrier ( PIP3 + Carr Ctrl ) or Carr Ctrl were provided to Calu3 over - expressing IFITM3 , control or KO for IFITM3 prior to infection with SARS - CoV2 Omicron . Infectious supernatants were collected after three days and tittered in Vero E6 cells ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for Wilcoxon signed ranked test , * for p < 0 . 05 . The inhibitory effect of exogenous PIP3 on infection is represented as Fold versus Carr Ctrl ( 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) IFITM3 and PIP3 level were measured by immunofluorescence (scale bar 100µm) and quantified as integrated density with ImageJ software in Calu3 infected or not with Omicron (n=3 biological replicates). p values are for Mann Whitney test, ** for p<0.01. (J) PIP3 coupled to a specific PIP carrier (PIP3+Carr Ctrl) or Carr Ctrl were provided to Calu3 over-expressing IFITM3, control or KO for IFITM3 prior to infection with SARS-CoV2 Omicron. Infectious supernatants were collected after three days and tittered in Vero E6 cells (mean ± SEM, n=4 biological replicates run in technical duplicate ). p values are for Wilcoxon signed ranked test, * for p<0.05. The inhibitory effect of exogenous PIP3 on infection is represented as Fold versus Carr Ctrl (mean ± SEM, n=4 biological replicates run in technical duplicate ). p values are for Mann Whitney test, * for p<0.05.


Western blot analysis was performed using SETD1A fragment - expressing 293T cells . These constructs were used for the following rescue experiments . Representative images from 2 independent experiments are shown .

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

Western blot analysis was performed using SETD1A fragment-expressing 293T cells. These constructs were used for the following rescue experiments. Representative images from 2 independent experiments are shown.


Setd1afl / fl ; CreER cells were transfected with SETD1A fragment and endogenous Setd1a was deleted by tamoxifen , then assayed for colony - forming potential . Representative data from 1 out of 3 independent experiments with 3 biological replicates are shown . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 , * P ≤ 0 . 05 ( Student ' s t - test ) .

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

Setd1afl/fl;CreER cells were transfected with SETD1A fragment and endogenous Setd1a was deleted by tamoxifen, then assayed for colony-forming potential. Representative data from 1 out of 3 independent experiments with 3 biological replicates are shown. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).


qRT - PCR was performed at 3 days post - tamoxifen to analyze Fancd2 expression in human SETD1A - expressing Setd1afl / fl ; CreER MLL - AF9 leukemia cells . qRT - PCR with 3 biological replicates was performed . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 , * P ≤ 0 . 05 ( Student ' s t - test ) .

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

qRT-PCR was performed at 3 days post-tamoxifen to analyze Fancd2 expression in human SETD1A-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells. qRT-PCR with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).


293T cells were transfected with SETD1A - GFP fusion constructs . Cell nucleus was visualized with DAPI . The percentage of GFP nuclear localization is indicated in each image . Asterisks in images indicate the cells with nuclear specific SETD1A - GFP signals . Scale bars : 10 µm . G − H . Mouse Setd1afl / fl ; CreER MLL - AF9 leukemia cells were transfected with FO3 - GFP fusion constructs . ( G ) Subcellular localization of GFP fusion constructs was visualized by immunofluorescence and nucleus was stained with DAPI . The percentage of GFP nuclear localization is indicated in each image . Scale bars : 10 µm . ( H ) FO3 - GFP fusion expressing cells were treated with tamoxifen and colony assay with 3 biological replicates was performed . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 , * P ≤ 0 . 05 ( Student ' s t - test ) .

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

293T cells were transfected with SETD1A-GFP fusion constructs. Cell nucleus was visualized with DAPI. The percentage of GFP nuclear localization is indicated in each image. Asterisks in images indicate the cells with nuclear specific SETD1A-GFP signals. Scale bars: 10 µm. G−H. Mouse Setd1afl/fl;CreER MLL-AF9 leukemia cells were transfected with FO3-GFP fusion constructs. (G) Subcellular localization of GFP fusion constructs was visualized by immunofluorescence and nucleus was stained with DAPI. The percentage of GFP nuclear localization is indicated in each image. Scale bars: 10 µm. (H) FO3-GFP fusion expressing cells were treated with tamoxifen and colony assay with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).


Relative expression of Mlh1 in SETD1A rescued MLL - AF9 leukemia cells . qRT - PCR with 3 biological replicates was performed . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 , * P ≤ 0 . 05 ( Student ' s t - test for ( D )

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

Relative expression of Mlh1 in SETD1A rescued MLL-AF9 leukemia cells. qRT-PCR with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test for (D)


Percentage of GFP + SETD1A mutant - expressing leukemia cells in PB was analyzed at 3 weeks post - transplant ( n = 5 / group ) . Data information : * * P ≤ 0 . 01 , * P ≤ 0 . 05 One - way

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

Percentage of GFP+ SETD1A mutant-expressing leukemia cells in PB was analyzed at 3 weeks post-transplant (n = 5 / group). Data information: **P ≤ 0.01, *P ≤ 0.05 One-way ANOVA


Survival of recipient mice harboring SETD1A mutant - expressing leukemia cells after transplantation ( TX ) was plotted ( n = 5 / group ) . Data information : * * P ≤ 0 . 01 , * P ≤ 0 . 05 Kaplan - Meier for ( F ) ) .

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

Survival of recipient mice harboring SETD1A mutant-expressing leukemia cells after transplantation (TX) was plotted (n = 5 / group). Data information: **P ≤ 0.01, *P ≤ 0.05 Kaplan-Meier for (F)).


SETD1A FLOS domain - binding proteins were identified by Co - IP MS . Full - length SETD1A and FO3 fragment were used as baits and overlapped proteins ( red dots ) were identified as SETD1A FLOS domain - binding proteins .

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

SETD1A FLOS domain-binding proteins were identified by Co-IP MS. Full-length SETD1A and FO3 fragment were used as baits and overlapped proteins (red dots) were identified as SETD1A FLOS domain-binding proteins.


293T cells were transfected with full - length SETD1A alanine mutants and protein extracts were used for the Co - IP experiments .

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

293T cells were transfected with full-length SETD1A alanine mutants and protein extracts were used for the Co-IP experiments.


F . MOLM - 13 leukemia cells were examined by ChIP - seq analyses against HA - tagged SETD1A , BuGZ , BUB3 , H3K4me3 and H3K4me1 . Heatmaps of SETD1A ( + ) / BuGZ ( + ) regions ( upper ) and SETD1A ( - ) / BuGZ ( + ) regions ( lower ) are shown .

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

F. MOLM-13 leukemia cells were examined by ChIP-seq analyses against HA-tagged SETD1A, BuGZ, BUB3, H3K4me3 and H3K4me1. Heatmaps of SETD1A(+)/BuGZ(+) regions (upper) and SETD1A(-)/BuGZ(+) regions (lower) are shown.


Relative expression levels of Bugz were analyzed in Bugz shRNA - expressing mouse MLL - AF9 leukemia cells at day 3 post - treatment , respectively . Data from 3 biological replicates are shown . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 ( Student ' s t -

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

Relative expression levels of Bugz were analyzed in Bugz shRNA-expressing mouse MLL-AF9 leukemia cells at day 3 post-treatment, respectively. Data from 3 biological replicates are shown. Data information: data are presented as mean ± SD. **P ≤ 0.01 (Student's t-test


Survival of recipient mice harboring dox - inducible shRNA - expressing leukemia cells was plotted ( n = 8 - 10 / group ) . Data information : * * P ≤ 0 . 01 Kaplan - Meier for ( H ) ) .

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

Survival of recipient mice harboring dox-inducible shRNA-expressing leukemia cells was plotted (n = 8-10 / group). Data information: **P ≤ 0.01 Kaplan-Meier for (H)).


Images are shown of representative karyotypes in control , Bugz and Bub3 shRNA - expressing leukemia cells . Asterisks in images indicate the trisomy 15 in all cells analyzed . Red arrows indicate chromosomal aneuploidies . Representative images of sister - chromatid separation or aneuploidy in Bub3 shRNAs are shown in lower panels .

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

Images are shown of representative karyotypes in control, Bugz and Bub3 shRNA-expressing leukemia cells. Asterisks in images indicate the trisomy 15 in all cells analyzed. Red arrows indicate chromosomal aneuploidies. Representative images of sister-chromatid separation or aneuploidy in Bub3 shRNAs are shown in lower panels.


293T cells were transfected with SETD1A F2 alanine mutant constructs . Myc - DDK - tagged SETD1A was used for Co - IP .

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

293T cells were transfected with SETD1A F2 alanine mutant constructs. Myc-DDK-tagged SETD1A was used for Co-IP.


293T cells were transfected with BuGZ mutants . Myc - DDK - tagged BuGZ mutants illustrated on Figure S5B were used for Co - IP .

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

293T cells were transfected with BuGZ mutants. Myc-DDK-tagged BuGZ mutants illustrated on Figure S5B were used for Co-IP.


293T cells were transfected with BUB3 mutants and protein extracts were used for the Co - IP experiments . Myc - DDK - tagged BUB3 mutants illustrated on Figure S5C were used .

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

293T cells were transfected with BUB3 mutants and protein extracts were used for the Co-IP experiments. Myc-DDK-tagged BUB3 mutants illustrated on Figure S5C were used.


293T cells were transfected with Myc - DDK - tagged human BuGZ constructs and the protein extracts were used for a Co - IP experiment .

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

293T cells were transfected with Myc-DDK-tagged human BuGZ constructs and the protein extracts were used for a Co-IP experiment.


293T cells were transfected with Myc - DDK - tagged BuGZ variant constructs and the protein extracts were used for the Co - IP experiment .

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

293T cells were transfected with Myc-DDK-tagged BuGZ variant constructs and the protein extracts were used for the Co-IP experiment.


293T cells were transfected with Myc - DDK - tagged BuGZ constructs and the protein extracts were used for a Co - IP experiment .

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

293T cells were transfected with Myc-DDK-tagged BuGZ constructs and the protein extracts were used for a Co-IP experiment.


293T cells were transfected with Myc - DDK - tagged wild - type BuGZ and the cells were treated with 1 , 6 - hexanediol for 3 h . The protein extracts were used for the Co - IP experiments .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

293T cells were transfected with Myc-DDK-tagged wild-type BuGZ and the cells were treated with 1,6-hexanediol for 3 h. The protein extracts were used for the Co-IP experiments.


SETD1A - GFP fusion - expressing Setd1afl / fl ; CreER MLL - AF9 leukemia cells were treated with tamoxifen , then assayed for colony - forming potential . The FUCCI reporter ( mCherry - Cdt1 ( 30 - 120 ) / Citrine - Geminin ( 1 - 110 ) ) - expressing cells were used as control . Data from 3 biological replicates are shown . Data are presented as mean ± SD .

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

SETD1A-GFP fusion-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells were treated with tamoxifen, then assayed for colony-forming potential. The FUCCI reporter (mCherry-Cdt1(30-120)/Citrine-Geminin(1-110))-expressing cells were used as control. Data from 3 biological replicates are shown. Data are presented as mean ± SD.


SGC - expressing Setd1afl / fl ; CreER MLL - AF9 leukemia cells were treated with tamoxifen , and RNA - seq analysis was performed with sorted cells . Data are shown as log2 fold change over tamoxifen non - treated GFP - expressing control .

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

SGC-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells were treated with tamoxifen, and RNA-seq analysis was performed with sorted cells. Data are shown as log2 fold change over tamoxifen non-treated GFP-expressing control.


( A , B ) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice . Brain sections were prepared 1 day after BrdU injection . The white box area is magnified in the lower panels : DAPI ( blue ) , Ki67 ( green ) , and BrdU ( red ) . Arrow heads indicate BrdU and Ki - 67 double positive cells in the SGZ .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

(A, B) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice. Brain sections were prepared 1 day after BrdU injection. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ.


( C , D ) Differentiating cells are identified by staining with BrdU and DCX . Brain sections were prepared 10 days after BrdU injection .

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

(C, D) Differentiating cells are identified by staining with BrdU and DCX. Brain sections were prepared 10 days after BrdU injection.


( E , F ) Maturation of progenitor cells are assessed by staining with BrdU and NeuN . Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness , and a total of 24 sections were obtained from one hippocampus . Scale bars : 100 μm in the large panel , and 10 μm in the magnified images . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animals for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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

(E, F) Maturation of progenitor cells are assessed by staining with BrdU and NeuN. Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness, and a total of 24 sections were obtained from one hippocampus. Scale bars:100 μm in the large panel, and 10 μm in the magnified images. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.05, **P< 0.01.


( A ) miR - 124 expression is altered in DSCR1 mutants . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animals for each condition , * P < 0 . 01 .

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

(A) miR-124 expression is altered in DSCR1 mutants. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.01.


( B ) Promoter activity of miR - 124 in Neuro2 A cells with DSCR1 reduction or overexpression . Firefly luciferase reporter under the control of miR - 124 promoter is measured . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Promoter activity of miR-124 in Neuro2 A cells with DSCR1 reduction or overexpression. Firefly luciferase reporter under the control of miR-124 promoter is measured. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01.


( C ) Bisulfite sequencing analyses of the miR - 124 promoter . Each CpG site is indicated , and the methylation status of two different regions are shown . Open and filled circles represent unmethylated and methylated CpG sites , respectively . The percentage of methylated CpGs among total number of CpGs is also shown .

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

(C) Bisulfite sequencing analyses of the miR-124 promoter. Each CpG site is indicated, and the methylation status of two different regions are shown. Open and filled circles represent unmethylated and methylated CpG sites, respectively. The percentage of methylated CpGs among total number of CpGs is also shown.


( D ) Neuro2A cells containing DSCR1shRNA show increased activity of miR - 124 promoter measured . The miR - 124 promoter was inserted in front of the luciferase reporter . Site - directed mutation of two methylation sites ( 31 and 58 ) in the promoter shows the luciferase activity similar to that of DSCR1 reduction . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Neuro2A cells containing DSCR1shRNA show increased activity of miR-124 promoter measured. The miR-124 promoter was inserted in front of the luciferase reporter. Site-directed mutation of two methylation sites (31 and 58) in the promoter shows the luciferase activity similar to that of DSCR1 reduction. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.


( E ) Overexpression of DSCR1 reduced the luciferase activity , while removing all methylation sites in the miR - 124 promoter increases it . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Overexpression of DSCR1 reduced the luciferase activity, while removing all methylation sites in the miR-124 promoter increases it. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.


( A ) DSCR1 binds to TET1 introns . The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA .

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

(A) DSCR1 binds to TET1 introns. The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA.


( B ) DSCR1 missing RNA recognition motif ( △ RRM ) does not bind to the TET1 intron 8 .

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

(B) DSCR1 missing RNA recognition motif (△RRM) does not bind to the TET1 intron 8.


( C , D ) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4 . DSCR1 reduction increases the activity of this luciferase construct , while DSCR1 overexpression decreases its activity . In contrast , altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 . * * * P < 0 . 001 .

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

(C, D) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4. DSCR1 reduction increases the activity of this luciferase construct, while DSCR1 overexpression decreases its activity. In contrast, altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01. ***P< 0.001.


( E ) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants . TET1 is increased in DSCR1 knockout , while it is decreased in DSCR1 transgenic mice . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animal for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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

(E) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants. TET1 is increased in DSCR1 knockout, while it is decreased in DSCR1 transgenic mice. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animal for each condition, *P< 0.05, **P< 0.01.


( B ) 3 weeks after lentivirus injection , neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region . The white arrow indicates Sox2 and GFP double positive cells . Scale bar , 10 μm . ( C ) The number of GFP and Sox2 double positive cells is restored to that of wild type . Scale bar : 10μm . Values are shown as mean ± SEM and tested for statistical significance by paired t - test . N = 3 animal for each condition , * P < 0 . 05 .

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

(B) 3 weeks after lentivirus injection, neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region. The white arrow indicates Sox2 and GFP double positive cells. Scale bar, 10 μm. (C) The number of GFP and Sox2 double positive cells is restored to that of wild type. Scale bar: 10μm. Values are shown as mean ± SEM and tested for statistical significance by paired t-test. N = 3 animal for each condition, *P< 0.05.


( A ) Genotype of Ts65Dn / DSCR1 + / - mouse was confirmed by detecting markers for Ts65Dn and DSCR1

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

(A) Genotype of Ts65Dn/DSCR1+/- mouse was confirmed by detecting markers for Ts65Dn and DSCR1 KO


( B ) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2 . 5 folds compared to that of wild type . However , Ts65Dn / DSCR1 + / - mouse showed normal level of DSCR1 mRNA transcript . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

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

(B) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2.5 folds compared to that of wild type. However, Ts65Dn/DSCR1+/- mouse showed normal level of DSCR1 mRNA transcript. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N=3 for each condition, *P< 0.05.


As expected , the levels of TET1 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores the TET1 mRNA expression . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 .

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

As expected, the levels of TET1 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the TET1 mRNA expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.


As expected , the levels of miR - 124 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores the miR - 124 expression . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

As expected, the levels of miR-124 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the miR-124 expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.