Representative image of transduced with GCaMP6 ( Left ) . Yellow arrows ( mesenchymal ) , red arrows ( epithelial ) , scale bar = 50μm . GCaMP6 mean ( MFI ) in ECADHIGH or ECADLOW cultured in indicated medium ( 0 Ca2 + or 2 Ca2 + ) for 1h ( Right ) . Representative of 3 independent murine PDA lines run in triplicate . Statistical by Student ' s unpaired t - ( * , p < 0 . 05 ; non - significant ( NS ) ; ± SD ) .

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

Representative image of tumor cells transduced with GCaMP6 (Left). Yellow arrows (mesenchymal cells), red arrows (epithelial cells), scale bar = 50μm. GCaMP6 mean fluorescent intensity (MFI) in ECADHIGH or ECADLOW cells cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h (Right). Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (*, p<0.05; non-significant (NS); ±SD).


Flow cytometry of ECADHIGH or ECADLOW loaded with Indo - 1 cultured in indicated medium ( 0 Ca2 + or 2 Ca2 + ) for 1h . Representative of 2 independent lines ( n indicates number of analyzed in each group , ECADHIGH 0 Ca2 + n = 4963 , ECADLOW 0 Ca2 + n = 14 , ECADHIGH 2 Ca2 + n = 6575 , ECADLOW 2 Ca2 + n = 29 ) . Representative of 2 independent murine PDA lines run in triplicate . Statistical by Student ' s unpaired t - ( * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

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

Flow cytometry analysis of ECADHIGH or ECADLOW cells loaded with Indo-1 and cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h. Representative of 2 independent cell lines (n indicates the number of cells analyzed in each group, ECADHIGH 0 Ca2+ n=4963, ECADLOW 0 Ca2+ n=14, ECADHIGH 2 Ca2+ n=6575, ECADLOW 2 Ca2+ n=29). Representative of 2 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (****, p<0.0001; non-significant (NS); ±SD).


Representative 20X brightfield of murine YFP + treated for 72h with vehicle control ( DMSO ) , 2 . 5μM Ionomycin , or 10ng / ml TGFβ co - stained for E - cadherin ( Red ) DAPI ( Blue ) . Scale bar is 100μm .

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Representative 20X brightfield and fluorescent images of murine YFP+ tumor cells treated for 72h with vehicle control (DMSO), 2.5μM Ionomycin, or 10ng/ml TGFβ and co-stained for E-cadherin (Red) and DAPI (Blue). Scale bar is 100μm.


Representative flow cytometry of surface ECAD ( Left ) , MFI of surface ECAD ( Middle ) , of surface ECAD negative ( Right ) in murine PDA , after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , 10ng / ml TGFβ for 72h . Representative of 3 independent murine PDA lines run in triplicate . Statistical by ANOVA ( * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR 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

Representative flow cytometry analysis of surface ECAD (Left), MFI of surface ECAD (Middle), and percentage of surface ECAD negative cells (Right) in murine PDA cells, after treatment with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 72h. Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by ANOVA (****, p<0.0001; non-significant (NS); ±SD).


Top : Relative mRNA expression of Ecad after treatment with 2 . 5μM ionomycin or 10ng / ml TGFβ for denoted time . Bottom : Western blot of total E - cadherin Vimentin for denoted time . Experiment was run in duplicate with three different murine PDA lines . Statistical by ANOVA ( * * * , p < 0 . 001 ; non - significant ( NS ) ; ± ) .

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

Top: Relative mRNA expression of Ecad after treatment with 2.5μM ionomycin or 10ng/ml TGFβ for denoted time. Bottom: Western blot analysis of total E-cadherin and Vimentin protein for denoted time. Experiment was run in duplicate with three different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; non-significant (NS); ±SEM).


Normalized MFI of surface E - cadherin in PDA treated with DMSO , 2 . 5μM Ionomycin ( IONO ) , 10ng / ml TGFβ for 7 days , followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on x - axis . Treated were analyzed at day 2 , 3 , 7 . Withdrawn were analyzed on days 1 , 2 , 7 . Experiment was performed in duplicate in two different murine PDA lines . Error bars indicate SD .

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

Normalized MFI of surface E-cadherin in PDA cells treated with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 7 days, followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on the x-axis. Treated cells were analyzed at day 2, 3, and 7. Withdrawn cells were analyzed on days 1, 2, and 7. Experiment was performed in duplicate in two different murine PDA cell lines. Error bars indicate SD.


of live migration over 4h after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , or 10ng / ml TGFβ for 48hrs . ( n = 16 per condition . Experiment was run in triplicate in two murine PDA different lines ) ( Right ) . of transwell migration after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , or 10ng / ml TGFβ for 72h ( n = 2 lines , 3 replicates per with 3 20X taken per transwell ) ( Left ) . Statistical by ANOVA ( * , p < 0 . 05 ; * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

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 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 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

Quantification of live cellular migration over 4h after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 48hrs. (n=16 cells per condition. Experiment was run in triplicate in two murine PDA different cell lines) (Right). Quantification of transwell migration after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 72h (n= 2 cell lines, 3 replicates per cell line with 3 20X images taken per transwell) (Left). Statistical analysis by ANOVA (*, p<0.05; ****, p<0.0001; non-significant (NS); ±SD).


Schematic for sequencing after EMT induction for 48h . illustration of EMT related genes in three independent murine PDA lines , sequenced in duplicate , treated with DMSO , 2 . 5μM Ionomycin , or 10ng / ml TGFβ . Scale indicates Z - score .

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Schematic for RNA sequencing after EMT induction for 48h. Heatmap illustration of EMT related genes in three independent murine PDA cell lines, sequenced in duplicate, treated with DMSO, 2.5μM Ionomycin, or 10ng/ml TGFβ. Scale indicates Z-score.


GSEA of ionomycin versus DMSO ( Left ) or TGFβ versus DMSO ( Right ) for EMT signatures . NES , nominal P - value , FDR are shown . Statistical performed in GSEA .

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GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for EMT signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


GSEA of ionomycin versus DMSO ( Left ) or TGFβ versus DMSO ( Right ) for calcium signatures . NES , nominal P - value , FDR are shown . Statistical performed in GSEA .

GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for calcium signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


Leading edge plots showing enrichment of C - EMT gene signatures in TGFβ treated ( Left ) or P - EMT gene signatures in ionomycin ( IONO ) treated ( Right ) , on GSEA . Enrichment scores ( ES ) , NES , nominal P - value , FDR are shown . Statistical performed in GSEA .

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-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

Leading edge plots showing enrichment of C-EMT gene signatures in TGFβ treated cells (Left) or P-EMT gene signatures in ionomycin (IONO) treated cells (Right), based on GSEA. Enrichment scores (ES), NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


Fura - 2 Ca2 + measurements after addition of 5μM CNO ( Top ) or 10μM ATP ( Bottom ) in murine PDA expressing a Gαq - DREADD . Blue or red indicates average of n = 22 . ± SD . Tracings are representative of experiments performed twice in 2 independent lines .

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Fura-2 Ca2+ measurements after addition of 5μM CNO (Top) or 10μM ATP (Bottom) in murine PDA cells expressing a Gαq-DREADD. Blue or red line indicates average of n=22 cells. ±SD. Tracings are representative of experiments performed twice in 2 independent cell lines.


Representative 20X brightfield of Gαq - DREADD - expressing 48h after addition of CNO ( Left ) . Scale bar = 100μm . MFI of surface E - cadherin in expressing a Gαq - DREADD after treatment with DMSO , 5μM CNO , or 2 . 5μM ionomycin ( Right ) . Both experiments performed in triplicate in 2 independent lines . Statistical by ANOVA ( * * , p < 0 . 01 ; ± SD ) .

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 I-MEASURED_VAR I-MEASURED_VAR 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

Representative 20X brightfield images of Gαq-DREADD-expressing cells 48h after addition of CNO (Left). Scale bar = 100μm. MFI of surface E-cadherin in cells expressing a Gαq-DREADD after treatment with DMSO, 5μM CNO, or 2.5μM ionomycin (Right). Both experiments performed in triplicate in 2 independent cell lines. Statistical analysis by ANOVA (**, p<0.01; ±SD).


MFI of surface E - cadherin in CnB knockout lines after 48h treatment with DMSO , 2 . 5μM ionomycin , or 10ng / ml TGFβ . Experiment was repeated in two different murine PDA lines in triplicate . Statistical by ANOVA ( * * * , p < 0 . 001 ; * * * * , p < 0 . 00001 ; ± SD ) .

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 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

MFI of surface E-cadherin in CnB knockout lines after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).


MFI of surface E - cadherin of murine PDA pretreated with 20μM W7 for 24h then DMSO , 2 . 5μM ionomycin , or 20μM W7 with 2 . 5μM ionomycin for 24h . Experiment was repeated in two different murine PDA lines in triplicate . Statistical by ANOVA ( * * , p < 0 . 01 ; * * * , p < 0 . 001 ; non - significant ( NS ) ; ± SD ) .

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-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 O O O O O O O O O O O

MFI of surface E-cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO, 2.5μM ionomycin, or 20μM W7 with 2.5μM ionomycin for 24h. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (**, p<0.01; ***, p<0.001; non-significant (NS); ±SD).


MFI of surface E - cadherin of shRNA non - targeting ( shNT ) ( Left ) or shCAMK2B ( Right ) after 48h treatment with DMSO , 2 . 5μM ionomycin , or 10ng / ml TGFβ . Experiment was repeated in two different murine PDA lines . Statistical by ANOVA ( * * * , p < 0 . 001 ; * * * * , p < 0 . 00001 ; ± SD ) .

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 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

MFI of surface E-cadherin of shRNA non-targeting (shNT) (Left) or shCAMK2B (Right) after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).


( a ) [ 3H ] thymidine incorporation at 72 h by wild - type ( WT ) Irgm1 - / - CD4 + T activated with soluble mAb to CD3 irradiated syngenic wild - type splenocytes .

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-CONTROLLED_VAR O O O O O O O

(a) [3H]thymidine incorporation at 72 h by wild-type (WT) and Irgm1-/- CD4+ T cells activated with soluble mAb to CD3 and irradiated syngenic wild-type splenocytes.


( b ) [ 3H ] thymidine incorporation at 72 h by OT - II Irgm1 - / - OT - II CD4 + T stimulated with OVA ( OVA ) or OVA peptide of 323 - 339 ( OVA ( 323 - 339 ) ) in presence of syngenic splenic DCs .

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-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

(b) [3H]thymidine incorporation at 72 h by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA protein (OVA) or OVA peptide of amino acids 323-339 (OVA(323-339)) in the presence of syngenic splenic DCs.


( c , d ) Kinetic of [ 3H ] thymidine incorporation ( c ) numbers ( d ) in cultures of OT - II Irgm1 − / − OT - II spleen stimulated with OVA peptide .

O 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

(c,d) Kinetic analysis of [3H]thymidine incorporation (c) and cell numbers (d) in cultures of OT-II and Irgm1−/− OT-II spleen cells stimulated with OVA peptide.


( e ) Flow cytometric of activation proliferation of OVA peptide - stimulated OT - II Irgm1 - / - OT - II CD4 + T pulsed with BrdU during final 4 h of 72 h of culture then stained with mAb to CD25 BrdU . Numbers in quadrants indicate viable CD4 + .

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(e) Flow cytometric analysis of the activation and proliferation of OVA peptide-stimulated OT-II and Irgm1-/- OT-II CD4+ T cells pulsed with BrdU during the final 4 h of 72 h of culture and then stained with mAb to CD25 and BrdU. Numbers in quadrants indicate percent viable CD4+ cells.


( f ) Enzyme - linked immunosorbent of IL - 2 produced by OT - II Irgm1 - / - OT - II CD4 + T stimulated with OVA peptide in presence of splenic DCs , measured at 72 h . Data are representative of more than five ( a , b ) or three ( c - f ) independent experiments with similar results ( mean s . d . of triplicate cultures , a - d , f ) .

O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Enzyme-linked immunosorbent assay of IL-2 produced by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DCs, measured at 72 h. Data are representative of more than five (a,b) or three (c-f) independent experiments with similar results (mean and s.d. of triplicate cultures, a-d,f).


( a ) Immunoblot of Irgm1 in lysates of flow cytometry - sorted naive wild - type CD4 + lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 ( 10 μg / ml ) alone or with mAb to CD28 ( 2 μg / ml ) in presence or absence of IFN - γ - neutralizing mAb . Actin , loading control .

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 B-CONTROLLED_VAR 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

(a) Immunoblot analysis of Irgm1 in lysates of flow cytometry-sorted naive wild-type CD4+ lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 (10 μg/ml) alone or with mAb to CD28 (2 μg/ml) in the presence or absence of IFN-γ-neutralizing mAb. Actin, loading control.


( b ) Enzyme - linked immunosorbent of IFN - γ produced by OT - II Irgm1 − / − OT - II CD4 + T stimulated with OVA peptide in presence of splenic DC in polarized conditions ( TH1 or TH2 ) or nonpolarized conditions ( N ) , assessed in 72 - hour cultures . ND , not detected .

O O 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 O O O O O O O O O O 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) Enzyme-linked immunosorbent assay of IFN-γ produced by OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions (TH1 or TH2) or nonpolarized conditions (N), assessed in 72-hour cultures. ND, not detected.


( c ) Accumulation of polarized nonpolarized OT - II ( open symbols ) or Irgm1 − / − OT - II ( filled symbols ) CD4 + T after peptide stimulation , quantified by counting of viable .

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(c) Accumulation of polarized and nonpolarized OT-II (open symbols) or Irgm1−/− OT-II (filled symbols) CD4+ T cells after peptide stimulation, quantified by counting of viable cells.


( d ) Viable OT - II Irgm1 − / − OT - II CD4 + T 72 h after restimulation with OVA peptide in presence of T - depleted APC samples .

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

(d) Viable OT-II and Irgm1−/− OT-II CD4+ T cells 72 h after restimulation with OVA peptide in the presence of T cell-depleted APC samples.


( e ) Flow cytometry of viability division of OT - II Irgm1 − / − OT - II CD4 + T stimulated with peptide , allowed to ' ' for 7 d in IL - 2 , then labeled with CFSE restimulated with OVA peptide in presence of T - depleted APC samples assessed 72 h later .

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(e) Flow cytometry of the viability and division of OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with peptide, allowed to 'rest' for 7 d in IL-2, then labeled with CFSE and restimulated with OVA peptide in the presence of T cell-depleted APC samples and assessed 72 h later.


( f ) Flow cytometric of population expansion of CFSE - labeled OT - II Irgm1 − / − OT - II CD4 + T activated with OVA peptide in presence or absence of IFN - γ - neutralizing mAb . Results in e , f obtained after gating on CD4 + T ; numbers in outlined areas ( e ) in top left corners ( f ) indicate CD4 + . PI , propidium iodide .

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-MEASURED_VAR 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 B-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 O O O O O

(f) Flow cytometric analysis of the population expansion of CFSE-labeled OT-II and Irgm1−/− OT-II CD4+ T cells activated with OVA peptide in the presence or absence of IFN-γ-neutralizing mAb. Results in e,f obtained after gating on CD4+ T cells; numbers in outlined areas (e) and in top left corners (f) indicate percent CD4+ cells. PI, propidium iodide.


( g ) [ 3H ] thymidine incorporation by wild - type , Irgm1 − / − , Ifng − / − Irgm1 − / − Ifng − / − CD4 + T stimulated for 72 h with soluble agonistic mAb to CD3 irradiated syngenic Ifng − / − splenocytes . Data are representative of two independent experiments with similar results ( mean s . d . of triplicate cultures , b - d , g ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR 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 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) [3H]thymidine incorporation by wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng−/− splenocytes. Data are representative of two independent experiments with similar results (mean and s.d. of triplicate cultures, b-d,g).


( a ) Flow cytometric of survival of CD45 . 2 + Ifng − / − ( open symbols ) or Irgm1 − / − Ifng − / − ( filled symbols ) CD4 + T lymphocytes preactivated with agonistic mAb to CD3 in vitro , then transferred intraperitoneally into congenic CD45 . 1 + C57BL / 6 . SJL recipients left untreated or inoculated intraperitoneally with T . gondii or M . avium 3 d earlier , assessed in peritoneal exudate samples collected at day 4 after transfer .

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 B-CONTROLLED_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 O O O O O O

(a) Flow cytometric analysis of the survival of CD45.2+ Ifng−/− (open symbols) or Irgm1−/−Ifng−/− (filled symbols) CD4+ T lymphocytes preactivated with agonistic mAb to CD3 in vitro, then transferred intraperitoneally into congenic CD45.1+ C57BL/6.SJL recipients left untreated or inoculated intraperitoneally with T. gondii or M. avium 3 d earlier, assessed in peritoneal exudate cell samples collected at day 4 after transfer.


( b ) Survival of M . avium - infected wild - type , Irgm1 − / − , Ifng − / − Irgm1 − / − Ifng − / − mice ( n = 5 mice per genotype ) .

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(b) Survival of M. avium-infected wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− mice (n = 5 mice per genotype).


( c - e ) Splenic loads ( c ) , circulating lymphocytes ( d ) splenic lymphocytes ( e ) , assessed at week 4 after M . avium infection ( n = 5 mice per genotype ) . Each symbol in a , c - e represents an mouse . Data are representative of two independent experiments .

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(c-e) Splenic bacterial loads (c), circulating lymphocytes (d) and splenic lymphocytes (e), assessed at week 4 after M. avium infection (n = 5 mice per genotype). Each symbol in a,c-e represents an individual mouse. Data are representative of two independent experiments.


( a ) [ 3H ] thymidine incorporation by naive CD4 + lymphocytes stimulated for 72 h with agonistic mAb to CD3 IFN - γ - deficient irradiated APCs , with IL - 2 ( 10 U / ml ) added 24 h after initiation of culture .

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(a) [3H]thymidine incorporation by naive CD4+ lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN-γ-deficient irradiated APCs, with IL-2 (10 U/ml) added 24 h after the initiation of culture.


( b ) Accumulation of viable CD4 + T at 72 h in presence of medium alone ( Med ) or IFN - γ ( 5 U / ml ) .

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(b) Accumulation of viable CD4+ T cells at 72 h in the presence of medium alone (Med) or IFN-γ (5 U/ml).


( c ) Flow cytometric of viability division of CD4 + T labeled with CFSE activated as described in a with T - depleted Ifng − / − APC samples in presence or absence of IFN - γ ( 5 U / ml ) . Numbers in plots indicate CFSE + CD4 + T populations .

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(c) Flow cytometric analysis of the viability and division of CD4+ T cells labeled with CFSE and activated as described in a with T cell-depleted Ifng−/− APC samples in the presence or absence of IFN-γ (5 U/ml). Numbers in plots indicate percent CFSE+CD4+ T cell populations.


( d - f ) of Ifng − / − Irgm1 − / − Ifng − / − T populations activated with mAb to CD3 expanded in IL - 2 , then treated with IFN - γ ( d ) , agonistic mAb to CD3 ( e ) or Fas ligand ( FasL ; f ) , assessed by propidium iodide at 48 h after treatment . Open bars , Ifng − / − ; filled bars , Irgm1 − / − Ifng − / − ( a , b , d - f ) . Data are representative of three independent experiments with similar results ( mean s . d . of triplicate cultures a , b , d - f ) .

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(d-f) Death of Ifng−/− and Irgm1−/−Ifng−/− T cell populations activated with mAb to CD3 and expanded in IL-2, then treated with IFN-γ (d), agonistic mAb to CD3 (e) or Fas ligand (FasL; f), assessed by propidium iodide staining at 48 h after treatment. Open bars, Ifng−/−; filled bars, Irgm1−/−Ifng−/− (a,b,d-f). Data are representative of three independent experiments with similar results (mean and s.d. of triplicate cultures a,b,d-f).


( a - f ) Electron microscopy of Ifng − / − CD4 + lymphocytes ( a ) Irgm1 − / − Ifng − / − CD4 + lymphocytes ( b - f ) exposed for 30 h to IFN - γ ( 5 U / ml ) . ( c ) Enlargement of outlined in b ; arrowheads indicate two autophagic vacuoles . ( d ) of vacuoles in 50 randomly selected electron microscopy sections of more than 400 . Each symbol represents one section . ( e ) Irgm1 − / − CD4 + T with many vacuoles containing dense materials . ( f ) Two Irgm1 − / − CD4 + with vacuolated cytoplasma . Original magnification ( a - c , e , f ) , × 1 , 000 . Data are representative of two experiments .

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(a-f) Electron microscopy analysis of Ifng−/− CD4+ lymphocytes (a) and Irgm1−/−Ifng−/− CD4+ lymphocytes (b-f) exposed for 30 h to IFN-γ (5 U/ml). (c) Enlargement of the area outlined in b; arrowheads indicate two autophagic vacuoles. (d) Quantification of vacuoles in 50 randomly selected electron microscopy sections of more than 400 cells. Each symbol represents one section. (e) Irgm1−/− CD4+ T cell with many vacuoles containing dense materials. (f) Two Irgm1−/− CD4+ cells with vacuolated cytoplasma. Original magnification (a-c,e,f), × 1,000. Data are representative of two experiments.


( g , h ) of Ifng − / − Irgm1 − / − Ifng − / − CD4 + T in presence or absence of wortmannin or Ly294002 ( g ) , assessed by flow cytometry with propidium iodide at 48 h after IFN - γ exposure . Data are representative of three independent experiments ( mean s . d . of triplicate cultures ) .

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(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells in the presence or absence of wortmannin or Ly294002 (g), assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).


( g , h ) of Ifng − / − Irgm1 − / − Ifng − / − CD4 + T transfected with control or beclin1 - specific small interfering , assessed by flow cytometry with propidium iodide at 48 h after IFN - γ exposure . Right ( h ) , immunoblot of beclin1 expression . C , control small interfering ; B , beclin1 - specific small interfering . Data are representative of three independent experiments ( mean s . d . of triplicate cultures ) .

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(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells transfected with control or beclin1-specific small interfering RNA, assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Right (h), immunoblot analysis of beclin1 expression. C, control small interfering RNA; B, beclin1-specific small interfering RNA. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).


Testicular maturation arrest in ( WHT3759 ) . Testicular tissue obtained by biopsy was sectioned stained with hematoxylin / eosin . Seminiferous tubules contained meiotic germ such as zygotene ( ZS , arrowheads ) pachytene spermatocytes ( PS , arrows ) , but no post - meiotic germ such as round spermatids were detectable . Scale bar , 25 μm .

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Testicular maturation arrest in the patient (WHT3759). Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin/eosin. Seminiferous tubules contained meiotic germ cells such as zygotene (ZS, arrowheads) and pachytene spermatocytes (PS, arrows), but no post-meiotic germ cells such as round spermatids were detectable. Scale bar, 25 μm.


Western blot of TEX11 in testes of 3 - month - old mice with different Tex11 gene dosages . ACTB serves as a loading control .

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Western blot analysis of TEX11 in the testes of 3-month-old mice with different Tex11 gene dosages. ACTB serves as a loading control.


Testis weight of 3-month-old males.


Sperm count of 3-month-old males.


Histological of testes from adult wild - type , Tex11 KI / KO , Tex11 − / Y . RS , round spermatids ; ES , elongating spermatids ; Pa , pachytene spermatocytes . Scale bar , 50 μm .

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Histological analysis of testes from adult wild-type, Tex11 KI/KO, and Tex11−/Y males. RS, round spermatids; ES, elongating spermatids; Pa, pachytene spermatocytes. Scale bar, 50 μm.


Tex11 knockin allele rescues chromosomal synapsis defects in Tex11 − / Y spermatocytes in 3 - month - old . Chromosomal synapsis defects were assessed by SYCP1 SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per ; for each genotype , three were analyzed . Abbreviations for Tex11 genotypes : − / Y , Tex11 knockout ( Yang et al , 2008 ) ; KI ; − / Y , Tex11 knockin knockout ; + / Y , wild type ; KI ; + / Y , Tex11 knockin plus wild - type Tex11 .

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The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11−/Y spermatocytes in 3-month-old males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed. Abbreviations for Tex11 genotypes: −/Y, Tex11 knockout (Yang et al, 2008); KI;−/Y, Tex11 knockin and knockout; +/Y, wild type; KI;+/Y, Tex11 knockin plus wild-type Tex11.


Reduced testis in juvenile but not adult Tex11 KI ; − / Y . time frames of first wave adult waves of spermatogenesis are indicated .

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Reduced testis weight in juvenile but not adult Tex11 KI; −/Y males. The time frames of the first wave and adult waves of spermatogenesis are indicated.


Increased chromosomal asynapsis in spermatocytes reduced of TEX11 in testes of 25 - day - old Tex11 KI / KO . Chromosomal synapsis defects were assessed by SYCP1 SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per ; for each genotype , three were analyzed .

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Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25-day-old Tex11 KI/KO males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed.


Pachytene spermatocytes from 25 - day - old Tex11 KI / KO contain significantly fewer MLH1 foci compared to wild - type spermatocytes . Values are shown as average ± standard deviation . Error bar is standard deviation .

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Pachytene spermatocytes from 25-day-old Tex11 KI/KO males contain significantly fewer MLH1 foci compared to wild-type spermatocytes. Values are shown as average ± standard deviation. Error bar is standard deviation.


number of MLH1 foci in pachytene spermatocytes from 3 - month - old positively correlates with increasing Tex11 gene dosage .

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The number of MLH1 foci in pachytene spermatocytes from 3-month-old males positively correlates with increasing Tex11 gene dosage.


of MLH1 foci in pachytene oocytes from ovaries of Tex11 − / − , Tex11 + / − , wild - type embryonic day - 17 . 5 ( E17 . 5 ) fetuses reveals a positive correlation of Tex11 gene dosage number of MLH1 foci .

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Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11−/−, Tex11+/−, and wild-type embryonic day-17.5 (E17.5) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci.


A Western blot reveals comparable of TEX11 in testes of wild - type point mutant KI / KO . ACTB serves as a control .

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A Western blot analysis reveals comparable levels of TEX11 protein in the testes of wild-type and point mutant KI/KO males. ACTB serves as a control.


B - D ( B ) , testisweight ( C ) in Tex11 KI ( V749A ) / KO .

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B-D Body weight (B), testisweight (C) in Tex11 KI(V749A)/KO males.


B - D Significantly reduced sperm ( per pair of epididymides ) in Tex11 KI ( V749A ) / KO ( D ) .

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B-D Significantly reduced sperm count (per pair of epididymides) in Tex11 KI(V749A)/KO males (D).


E Mating test of 3- to 5-month-old males.


F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI ( V749A ) / KO . 100 pachytene spermatocytes per mouse were examined by surface spread .

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F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI(V749A)/KO males. 100 pachytene spermatocytes per mouse were examined by surface spread analysis.


G Number of MLH1 foci in pachytene spermatocytes . Note a significantly higher number of MLH1 foci in spermatocytes from KI ( Q173R ) / KO .

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G Number of MLH1 foci in pachytene spermatocytes. Note a significantly higher number of MLH1 foci in spermatocytes from KI(Q173R)/KO males.


migration assays were performed using Transwell migration chambers . HCT116 were transfected with siRNAs targeting 11 candidate lincRNAs migration activities were evaluated by Transwell migration assays ( n = 3 ) . CALIC is lincRNA targeted by si - LINC00920 .

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Cell migration assays were performed using Transwell migration chambers. HCT116 cells were transfected with siRNAs targeting 11 candidate lincRNAs and cell migration activities were evaluated by Transwell migration assays (n = 3). CALIC is the lincRNA targeted by si-LINC00920.


Migration of HCT116 transfected with indicated siRNAs or negative control . ( n = 4 )

Migration of HCT116 cells transfected with the indicated siRNAs or negative control. (n = 4)


Migration of colon cancer treated with an siRNA targeting CALIC . ( n = 3 )

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Migration of colon cancer cells treated with an siRNA targeting CALIC. (n = 3)


Subcellular localization of CALIC . RNAs were isolated from fractions of HCT116 quantified by qRT - PCR ( n = 3 ) . controls : U6 , MALAT1 ; control : GAPDH .

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Subcellular localization analysis of CALIC. RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT-PCR (n = 3). Nuclear controls: U6, MALAT1; Cytoplasmic control: GAPDH.


CALIC expression in cancer tissues . N , normal mucosa ; T , .

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CALIC expression levels in cancer tissues. N, normal mucosa; T, tumor.


Expression of CALIC in skin tumors ( P ) their metastases ( M ) . Expression of CALIC in skin tumors of without ( w / o ) or with ( w ) metastases .

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Expression of CALIC in primary skin tumors (P) and their metastases (M). Expression of CALIC in primary skin tumors of patients without (w/o) or with (w) metastases.


qRT - PCR of CALIC expression in early ( I / II ) late ( III / IV ) stage colon cancers . [ Normal ( N ) n = 8 , stage I / II n = 14 , stage III / IV n = 26 ]

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qRT-PCR analysis of CALIC expression in early (I/II) and late (III/IV) stage colon cancers. [Normal (N) n = 8, stage I/II n = 14, stage III/IV n = 26]


Kaplan - Meier curves for disease - free survival of colon cancer whose tumors expressed low ( blue , n = 113 ) or high ( red , n = 250 ) of CALIC .

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Kaplan-Meier curves for disease-free survival of colon cancer patients whose primary tumors expressed low (blue, n = 113) or high (red, n = 250) levels of CALIC.


Biotinylated full - CALIC or antisense CALIC ( negative control ) generated in vitro were incubated with lysates from DLD - 1 colon cancer precipitated with streptavidin beads . Precipitated were resolved by SDS - PAGE followed by silver . band indicated by arrow was excised subjected to mass spectrometry .

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Biotinylated full-length CALIC or antisense CALIC (negative control) generated in vitro were incubated with lysates from DLD-1 colon cancer cells and precipitated with streptavidin beads. Precipitated proteins were resolved by SDS-PAGE followed by silver staining. The protein band indicated by the arrow was excised and subjected to mass spectrometry.


( Top ) schematic of full - mutants of CALIC used for precipitation of hnRNP - L from HCT116 lysates . mutated elements are indicated by cross marks . Se , sense transcript ; AS , antisense transcript . + , detectable binding activity ; - , no detectable activity . ( Bottom ) precipitated hnRNP - L biotin - labeled fragments of CALIC are shown .

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(Top) schematic of the full-length and mutants of CALIC used for the precipitation of hnRNP-L from HCT116 cell lysates. The mutated elements are indicated by the cross marks. Se, sense transcript; AS, antisense transcript. +, detectable binding activity; -, no detectable activity. (Bottom) precipitated hnRNP-L and biotin-labeled fragments of CALIC are shown.


RIP was performed using control rabbit IgG or anti - hnRNP - L antibody . HPRT1 lncRNA MALAT1 were used as negative controls .

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RIP analysis was performed using control rabbit IgG or anti-hnRNP-L antibody. HPRT1 and the lncRNA MALAT1 were used as negative controls.


Immunoblotting of precipitated by RIP assays using anti - hnRNP - L antibody are shown ( D ) .

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Immunoblotting analysis of proteins precipitated by RIP assays using anti-hnRNP-L antibody are shown (D).


ChIRP - immunoblotting of hnRNP - L association with endogeneous CALIC in HCT116 .

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ChIRP-immunoblotting analysis of hnRNP-L association with endogeneous CALIC in HCT116 cells.


- Seq of hnRNP - L - CALIC - regulated genes in HCT116 . Venn diagram showing overlap of genes differentially expressed by hnRNP - L ( blue ) or CALIC ( purple ) knockdown ( B , top ) . Expression of 59 genes regulated in common in both hnRNP - L CALIC knockdown relative to control siRNA experiments ( B , bottom ) . Two biological replicates ( R1 R2 ) were included for each siRNA treatment . number of genes within each category is indicated .

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RNA-Seq analysis of hnRNP-L- and CALIC-regulated genes in HCT116 cells. Venn diagram showing the overlap of genes differentially expressed by hnRNP-L (blue) or CALIC (purple) knockdown (B, top). Expression levels of 59 genes regulated in common in both hnRNP-L and CALIC knockdown cells relative to control siRNA experiments (B, bottom). Two biological replicates (R1 and R2) were included for each siRNA treatment. The number of genes within each category is indicated.


Scatterplots showing correlations between CALIC - hnRNP - L - regulated genes in ( B ) . Left , co - regulated 59 genes ; Right , differentially regulated genes ( DEGs ) . correlation was calculated using data except for gene indicated by asterisk .

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Scatterplots showing correlations between CALIC- and hnRNP-L-regulated genes in (B). Left, co-regulated 59 genes; Right, all differentially regulated genes (DEGs). The correlation was calculated using the data except for the gene indicated by the asterisk.


Genes induced by CALIC are significantly enriched in those upregulated by hnRNP - L . GSEA comparing genes repressed by CALIC hnRNP - L knockdown . NES , normalized enrichment score .

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Genes induced by CALIC are significantly enriched in those upregulated by hnRNP-L. GSEA comparing the genes repressed by CALIC and hnRNP-L knockdown. NES, normalized enrichment score.


showing expression of genes positively regulated in common by both CALIC hnRNP - L in colon rectal cancers . Samples are ordered on expression of AXL .

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Heatmap showing the expression levels of genes positively regulated in common by both CALIC and hnRNP-L in colon and rectal cancers. Samples are ordered based on the expression levels of AXL.


qRT - PCR analyses of AXL in HCT116 LS180 transfected with an siRNAs targeting

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qRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALIC


immunoblotting ( B , D ) analyses of AXL in HCT116 LS180 transfected with an siRNAs targeting

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immunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALIC


qRT - PCR analyses of AXL in HCT116 LS180 transfected with an siRNAs targeting hnRNP - L .

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qRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.


immunoblotting ( B , D ) analyses of AXL in HCT116 LS180 transfected with an siRNAs targeting hnRNP - L .

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immunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.


qRT - PCR analyses of AXL in HCT116 infected with a lentivirus expressing wild - type CALIC ( CALIC - full ) , CALIC - 296 - 913 or CALIC -

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qRT-PCR analyses of AXL in HCT116 cells infected with a lentivirus expressing wild-type CALIC (CALIC-full), CALIC-296-913 or CALIC-mut


HCT116 that had been treated with an siRNA targeting hnRNP - L or CALIC were transfected with an AXL reporter construct subjected to luciferase assays .

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HCT116 cells that had been treated with an siRNA targeting hnRNP-L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays.


ChIP assays were performed using anti - hnRNP - L antibody or control mouse IgG . promoter regions of GAPDH Egr1 were used as negative positive controls , respectively .

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ChIP assays were performed using anti-hnRNP-L antibody or control mouse IgG. The promoter regions of GAPDH and Egr1 were used as negative and positive controls, respectively.


Immunoblotting of precipitated by ChIP assays using anti - hnRNP - L

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Immunoblotting analysis of proteins precipitated by ChIP assays using anti-hnRNP-L antibody


ChIP of hnRNP - L association with AXL promoter region in CALIC knockdown HCT116 .

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ChIP analysis of hnRNP-L association with the AXL promoter region in CALIC knockdown HCT116 cells.


ChIP of hnRNP - L association with AXL promoter region in HCT116 expressing lentiviral CALIC - full .

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ChIP analysis of hnRNP-L association with the AXL promoter region in HCT116 cells expressing lentiviral CALIC-full.


ChIRP of CALIC binding to AXL promoter . Binding of CALIC to promoter regions of indicated genes was compared with that of LacZ .

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ChIRP analysis of CALIC binding to the AXL promoter. Binding of CALIC to the promoter regions of the indicated genes was compared with that of LacZ.


Migratory activity of AXL knockdown HCT116 .

Migratory activity of AXL knockdown HCT116 cells.


Lentiviral expression of AXL restores migration activity of HCT116 transfected with an siRNA targeting CALIC . Immunoblotting qRT - PCR analyses of HA - tagged AXL CALIC expression , respectively ( left ) . α - Tubulin was used as a loading control .

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Lentiviral expression of AXL restores the migration activity of HCT116 cells transfected with an siRNA targeting CALIC. Immunoblotting and qRT-PCR analyses of HA-tagged AXL and CALIC expression, respectively (left). α-Tubulin was used as a loading control.


Representative of gross specimens ( top ) H & E stained sections ( bottom ) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 . Tumors were marked with yellow arrows . dashed lines depict boundary between normal tissues . ( Right ) of mice with lung metastases .

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Representative images of gross specimens (top) and H&E stained sections (bottom) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 cells. Tumors were marked with yellow arrows. The dashed lines depict the boundary between normal and tumor tissues. (Right) Percentage of mice with lung metastases.


Representative of gross specimens of cecal tumors ( top ) liver metastatic lesions ( bottom ) in mice orthotopically ( cecal ) injected with CALIC knockdown HCT116 . Tumors are marked with yellow arrows . ( Right ) of mice with tumors liver metastases .

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Representative images of gross specimens of primary cecal tumors (top) and liver metastatic lesions (bottom) in mice orthotopically (cecal) injected with CALIC knockdown HCT116 cells. Tumors are marked with yellow arrows. (Right) Percentage of mice with primary tumors and liver metastases.


B . RT - PCR for forebrain ( FOXG1 ) , optic cup ( VSX2 ) , dorsal forebrain ( PAX6 EMX2 ) ventral forebrain ( NKX2 - 1 ) identity in organoids . expression values ( 2 - ΔCt ) calculated relative to reference gene TBP . was performed on 5 - 6 organoids from multiple independent differentiations - control ( 7 ) , dorsal ( 7 ) , extended ventral ( 9 ) short ventral ( 5 ) . Significance values , * = < 0 . 05 , * * = < 0 . 01 , * * * = < 0 . 001 . Statistical was performed using one - way ANOVA post - hoc Tukey ' s comparison of means . central band depicts median , boxes depict values between lower upper quartile whiskers display minimum maximum values .

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B. RT-PCR analysis for forebrain (FOXG1), optic cup (VSX2), dorsal forebrain (PAX6 and EMX2) and ventral forebrain (NKX2-1) identity in organoids. All expression values (2-ΔCt) calculated relative to reference gene TBP. Analysis was performed on 5-6 organoids from multiple independent differentiations - control (7), dorsal (7), extended ventral (9) and short ventral (5). Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's comparison of means. The central band depicts the median, the boxes depict values between lower and upper quartile and the whiskers display the minimum and maximum values.


F . Magnified ( 60x ) dorsal regions from organoids fusions at day 60 ; Co - immunostaining of GFP with markers of interneuronal ( GABA ) , GABAergic ( DLX2 ) mature neuronal ( NeuN ) identity . Scale bars , 10μm . arrows indicate GFP + with positive labeling . G . of co - expression of GFP with markers mentioned in 1F in dorsal ventral regions of organoid fusions at day 60 . was performed on a total of 3 organoid fusions ( technical replicates ) from two independent differentiations each ( biological replicates ) , with 2 regions of interest ( ROI ; 10x field of view ) in dorsal ventral regions of each fusion analyzed . Each dot represents of co - expression for both ROI analyzed per organoid fusion . Significance values , * = < 0 . 05 , * * = < 0 . 01 , * * * = < 0 . 001 . Statistical was performed using an unpaired two - Student ' s t - . central band displays mean error bars depict SD .

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F. Magnified (60x) dorsal regions from organoids fusions at day 60; Co-immunostaining of GFP with markers of interneuronal (GABA), GABAergic (DLX2) and mature neuronal (NeuN) identity. Scale bars, 10μm. White arrows indicate GFP+ cells with positive labeling. G. Quantification of co-expression of GFP with markers mentioned in 1F in dorsal and ventral regions of organoid fusions at day 60. Quantification was performed on a total of 3 organoid fusions (technical replicates) from two independent differentiations each (biological replicates), with 2 regions of interest (ROI; 10x field of view) in the dorsal and ventral regions of each fusion analyzed. Each dot represents the quantification of co-expression for both ROI analyzed per organoid fusion. Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using an unpaired two-tailed Student's t-test. The central band displays the mean and error bars depict the SD.


C . UMAPs depicting expression of GABAergic markers DLX2 DLX5 , progenitor marker TOP2A , MGE marker SOX6 , CGE marker NR2F2 , striatal marker ZFHX3 .

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C. UMAPs depicting the expression of GABAergic markers DLX2 and DLX5, progenitor marker TOP2A, MGE marker SOX6, CGE marker NR2F2, and striatal marker ZFHX3.


C . UMAPs depicting relative expression of migration genes CXCR4 EPHA5 . Visualization of gene expression of migration genes CXCR4 EPHA5 along pseudotime axis . D . UMAPs depicting their relative expression of neurotransmitter genes GRIA4 GRIA2 along with visualization of their gene expression along pseudotime axis .

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C. UMAPs depicting the relative expression of migration genes CXCR4 and EPHA5. Visualization of the gene expression of migration genes CXCR4 and EPHA5 along the pseudotime axis. D. UMAPs depicting their relative expression of neurotransmitter genes GRIA4 and GRIA2 along with visualization of their gene expression along the pseudotime axis.


B . Illustration of saltatory tangential migration performed by an interneuron . Nucleus is marked with a red asterisk . taken on a spinning disc confocal microscope at 10x magnification .

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B. Illustration of saltatory tangential migration performed by an interneuron. Nucleus is marked with a red asterisk. All images taken on a spinning disc confocal microscope at 10x magnification.


C . depicting control - normalized values ( log2 - scaled ) for selected parameters ( from Figure S9A ) for 8 receptors analyzed in 5B . Grey values indicated non - significant values . In total 3979 moving tracks with following number of tracks per group : Control - 696 , Bicuculline - Methiodide - 1189 , Saclofen - 527 , TPMPA - 138 , AP - 5 - 429 , CNQX - 500 , Strychnine - 98 , Serotonin - 180 , SR57227 - 222 , were analyzed .

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C. Heatmap depicting the control-normalized values (log2-scaled) for selected parameters (from Figure S9A) for the 8 receptors analyzed in 5B. Grey values indicated non-significant values. In total 3979 moving cell tracks with the following number of tracks per group: Control - 696, Bicuculline-Methiodide - 1189, Saclofen - 527, TPMPA - 138, AP-5 - 429, CNQX - 500, Strychnine - 98, Serotonin - 180, and SR57227 - 222, were analyzed.


C . Mean square displacement ( MSD ) in μm2 over delay in minutes ( min ) for tracks in a cluster . curves are color - coded to denote corresponding cluster . For visualization purposes , MSD is shown for a time delay corresponding to 24 hours of imaging .

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C. Mean square displacement (MSD) in μm2 over the delay in minutes (min) for all cell tracks in a cluster. The curves are color-coded to denote the corresponding cluster. For visualization purposes, the MSD is shown for a time delay corresponding to 24 hours of imaging.


F . Bar plot depicting fold - change in composition of cluster groups - directed , exploratory or confined - of treatment groups relative to control distribution . Bars are color - coded to denote corresponding cluster group . dotted represents control baseline for visualization of fold changes between groups .

F. Bar plot depicting the fold-change in composition of cluster groups - directed, exploratory or confined - of treatment groups relative to the control distribution. Bars are color-coded to denote the corresponding cluster group. The dotted line represents the control baseline for visualization of the fold changes between the groups.


Growth curves of MCF7 ( left ) T47D ( right ) upon gene silencing with siRNAs . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA 1 for HNRNPC , siHN - 2 : siRNA 2 for HNRNPC , siLMNA : siRNA for LMNA as another negative control . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the MCF7 (left) and T47D cells (right) upon gene silencing with siRNAs. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.


Relative of HNRNPC upon siRNA - mediated silencing , quantified from Western blots of 3 replicates . error bars represent ± SD .

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Relative protein levels of HNRNPC upon siRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD.


Growth curves of Tet - on Crispr - Cas9 - MCF7 ( left ) Tet - on Crispr - Cas9 - T47D ( right ) . Expression of Cas9 was induced with 5uM doxycycline after transfection of sgRNAs . sgNC : non - targeting sgRNA as a negative control , sgHN - 1 : sgRNA 1 for HNRNPC , sgHN - 2 : sgRNA 2 for HNRNPC , sgLMNA : sgRNA for LMNA as another negative control . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the Tet-on Crispr-Cas9-MCF7 (left) and Tet-on Crispr-Cas9-T47D cells (right). Expression of Cas9 was induced with 5uM doxycycline after transfection of the sgRNAs. sgNC: non-targeting sgRNA as a negative control, sgHN-1: sgRNA sequence 1 for HNRNPC, sgHN-2: sgRNA sequence 2 for HNRNPC, sgLMNA: sgRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.


Relative of HNRNPC upon CRISPR - mediated gene knock - down , quantified from Western blots of 3 replicates . error bars represent ± SD .

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Relative protein levels of HNRNPC upon CRISPR-mediated gene knock-down, quantified from Western blots of 3 replicates. The error bars represent the ± SD.


Anchorage independent growth assays of HNRNPC - deficient MCF7 ( top ) T47D ( bottom ) .

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Anchorage independent cell growth assays of the HNRNPC-deficient MCF7 (top) and T47D (bottom) cells.


Relative of HNRNPC upon shRNA - mediated silencing , quantified from Western blots of 3 replicates . error bars represent ± SD .

Relative protein levels of HNRNPC upon shRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD.


, weights , growth records of xenograft models in NSG mice established from MCF7 with lentivirus - mediated stable gene knock - down . Each group has 6 mice . error bars represent ± .

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Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the MCF7 cells with lentivirus-mediated stable gene knock-down. Each group has 6 mice. The error bars represent the ± SEM.


, weights , growth records of MCF7 - derived xenograft tumors , which were subjected to periodic siRNA injection , starting from 2 after transplantation of . Each group has 4 mice . error bars represent ± .

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Images, weights, and growth records of the MCF7-derived xenograft tumors, which were subjected to periodic siRNA injection, starting from 2 weeks after transplantation of the cells. Each group has 4 mice. The error bars represent the ± SEM.


Volcano plots showing differential expression of genes after HNRNPC knock - down in MCF7 ( A ) T47D ( B ) . ISGs with significant differential expression were marked as red dots . cutoffs ( dashed ) were set at log2 fold - change > 1 . 5 or < - 1 . 5 P - value < 0 . 001 .

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Volcano plots showing the differential expression of the genes after HNRNPC knock-down in the MCF7 (A) and T47D (B) cells. The ISGs with significant differential expression were marked as red dots. The cutoffs (dashed line) were set at the log2 fold-change > 1.5 or < -1.5 and the P-value < 0.001.


qPCR measurements of expressions of ISGs upon knock - down of HNRNPC in MCF7 T47D using

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qPCR measurements of the expressions of ISGs upon knock-down of HNRNPC in MCF7 and T47D cells using siRNA


qPCR measurements of expressions of ISGs upon knock - down of HNRNPC in MCF7 T47D using CRISPR -

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qPCR measurements of the expressions of ISGs upon knock-down of HNRNPC in MCF7 and T47D cells using CRISPR-Cas9


11 TFs involved in interferon response signaling , which were identified by MARINa as master TFs upon HNRNPC knock - down . In each row , genes were sorted ( from left to right ) by their differential expressions in siHNRNPC vs . control . predicted target genes that are positive or negative regulated by TF are marked as red or blue bars . TFs were sorted by P - values ( FDR - corrected ) from MARINa . Ranks of these 11 TFs among master TFs identified by MARINa were provided to right .

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11 TFs involved in the interferon response and signaling, which were identified by MARINa as master TFs upon HNRNPC knock-down. In each row, all genes were sorted (from left to right) by their differential expressions in siHNRNPC vs. control cells. The predicted target genes that are positive or negative regulated by the TF are marked as red or blue bars. All the TFs were sorted by the P-values (FDR-corrected) from the MARINa analysis. Ranks of these 11 TFs among all the master TFs identified by MARINa were provided to the right.


Concentrations of IFNβ , measured by ELISA , in culturing media of MCF7 T47D 48 hours after siRNA transfections . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA 1 for HNRNPC , siHN - 2 : siRNA 2 for HNRNPC , siLMNA : siRNA for LMNA as another negative control . Each sample has 3 replicates . Error bars represent mean ± SD .

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Concentrations of IFNβ, measured by ELISA, in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.


normal MCF7 ( left ) T47D ( right ) were cultured in media collected from corresponding 48 hours after siRNA - mediated gene knock - down . Expressions of ISGs in these normal were measured with qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

The normal MCF7 (left) and T47D (right) cells were cultured in the media collected from the corresponding cells 48 hours after siRNA-mediated gene knock-down. Expressions of the ISGs in these normal cells were measured with qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


Different doses of IFNβ antibody were added to media of siRNA - transfected MCF7 ( left ) or T47D ( right ) right before media were transferred to wild type . wild type MCF7 or T47D were then cultured in these media for 48 hours , expressions of ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

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Different doses of the IFNβ antibody were added to the media of the siRNA-transfected MCF7 (left) or T47D (right) cells right before the media were transferred to the wild type cells. The wild type MCF7 or T47D cells were then cultured in these media for 48 hours, and the expressions of ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


Growth curves of MCF7 ( left ) T47D ( right ) cultured in media collected from siRNA - transfected . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the MCF7 (left) and T47D (right) cells cultured in the media collected from the siRNA-transfected cells. Each sample has 3 replicates. Error bars represent mean ± SD.


In siRNA - transfected MCF7 ( left ) or T47D ( right ) , interferon signaling pathway was blocked at different stages by means of IFNβ neutralization expressions of ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

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In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of IFNβ neutralization The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


In siRNA - transfected MCF7 ( left ) or T47D ( right ) , interferon signaling pathway was blocked at different stages by means of , IFNAR2 neutralization expressions of ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

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In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of , IFNAR2 neutralization The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


In siRNA - transfected MCF7 ( left ) or T47D ( right ) , interferon signaling pathway was blocked at different stages by means of JAK - STAT inhibition ( C ) . expressions of ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

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In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of JAK-STAT inhibition (C). The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


Growth curves of MCF7 ( D ) or T47D ( E ) upon HNRNPC knock - down but with interferon response blocked with IFNβ antibody . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the MCF7 (D) or T47D cells (E) upon HNRNPC knock-down but with the interferon response blocked with the IFNβ antibody. Each sample has 3 replicates. Error bars represent mean ± SD.


Growth curves of MCF7 ( left ) or T47D ( right ) upon HNRNPC knock - down JAK - STAT inhibition with Ruxolitinib ( 5uM ) . Error bars represent mean ± SD . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA 1 for HNRNPC , siHN - 2 : siRNA 2 for HNRNPC . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the MCF7 (left) or T47D (right) cells upon HNRNPC knock-down and JAK-STAT inhibition with Ruxolitinib (5uM). Error bars represent mean ± SD. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. Each sample has 3 replicates. Error bars represent mean ± SD.


Expression of ISGs ( A ) upon HNRNPC silencing in with lentiviral shRNA - mediated stable knock - down of DDX58 or IFIH1 . Each sample has 3 replicates . Error bars represent mean ± SD .

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Expression levels of ISGs (A) upon HNRNPC silencing in the cells with lentiviral shRNA-mediated stable knock-down of DDX58 or IFIH1. Each sample has 3 replicates. Error bars represent mean ± SD.


Expression of IFNβ in media ( B ) upon HNRNPC silencing in with lentiviral shRNA - mediated stable knock - down of DDX58 or IFIH1 . Each sample has 3 replicates . Error bars represent mean ± SD .

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Expression levels of IFNβ concentration in the media (B) upon HNRNPC silencing in the cells with lentiviral shRNA-mediated stable knock-down of DDX58 or IFIH1. Each sample has 3 replicates. Error bars represent mean ± SD.


Growth curves of DDX58 - IFIH1 - deficient MCF7 ( left ) T47D ( right ) upon HNRNPC knock - down . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the DDX58- and IFIH1-deficient MCF7 (left) and T47D (right) cells upon HNRNPC knock-down. Each sample has 3 replicates. Error bars represent mean ± SD.


Xenograft models with DDX58 - deficient MCF7 . , weights , growth records of xenograft models in NSG mice established from DDX58 - deficient MCF7 upon lentivirus - mediated stable knock - down of LMNA or HNRNPC . Each group has 6 mice . error bars represent ± .

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Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the DDX58-deficient MCF7 cells upon lentivirus-mediated stable knock-down of LMNA or HNRNPC. Each group has 6 mice. The error bars represent the ± SEM.


Xenograft models with DDX58 - deficient MCF7 . , weights , growth records of xenograft tumors derived from DDX58 - deficient MCF7 , which were subjected to periodic siRNA injection , starting from 2 after transplantation of . Each group has 4 mice . error bars represent ± .

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Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumors derived from DDX58-deficient MCF7 cells, which were subjected to periodic siRNA injection, starting from 2 weeks after transplantation of the cells. Each group has 4 mice. The error bars represent the ± SEM.


Immunofluorescence of dsRNA in MCF7 after knock - down of HNRNPC , with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) ( blue ) anti - dsRNA antibody J2 ( green ) . transfected with poly I : C was included as a positive control of dsRNA , treated with RNase III was used as a negative control . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA 1 for HNRNPC , siHN - 2 : siRNA 2 for HNRNPC . of scale bar is 10um .

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Immunofluorescence analysis of the dsRNA in MCF7 cells after knock-down of HNRNPC, with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) and anti-dsRNA antibody J2 (green). Cells transfected with poly I:C was included as a positive control of dsRNA, and the cells treated with RNase III was used as a negative control. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. The size of scale bar is 10um.


of dsRNA regions in siNC control or in with siHNRNPC , identified in dsRNA - enriched libraries with anti - dsRNA J2 pull - down or in control libraries including IgG control input control for dsRNA pull - down . Each bar represents overlapping dsRNA species from two replicate experiments . Among each set of dsRNA regions , dsRNAs from Alus were marked in grey .

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Counts of dsRNA regions in the siNC control cells or in the cells with siHNRNPC, identified in the dsRNA-enriched libraries with anti-dsRNA J2 pull-down or in the control libraries including the IgG control and the input control for dsRNA pull-down. Each bar represents the overlapping dsRNA species from two replicate experiments. Among each set of the dsRNA regions, the dsRNAs from Alus were marked in grey.


MA plot showing dsRNA regions identified in dsRNA - enriched libraries . For each dsRNA region , mean of read ( log2 ) for two replicates of siHNRNPC siNC was shown on x - axis , average fold change ( log2 ) in siHNRNPC vs . siNC on y - axis . up - regulated dsRNA regions were marked in red down - regulated regions in black .

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MA plot showing all the dsRNA regions identified in the dsRNA-enriched libraries. For each dsRNA region, mean of the read counts (log2) for two replicates of siHNRNPC and siNC cells was shown on the x-axis, and the average fold change (log2) in siHNRNPC vs. siNC on the y-axis. The up-regulated dsRNA regions were marked in red and down-regulated regions in black.


( A ) Internalization of apoptotic or necrotic GFP - expressing RAW264 . 7 . ( B ) Phagocytosis of negatively charged carboxylated positively charged microspheres ( B ) in M2 ( IL - 4 ) M0 macrophages . Cytochalasin D ( Cyto ) ( 6 µM ) was used as an inhibitor of phagocytosis , 1 h before phagocytosis . Data are shown as means of relative units ( RFU ) ± standard error of mean ( ) .

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(A) Internalization of apoptotic or necrotic GFP-expressing RAW264.7 cells. (B) Phagocytosis of fluorescent negatively charged carboxylated and positively charged amino microspheres (B) in primary M2 (IL-4) and M0 macrophages. Cytochalasin D (Cyto) (6 µM) was used as an inhibitor of phagocytosis, 1 h before phagocytosis. Data are shown as means of relative fluorescence units (RFU) ± standard error of the mean (SEM).


( C - E ) Real - time assays for intraphagosomal proteolysis ( C ) , acidification ( D ) lipolysis ( E ) show substantially increased proteolysis , acidification lipolysis in phagosomes of M2 ( IL - 4 ) macrophages . kinetics of proteolysis , acidification lipolysis of phagocytosed beads were plotted as a of substrate to calibration . Beads were added to macrophages at 0 min . Shaded in ( C ) & ( D ) represents . ( E ) is a representative of three independent experiments . Leupeptin ( 100nM ) Bafilomycin ( 100nM ) treatment serve as negative controls in ( C ) ( D ) , respectively .

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(C-E) Real-time fluorescence assays for intraphagosomal proteolysis (C), acidification (D) and lipolysis (E) show substantially increased proteolysis, acidification and lipolysis in the phagosomes of M2(IL-4) macrophages. The kinetics of proteolysis, acidification and lipolysis of phagocytosed beads were plotted as a ratio of substrate fluorescence to calibration fluorescence. Beads were added to macrophages at 0 min. Shaded area in (C) & (D) represents SEM. (E) is a representative of three independent experiments. Leupeptin (100nM) and Bafilomycin (100nM) treatment serve as negative controls in (C) and (D), respectively.


( B ) Volcano plot of phagosome proteome data . 1 , 766 were quantified of which 121 were significantly up - regulated 62 were down - regulated in M2 ( IL - 4 ) macrophages . Selected are indicated .

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(B) Volcano plot of the phagosome proteome data. 1,766 proteins were quantified of which 121 proteins were significantly up-regulated and 62 proteins were down-regulated in M2(IL-4) macrophages. Selected proteins are indicated.


( C ) of proteomics data shows high reproducibility between biological replicates . Selected are highlighted .

(C) Heatmap of proteomics data shows high reproducibility between biological replicates. Selected proteins are highlighted.


( A ) Immunoblot ( IB ) showing recruitment of TAK1 , MKK7 , JNK TAB1 / TAB2 to phagosome in M2 ( IL - 4 ) macrophages , while MKK4 is not recruited . Rab7a , a phagosomal marker , was used as a loading control for phagosomes vimentin was used as a loading control for total lysates . in JNK blot shows other bands were cut out .

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(A) Immunoblot (IB) analysis showing the recruitment of TAK1, MKK7, JNK and TAB1/TAB2 to the phagosome in M2(IL-4) macrophages, while MKK4 is not recruited. Rab7a, a phagosomal marker, was used as a loading control for phagosomes and vimentin was used as a loading control for total cell lysates. The white line in the JNK blot shows other bands were cut out.


( B ) Quantitation of three independent IB experiments by imageJ of non - saturated blots for TAK1 , MKK7 , TAB1 TAB2 expression on phagosomes of resting M0 macrophages M2 ( IL - 4 ) macrophages . Error bars represent . * p < 0 . 01 , * * p < 0 . 001 ( Student ' s T - )

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(B) Quantitation of three independent IB experiments by imageJ of non-saturated blots for TAK1, MKK7, TAB1 and TAB2 expression on phagosomes of resting M0 macrophages and M2(IL-4) macrophages. Error bars represent SEM. *p<0.01, **p<0.001 (Student's T-test)


( C ) IB showing enrichment of K63 polyubiquitylated on phagosome of M2 ( IL - 4 ) macrophages compared to M0 macrophages .

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(C) IB showing enrichment of K63 polyubiquitylated proteins on the phagosome of M2(IL-4) macrophages compared to M0 macrophages.


Treatment with UBC13 inhibitor NSC697923 reduces recruitment of TAB1 , TAB2 , TAK1 MKK7 to phagosome of M2 ( IL - 4 ) macrophages , indicating a K63 - polyubiquitylation dependent translocation for these .

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Treatment with the UBC13 inhibitor NSC697923 reduces recruitment of TAB1, TAB2, TAK1 and MKK7 to the phagosome of M2(IL-4) macrophages, indicating a K63-polyubiquitylation dependent translocation for these proteins.


( B ) Selected ubiquitylated phagosomal identified by TUBE - MS approach .

(B) Selected ubiquitylated phagosomal proteins identified by TUBE-MS approach.


( D ) Tab2 - TUBE pull - downs from phagosome extracts of M2 ( IL - 4 ) macrophages treated with K63 specific deubiquitylase ( DUB ) AMSH - LP or unspecific DUB USP2 .

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(D) Tab2-TUBE pull-downs from phagosome extracts of M2(IL-4) macrophages treated with the K63 specific deubiquitylase (DUB) AMSH-LP or the unspecific DUB USP2.


MSR1 immunoprecipitation from M2 ( IL - 4 ) macrophage phagosomes shows that TAB1 , TAB2 , TAK1 MKK7 bind to polyubiquitylated MSR1 . TAK1 shows a specific pattern of post - translational modifications indicative of its activation .

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MSR1 immunoprecipitation from M2(IL-4) macrophage phagosomes shows that TAB1, TAB2, TAK1 and MKK7 bind to polyubiquitylated MSR1. TAK1 shows a specific pattern of post-translational modifications indicative of its activation.


( A ) Immunoblot against MSR1 of TUBE pulldowns of MSR1 WT KO BMDMs shows increasing amounts of ubiquitylated MSR1 upon alternative activation further increases upon MSR1 ligation with fucoidan or oxLDL .

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(A) Immunoblot against MSR1 of TUBE pulldowns of MSR1 WT and KO BMDMs shows increasing amounts of ubiquitylated MSR1 upon alternative activation and further increases upon MSR1 ligation with fucoidan or oxLDL.


( B ) Msr1 + / + ( WT ) Msr1 - / - ( KO ) M0 macrophages M2 ( IL - 4 ) macrophages untreated or stimulated with MSR1 ligands fucoidan or oxLDL ( 50 μg / ml , 30 min ) were analysed for phosphorylated total forms of JNK1 / 2 MSR1 . ITGAM ( CD11b ) serves as a loading control . Both fucoidan oxLDL activate JNK in a MSR1 - dependent manner .

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(B) Msr1+/+ (WT) and Msr1-/- (KO) M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan or oxLDL (50 μg/ml, 30 min) were analysed for the phosphorylated and the total forms of JNK1/2 and MSR1. ITGAM (CD11b) serves as a loading control. Both fucoidan and oxLDL activate JNK in a MSR1-dependent manner.


( C ) IL4 - activated MSR1 knock - out BMDMs were transfected with WT or K27R MSR1 treated with 50 μg / ml Fucoidan for 1 hr . Mutation of ubiquitylation site K27 abolishes MSR1 signalling .

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(C) IL4-activated MSR1 knock-out BMDMs were transfected with WT or K27R MSR1 and treated with 50 μg/ml Fucoidan for 1 hr. Mutation of the ubiquitylation site K27 abolishes MSR1 signalling.


( D ) qPCR data of Tnfa , Il1b Ccl2 mRNA in WT MSR1 KO M0 M2 ( IL - 4 ) BMDMs shows an MSR1 - dependent increase of pro - inflammatory cytokines in response to MSR1 ligation by fucoidan .

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(D) qPCR data of Tnfa, Il1b and Ccl2 mRNA levels in WT and MSR1 KO M0 and M2(IL-4) BMDMs shows an MSR1-dependent increase of pro-inflammatory cytokines in response to MSR1 ligation by fucoidan.


( E ) Inhibition of JNK by JNK - IN8 reduces expression of Tnfa Ccl2 upon MSR1 ligation , showing that it is JNK - dependent .

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(E) Inhibition of JNK by JNK-IN8 reduces expression of Tnfa and Ccl2 upon MSR1 ligation, showing that it is JNK-dependent.


( F ) Flow cytometry of surface markers in WT MSR1 - / - M0 macrophages M2 ( IL - 4 ) macrophages untreated or stimulated with MSR1 ligands fucoidan ( 50 μg / ml , 24 h ) . Data shows MSR1 - dependent increase of early activation markers CD54 , CD69 CD86 a decrease of M2 marker CD301b / Mgl2 . CD11b serves as a control .

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(F) Flow cytometry analysis of cell surface markers in WT and MSR1-/- M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan (50 μg/ml, 24 h). Data shows MSR1-dependent increase of the early activation markers CD54, CD69 and CD86 and a decrease of the M2 marker CD301b/Mgl2. CD11b serves as a control.


( A ) Immunohistochemistry of MSR1 in ovarian , uterus sarcoma cancers showing tumour - associated macrophages .

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(A) Immunohistochemistry analysis of MSR1 in patient ovarian, uterus and sarcoma cancers showing tumour-associated macrophages.


( B ) Pull - downs of K63 polyubiquitin chains IB of five human cancers shows a correlation between amount of polyubiquitylated MSR1 JNK activation in an ovarian tumour .

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(B) Pull-downs of K63 polyubiquitin chains and IB analysis of five human primary cancers shows a correlation between the amount of polyubiquitylated MSR1 and JNK activation in an ovarian tumour.


( B ) Schematic of marker - less GFP - tagging at endogenous locus representative live of Mad1 - , Mad2 - , Mad3 - GFP strains ( average projections ) .

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(B) Schematic of marker-less GFP-tagging at the endogenous locus and representative live cell images of Mad1-, Mad2-, and Mad3-GFP strains (average intensity projections).


( C ) Representative of single molecule mRNA FISH ( smFISH ) of S . pombe using probes against GFP ( red ) . was stained with DAPI ( blue ) . gamma - value was adjusted to make cytoplasm visible ; shapes are outlined in blue . ( D ) Frequency distribution of mRNA numbers per determined by smFISH ; n = number of . Curves show fit to a Poisson distribution .

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(C) Representative images of single molecule mRNA FISH (smFISH) staining of S. pombe using probes against GFP (red). DNA was stained with DAPI (blue). The gamma-value was adjusted to make the cytoplasm visible; cell shapes are outlined in blue. (D) Frequency distribution of mRNA numbers per cell determined by smFISH; n = number of cells. Curves show fit to a Poisson distribution.


( E ) Frequency distribution of mRNA numbers per using FISH probes against endogenous genes using either strains expressing GFP - tagged gene or endogenous , untagged gene . Curves show fit to a Poisson distribution . difference for mad1 + is statistically significant , that for mad2 + is not A lower mRNA number for untagged mad1 + was also observed in an independent .

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(E) Frequency distribution of mRNA numbers per cell using FISH probes against the endogenous genes and using either strains expressing the GFP-tagged gene or the endogenous, untagged gene. Curves show fit to a Poisson distribution. The difference for mad1+ is statistically significant, that for mad2+ is not A lower mRNA number for untagged mad1+ was also observed in an independent strain.


( F ) Co - by smFISH using probes against mad1 + GFP either in a expressing mad1 + - GFP as a positive control or in a expressing wild - type mad1 + mad2 + - GFP . mad1 + ( green ) or GFP mRNA spots ( magenta ) were quantified as co - localizing or not with respective other . For mad1 + - GFP , 544 a total of 1 , 641 mad1 spots 1 , 839 GFP spots were analyzed ; 48 were not considered since they did not contain at least one spot of each type in cytoplasm . For mad1 + mad2 + - GFP , 571 a total of 1 , 107 mad1 spots 1 , 537 GFP spots were analyzed ; 158 were not considered since they did not contain at least one spot of each type in cytoplasm .

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(F) Co-staining by smFISH using probes against mad1+ and GFP either in a strain expressing mad1+-GFP as a positive control or in a strain expressing wild-type mad1+ and mad2+-GFP. Cytoplasmic mad1+ (green) or GFP mRNA spots (magenta) were quantified as co-localizing or not with the respective other. For the mad1+-GFP strain, 544 cells and a total of 1,641 mad1 spots and 1,839 GFP spots were analyzed; 48 cells were not considered since they did not contain at least one spot of each type in the cytoplasm. For the mad1+ mad2+-GFP strain, 571 cells and a total of 1,107 mad1 spots and 1,537 GFP spots were analyzed; 158 cells were not considered since they did not contain at least one spot of each type in the cytoplasm.


( A ) noise ( coefficient of variation , CV = std / mean ) in live - microscopy of S . pombe ; n = 7 ( Nmt1 - GFP ) , 11 ( Mad1 - GFP ) , 19 ( Mad2 - GFP ) , 10 ( Mad3 - GFP ) ; single had 16 - 79 GFP - positive 6 - 94 GFP - negative ( control ) . Boxplots show median interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times IQR from first third quartile , respectively . Mad1 , Mad2 , Mad3 showed significantly lower noise than Nmt1 ( Wilcoxon rank sum ; p < 0 . 001 ) .

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(A) Cellular protein noise (coefficient of variation, CV = std / mean) in live-cell microscopy images of S. pombe; n = 7 images (Nmt1-GFP), 11 (Mad1-GFP), 19 (Mad2-GFP), 10 (Mad3-GFP); single images had 16-79 GFP-positive and 6-94 GFP-negative (control) cells. Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Mad1, Mad2, and Mad3 all showed significantly lower noise than Nmt1 (Wilcoxon rank sum test; all p < 0.001).


( B ) Simulations of stochastic gene expression noise from selected mRNA / half - life combinations assuming a constantly active promoter Synthesis rates were set to obtain a mean mRNA number of 4 per , a mean number of 6000 per . x - axis of each graph shows time , y - axis shows mRNA number per ( blue ) or number per ( black ) . ( C ) Theoretical prediction for coefficient of variation ( CV = std / mean ) of number per , assuming different mRNA half - lives , using same underlying model as in B . Synthesis rates were adjusted to maintain a mean mRNA number per of 3 . 5 , a mean number per of 6000 ( approx . 100 nM ) .

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(B) Simulations of stochastic gene expression noise from selected mRNA/protein half-life combinations assuming a constantly active promoter Synthesis rates were set to obtain a mean mRNA number of 4 per cell, and a mean protein number of 6000 per cell. The x-axis of each graph shows time, the y-axis shows mRNA number per cell (blue) or protein number per cell (black). (C) Theoretical prediction for the coefficient of variation (CV = std / mean) of the protein number per cell, assuming different mRNA and protein half-lives, using the same underlying model as in B. Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3.5, and a mean protein number per cell of 6000 (approx. 100 nM).


( D ) mRNA abundances by qPCR following metabolic labeling removal of labeled pool ( two independent experiments ) . Lines are regression curves from generalized linear mixed model fits , excluding measurements at t = 0 in order to accommodate for non - instantaneous labeling by 4tU . Act1 + ecm33 + were used as long short half - life controls , respectively ; qPCR was performed for endogenous mRNAs . Half - lives ( 95 % confidence interval ) : mad1 + 5 . 6 min ( 4 . 3 - 8 . 4 ) , mad2 + 7 . 7 min ( 6 . 2 - 10 . 4 ) , mad3 + 5 . 2 min ( 4 . 3 - 6 . 9 ) , act1 + 61 . 8 min ( 37 . 2 - 172 . 3 ) , ecm33 + 5 . 0 min ( 4 . 5 - 5 . 7 ) .

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(D) mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Lines are regression curves from generalized linear mixed model fits, excluding the measurements at t = 0 in order to accommodate for non-instantaneous labeling by 4tU. Act1+ and ecm33+ were used as long and short half-life controls, respectively; qPCR was performed for the endogenous mRNAs. Half-lives (95 % confidence interval): mad1+ 5.6 min (4.3 - 8.4), mad2+ 7.7 min (6.2 - 10.4), mad3+ 5.2 min (4.3 - 6.9), act1+ 61.8 min (37.2 - 172.3), ecm33+ 5.0 min (4.5 - 5.7).