g , In - gel fluorescence assay ( top panel ) visualizing Alexa Fluor 647 - labelled myristoylated ARF1 - strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated . The expressed amounts of ARF1 - strep are shown ( bottom panel ) .

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g, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated. The expressed amounts of ARF1-strep are shown (bottom panel).


h , In - gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid , azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry . Arrows indicate proteins that are proteolytically demyristoylated by IpaJ .

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h, In-gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid, azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry. Arrows indicate proteins that are proteolytically demyristoylated by IpaJ.


b , In - gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant . The peptide - eGFP and IpaJ inputs are indicated . * Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide , potentially owing to reduced mobility in SDS - polyacrylamide gel electrophoresis caused by proteolytic reaction .

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b, In-gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant. The peptide-eGFP and IpaJ inputs are indicated. *Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide, potentially owing to reduced mobility in SDS-polyacrylamide gel electrophoresis caused by proteolytic reaction.


c , In - gel fluorescence assay showing myristoylated ARF1 T31N mutant ( a GDP - locked mutant ) or ARF1 Q71L ( a GTP - locked mutant ) after in cells co - expressing IpaJ or catalytic mutants as indicated . Equal loading of ARF1 ( middle panel ) and IpaJ ( lower panel ) was determined by western blot analysis .

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c, In-gel fluorescence assay showing myristoylated ARF1 T31N mutant (a GDP-locked mutant) or ARF1 Q71L (a GTP-locked mutant) after in cells co-expressing IpaJ or catalytic mutants as indicated. Equal loading of ARF1 (middle panel) and IpaJ (lower panel) was determined by western blot analysis.


d , Fluorescence microscopy showing ARF1 - mCherry and the Golgi apparatus ( GM130 ) after cellular microinjection of recombinant IpaJ or IpaJ C64A , over the indicated period . Scale bar , 10 µm .

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d, Fluorescence microscopy showing ARF1-mCherry and the Golgi apparatus (GM130) after cellular microinjection of recombinant IpaJ or IpaJ C64A, over the indicated period. Scale bar, 10 µm.


e , Fluorescence microscopy showing ARF1 - eGFP ( green ) and Golgi membranes ( blue , GM130 ) after infection with Shigella M90T and the indicated mutants ( red ; mCherry - expressing bacteria ) . Scale bar , 10µm .

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e, Fluorescence microscopy showing ARF1-eGFP (green) and Golgi membranes (blue, GM130) after infection with Shigella M90T and the indicated mutants (red; mCherry-expressing bacteria). Scale bar, 10µm.


( A ) Mice bearing established A375 were treated with vehicle ( n = 7 ) , PLX4720 ( n = 5 ) bevacizumab ( n = 7 ) or COMBO ( n = 5 ) . Tumor growth is expressed as % change of the initial tumor , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( PLX4720 P = 0 . 0049 ; bevacizumab P = 0 . 0003 ; P = 4 . 86E - 10 ) ŦP < 0 . 05 compared to PLX4720 ( P = 0 . 015 ) . ( B ) Mice bearing established COLO205 were treated with vehicle ( n = 7 ) , PLX4720 ( n = 8 ) bevacizumab ( n = 7 ) or COMBO ( n = 8 ) . Tumor growth is expressed as % change of the initial tumor , * * * P < 0 . 001 versus vehicle ( PLX4720 P = 2 . 75E - 05 ; bevacizumab P = 2 . 96E - 05 ; COMBO P = 2 . 13E - 07 ) Ŧp < 0 . 05 compared to PLX4720 ( P = 0 . 039 ) .

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(A) Mice bearing established A375 were treated with vehicle (n=7), PLX4720 (n=5) bevacizumab (n=7) or COMBO (n=5). Tumor growth is expressed as % change of the initial tumor, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720 P = 0.0049; bevacizumab P = 0.0003; P = 4.86E-10) ŦP < 0.05 compared to PLX4720 (P = 0.015).(B) Mice bearing established COLO205 were treated with vehicle (n=7), PLX4720 (n=8) bevacizumab (n=7) or COMBO (n=8). Tumor growth is expressed as % change of the initial tumor, ***P < 0.001 versus vehicle (PLX4720 P = 2.75E-05; bevacizumab P = 2.96E-05; COMBO P = 2.13E-07) Ŧp < 0.05 compared to PLX4720 (P = 0.039).


( C ) Melanoma MC - 1 cells ( 5 x 105 ) were injected into the tail vein of CD1 mice . Lung colonization was assayed by HE staining and calculating the number of nodules and their total area normalized per the total area of the lungs . The mice analyzed were : start point , n = 6 ; vehicle , n = 7 ; PLX4720 , n = 6 ; bevacizumab , n = 6 ; COMBO , n = 7 , * P < 0 . 001 versus vehicle ( P = 0 . 031 ) .

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(C) Melanoma MC-1 cells (5 x 105) were injected into the tail vein of CD1 mice. Lung colonization was assayed by HE staining and calculating the number of nodules and their total area normalized per the total area of the lungs. The mice analyzed were: start point, n=6; vehicle, n=7; PLX4720, n=6; bevacizumab, n=6; COMBO, n=7, *P < 0.001 versus vehicle (P = 0.031).


( D ) Representative images of tumor cell proliferation determined by immunofluorescence Ki67 staining in A375 xenografts treated as indicated . Bar graphs indicate the Ki67 + area / tumor area ( n = 4 tumors ) , * * * P < 0 . 001 versus vehicle ( PLX4720 P = 2 . 83E - 08 ; bevacizumab P = 4 . 25E - 13 ; COMBO P = 6 . 16E - 08 ) . ( E ) Representative images of tumor cell apoptosis determined by immunofluorescence staining with TUNEL in A375 xenografts treated as indicated . Bar graphs indicate the TUNEL + area / tumor area ( n = 3 tumors ) . * * * P < 0 . 001 versus vehicle ( P = 6 . 2 . 7E - 10 ) .

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(D) Representative images of tumor cell proliferation determined by immunofluorescence Ki67 staining in A375 xenografts treated as indicated. Bar graphs indicate the Ki67+ area/tumor area (n=4 tumors), ***P < 0.001 versus vehicle (PLX4720 P = 2.83E-08; bevacizumab P = 4.25E-13; COMBO P = 6.16E-08).(E) Representative images of tumor cell apoptosis determined by immunofluorescence staining with TUNEL in A375 xenografts treated as indicated. Bar graphs indicate the TUNEL+ area/tumor area (n=3 tumors). ***P < 0.001 versus vehicle (P = 6.2.7E-10).


( A ) Representative images of vasculature stained by an antibody anti - CD31 in A375 xenografts treated as indicated . Bar graphs indicate quantitative micro vessel density ( MVD ) and micro vessel area ( MVA ) analysis ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( MVD : bevacizumab P = 5 . 94E - 25 ; COMBO P = 1 . 84E - 04 ) ( MVA : PLX4720 P = 4 . 98E - 09 ; bevacizumab P = 1 . 16E - 19 ; COMBO P = 3 . 00E - 08 ) .

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(A) Representative images of vasculature stained by an antibody anti-CD31 in A375 xenografts treated as indicated. Bar graphs indicate quantitative micro vessel density (MVD) and micro vessel area (MVA) analysis (n=5 tumors), ***P < 0.001 versus vehicle (MVD: bevacizumab P = 5.94E-25; COMBO P = 1.84E-04) (MVA: PLX4720 P = 4.98E-09; bevacizumab P = 1.16E-19; COMBO P = 3.00E-08).


( B ) Representative images of vessel lumen in A375 xenografts treated as indicated . Bar graphs indicate the quantitative analysis of lumen diameters in A375 and COLO205 xenografts ( n = 3 tumors ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( A375 : PLX4720 P = 0 . 0078 ; COMBO P = 0 . 0012 ) ( COLO205 : PLX4720 P = 2 . 01E - 06 ; COMBO P = 1 . 33E - 07 ) .

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(B) Representative images of vessel lumen in A375 xenografts treated as indicated. Bar graphs indicate the quantitative analysis of lumen diameters in A375 and COLO205 xenografts (n=3 tumors), *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 0.0078; COMBO P = 0.0012) (COLO205: PLX4720 P = 2.01E-06; COMBO P = 1.33E-07).


( C ) Representative images of perfusing fluorescent beads and their relationship with microvessels in A375 xenografts treated as indicated . Bar graphs indicate the % of CD31 + vessels co - stained with fluorescent beads ( n = 4 tumors ) , * * P < 0 . 01 versus vehicle ( PLX4720 P = 0 . 0031 ; COMBO P = 0 . 0033 ) .

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(C) Representative images of perfusing fluorescent beads and their relationship with microvessels in A375 xenografts treated as indicated. Bar graphs indicate the % of CD31+ vessels co-stained with fluorescent beads (n=4 tumors), **P < 0.01 versus vehicle (PLX4720 P = 0.0031; COMBO P = 0.0033).


( D ) Representative images of hypoxia marker pimonidazole in A375 xenografts treated as indicated . Bar graphs show the % of tumor hypoxic area in A375 and COLO205 xenografts ( n = 5 tumors ) , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( A375 : PLX4720 P = 6 . 94E - 12 ; COMBO P = 3 . 34E - 12 ) ( COLO205 : PLX4720 P = 0 . 0015 ; bevacizumab P = 0 . 0004 ; COMBO P = 0 . 0003 ) .

(D) Representative images of hypoxia marker pimonidazole in A375 xenografts treated as indicated. Bar graphs show the % of tumor hypoxic area in A375 and COLO205 xenografts (n=5 tumors), **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 6.94E-12; COMBO P = 3.34E-12) (COLO205: PLX4720 P = 0.0015; bevacizumab P = 0.0004; COMBO P = 0.0003).


( D ) GSEA enrichment plots for " Reactome Cytokine Signaling in Immune System " and " Reactome Innate Immune System " ( upper panels ) and " Reactome Extracellular Matrix Organization " and " Reactome Collagen Formation " ( lower panels ) highlight significant enrichment of the pathways relative to the immune response and a decreased expression of the pathways relative to extracellular matrix remodeling in COMBO treated tumors as compared to the other treatments ( vehicle , PLX4720 , bevacizumab ) .

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(D) GSEA enrichment plots for "Reactome Cytokine Signaling in Immune System" and "Reactome Innate Immune System" (upper panels) and "Reactome Extracellular Matrix Organization" and "Reactome Collagen Formation" (lower panels) highlight significant enrichment of the pathways relative to the immune response and a decreased expression of the pathways relative to extracellular matrix remodeling in COMBO treated tumors as compared to the other treatments (vehicle, PLX4720, bevacizumab).


( E ) Heatmap representation of gene expression changes within the " Reactome Innate Immune System " ( left panel ) and " Reactome Extracellular matrix Organization " ( right panel ) gene set . Genes in heatmaps are shown in rows and samples are shown in columns . Expression level is represented as a gradient from high ( red ) to low ( blue ) . V , P , B and C respectively indicate vehicle , PLX4720 , bevacizumab and COMBO .

(E) Heatmap representation of gene expression changes within the "Reactome Innate Immune System" (left panel) and "Reactome Extracellular matrix Organization" (right panel) gene set. Genes in heatmaps are shown in rows and samples are shown in columns. Expression level is represented as a gradient from high (red) to low (blue). V, P, B and C respectively indicate vehicle, PLX4720, bevacizumab and COMBO.


( A ) Representative images of leukocytes infiltration determined by immunofluorescence CD45 staining in of A375 xenografts treated as indicated . Bar graphs indicate the CD45 + area / tumor area in A375 and COLO205 xenografts ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( A375 : P = 2 . 19E - 20 ) ( COLO205 : P = 1 . 81E - 06 ) . ( B ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the F4 / 80 + area / tumor area in A375 and COLO205 xenografts ( n = 5 tumors ) * * * P < 0 . 001 versus vehicle ( A375 : COMBO P = 3 . 42E - 13 ) ( COLO205 : COMBO P = 1 . 94E - 06 ) .

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(A) Representative images of leukocytes infiltration determined by immunofluorescence CD45 staining in of A375 xenografts treated as indicated. Bar graphs indicate the CD45+ area/tumor area in A375 and COLO205 xenografts (n=5 tumors), ***P < 0.001 versus vehicle (A375: P = 2.19E-20) (COLO205: P = 1.81E-06).(B) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the F4/80+ area/tumor area in A375 and COLO205 xenografts (n=5 tumors) ***P < 0.001 versus vehicle (A375: COMBO P = 3.42E-13) (COLO205: COMBO P = 1.94E-06).


( C ) A375 xenograft tumors after 14 days of treatment were subjected to FACS and tumor infiltrate was analyzed . Gating strategy for CD45 + cells and graph showing the quantification of FACS analysis of infiltrating CD45 + leukocytes in vehicle ( n = 4 tumors ) compared to COMBO ( n = 6 tumors ) treated tumors , * * P < 0 . 01 versus vehicle ( P = 0 . 0064 ) .

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(C) A375 xenograft tumors after 14 days of treatment were subjected to FACS and tumor infiltrate was analyzed. Gating strategy for CD45+ cells and graph showing the quantification of FACS analysis of infiltrating CD45+ leukocytes in vehicle (n=4 tumors) compared to COMBO (n=6 tumors) treated tumors, **P < 0.01 versus vehicle (P = 0.0064).


( D ) Graph showing the quantification of FACS analysis of infiltrating F4 / 80 + macrophages and CD11c + MHCII + dendritic cells in vehicle ( n = 4 tumors ) compared to COMBO ( n = 6 tumors ) treated tumors , * * P < 0 . 01 versus vehicle ( TAM P = 0 . 0061 ; DC P = 0 . 0091 ) .

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(D) Graph showing the quantification of FACS analysis of infiltrating F4/80+ macrophages and CD11c+MHCII+ dendritic cells in vehicle (n=4 tumors) compared to COMBO (n=6 tumors) treated tumors, **P < 0.01 versus vehicle (TAM P = 0.0061; DC P = 0.0091).


( A ) Real - time quantitative PCR of the indicated genes ( M1 - like and M2 - like macrophages markers ) of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) compared with vehicle after normalization for housekeeping gene TBP ( n = 3 tumors ) , ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( PLX4720 : m - Arg1 P = 5 . 65E - 06 ) ( bevacizumab : m - Ccl5 P = 0 . 025 ) ( COMBO : m - Ccl5 P = 0 . 0070 ; m - Cd40 P = 0 . 00099 ; m - Cxcl10 P = 0 . 043 ; m - Cxcl9 P = 0 . 046 ; m - Il1b P = 0 . 0367 ; m - Stat1 P = 0 . 049 ; m - Tlr2 = 0 . 00182 ; m - Arg - 1 P = 0 . 0013 ) .

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(A) Real-time quantitative PCR of the indicated genes (M1-like and M2-like macrophages markers) of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for housekeeping gene TBP (n=3 tumors), (*P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720: m-Arg1 P = 5.65E-06) (bevacizumab: m-Ccl5 P = 0.025) (COMBO: m-Ccl5 P = 0.0070; m-Cd40 P = 0.00099; m-Cxcl10 P = 0.043; m-Cxcl9 P = 0.046; m-Il1b P = 0.0367; m-Stat1 P = 0.049; m-Tlr2 = 0.00182; m-Arg-1 P = 0.0013).


( B ) Representative images of COMBO - treated tumor sections co - stained with an antibody against CD68 and an antibody anti - CCR7 ( M1 - like polarization ) or anti - CD206 ( M2 - like polarization ) .

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(B) Representative images of COMBO-treated tumor sections co-stained with an antibody against CD68 and an antibody anti-CCR7 (M1-like polarization) or anti-CD206 (M2-like polarization).


( C ) Graphs showing the quantification of FACS analysis of infiltrating F4 / 80 + tumor macrophages and average percentage of median fluorescence intensity ( MFI ) of TNFα + or INOS + on F4 / 80 + cells after ex vivo stimulation with PMA and ionomycin in vehicle ( n = 4 tumors ) and COMBO ( n = 6 tumors ) , * P < 0 . 05 versus vehicle ( TNFα P = 0 . 037 ; INOS = 0 . 038 ) .

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(C) Graphs showing the quantification of FACS analysis of infiltrating F4/80+ tumor macrophages and average percentage of median fluorescence intensity (MFI) of TNFα+ or INOS+ on F4/80+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.037; INOS = 0.038).


( D ) Graphs showing the quantification of FACS analysis of infiltrating CD11b + Ly6C + tumor monocytes and average percentage of MFI of TNFα + or INOS + on CD11b + Ly6C + cells after ex vivo stimulation with PMA and ionomycin in vehicle ( n = 4 tumors ) and COMBO ( n = 6 tumors ) , * P < 0 . 05 versus vehicle ( TNFα P = 0 . 048 ; INOS = 0 . 038 ) .

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(D) Graphs showing the quantification of FACS analysis of infiltrating CD11b+Ly6C+ tumor monocytes and average percentage of MFI of TNFα+ or INOS+ on CD11b+Ly6C+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.048; INOS = 0.038).


( E ) Graph showing the quantification of circulating CD11b + Ly6C + monocytes in peripheral blood analyzed by FACS in vehicle - ( n = 4 tumors ) and COMBO - ( n = 6 tumors ) treated tumors .

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(E) Graph showing the quantification of circulating CD11b+Ly6C+ monocytes in peripheral blood analyzed by FACS in vehicle- (n=4 tumors) and COMBO- (n=6 tumors) treated tumors.


( F ) Cell viability measured as fluorescence intensity of ZsGreen - A375 cells co - cultured with cells isolated from xenograft treated with vehicle or COMBO with after 48h . Data are representative of one experiment out of two done in triplicate * P < 0 . 05 versus vehicle ( P = 0 . 046 ) .

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(F) Cell viability measured as fluorescence intensity of ZsGreen-A375 cells co-cultured with cells isolated from xenograft treated with vehicle or COMBO with after 48h. Data are representative of one experiment out of two done in triplicate *P < 0.05 versus vehicle (P = 0.046).


( A ) Representative images of collagen deposition determined by collagen I immunofluorescent staining in A375 xenografts treated as indicated . Bar graphs indicate the collagen I + area / tumor area ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 2 . 93E - 06 ) . ( B ) Representative images of CAF density determined by α - SMA immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate α - SMA + area / tumor area ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 0 . 0006 ) . ( C ) Representative images of Lysyl Oxidase enzyme determined by LOX immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the LOX + area / tumor area ( n = 3 tumors ) , * P < 0 . 05 versus vehicle ( bevacizumab P = 0 . 033 ; COMBO P = 0 . 040 ) .

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(A) Representative images of collagen deposition determined by collagen I immunofluorescent staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 2.93E-06).(B) Representative images of CAF density determined by α-SMA immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate α-SMA+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 0.0006).(C) Representative images of Lysyl Oxidase enzyme determined by LOX immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the LOX+ area/tumor area (n=3 tumors), *P < 0.05 versus vehicle (bevacizumab P = 0.033; COMBO P = 0.040).


( E ) Quantitative Real - time PCR of h - TGFB1 and m - Tgfb1 of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) compared with vehicle after normalization for the housekeeping gene TBP ( n = 3 tumors ) , * P < 0 . 05 versus vehicle ( P = 0 . 043 ) .

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(E) Quantitative Real-time PCR of h-TGFB1 and m-Tgfb1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for the housekeeping gene TBP (n=3 tumors), *P < 0.05 versus vehicle (P = 0.043).


( F ) Western blot for human TGFβ in total protein extract from A375 tumors treated as indicate . Vinculin was used as an internal control .

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(F) Western blot for human TGFβ in total protein extract from A375 tumors treated as indicate. Vinculin was used as an internal control.


( A ) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors . Tumor growth is expressed as % change of the initial tumor . Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab ( n = 5 mice / group ) , * P < 0 . 05 , * * * P < 0 . 001 versus vehicle ( vehicle Clodronate P = 0 . 049 , COMBO P = 5 . 72E - 06 ) ŦP < 0 . 05 compared to COMBO ( P = 0 . 033 ) .

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(A) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors. Tumor growth is expressed as % change of the initial tumor. Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab (n=5 mice/group), *P < 0.05, ***P < 0.001 versus vehicle (vehicle Clodronate P = 0.049, COMBO P = 5.72E-06) ŦP < 0.05 compared to COMBO (P = 0.033).


( B ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the F4 / 80 + area / tumor ( n = 3 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 1 . 53E - 25 ) . ( C ) Representative images of collagen deposition determined by collagen I immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the collagen I + area / tumor ( n = 3 tumors ) .

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(B) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the F4/80+ area/tumor (n=3 tumors), ***P < 0.001 versus vehicle (P = 1.53E-25).(C) Representative images of collagen deposition determined by collagen I immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor (n=3 tumors).


( D ) Waterfall plots showing the percent change in volume ( relative to the initial tumor volume ) for the individual A375 xenografts in each treatment group ( vehicle , PLX4720 , bevacizumab and COMBO ) from week 1 to week 6 .

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(D) Waterfall plots showing the percent change in volume (relative to the initial tumor volume) for the individual A375 xenografts in each treatment group (vehicle, PLX4720, bevacizumab and COMBO) from week 1 to week 6.


( E ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in a responder and a relapsing A375 xenografts . Bar graphs indicate the F4 / 80 + area / tumor ( n = 3 mice ) , * * * P < 0 . 001 versus vehicle ( P = 1 . 25E - 06 ) . ( F ) Representative images of collagen deposition determined by collagen I immunofluorescence staining in a responder and a relapsing A375 xenografts . Bar graphs indicate the collagen I + area / tumor ( n = 3 mice ) , * * * P < 0 . 001 versus vehicle ( P = 4 . 03E - 05 ) .

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(E) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the F4/80+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 1.25E-06).(F) Representative images of collagen deposition determined by collagen I immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the collagen I+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 4.03E-05).


( G ) Real - time quantitative PCR of h - TGFB1 of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP ( n = 3 tumors ) * * * P < 0 . 001 versus vehicle ( P = 0 . 0006 ) .

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(G) Real-time quantitative PCR of h-TGFB1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP (n=3 tumors) ***P < 0.001 versus vehicle (P = 0.0006).


A Isothermal calorimetry ( ITC ) assays using ADP - ribose ligand and purified macrodomains of human macroH2A1 . 1 and macroH2A2 .

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A Isothermal calorimetry (ITC) assays using ADP-ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2.


B In vitro PARP1 activity assay in the presence of increasing concentrations ( 10 , 25 and 50 μM ) of purified macrodomains of human macroH2A1 . 1 , macroH2A1 . 1 G224E mutant and macroH2A2 . PARP1 activity is assessed by its auto - PARylation detected by an anti - PAR immunoblot . The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction . A representative blot of one of three independent experiments is shown .

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B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.


A The diameter ( d ) of the fluorescence signaling of H2B tagged with photo - activatable GFP ( PAGFP - H2B ) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser .

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A The diameter (d) of the fluorescence signaling of H2B tagged with photo-activatable GFP (PAGFP-H2B) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser.


B Chromatin expansion in absence and presence of intercalating DNA dye ( - and + DNA damage ) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor . Boxplots represent single cell measurement of chromatin expansion at 120 sec . post DNA damage from 3 biological replicates with n > 30 cells each . The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values ( * , p < 0 . 05 using unpaired , two - tailed Student ' s T - test ) .

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B Chromatin expansion in absence and presence of intercalating DNA dye (- and + DNA damage) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage from 3 biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values (*, p < 0.05 using unpaired, two-tailed Student's T-test).


C Quantified chromatin expansion in cells not transfected ( control ) and cells transfected with each mCherry - tagged histone ( as indicated ) . Data is represented as in B ( n > 30 cells , 3 bio . repl . ; * , p < 0 . 05 compared to mCherry - H2A - expressing cells using unpaired , two - tailed Student ' s T - test ) .

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C Quantified chromatin expansion in cells not transfected (control) and cells transfected with each mCherry-tagged histone (as indicated). Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 compared to mCherry-H2A-expressing cells using unpaired, two-tailed Student's T-test).


D Chromatin expansion in Hela cells containing a doxocyclin ( doxo . ) - inducible macroH2A1 . 1 transgene . Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti - macroH2A1 . 1 - immunoblotting . Data is represented as in B ( n > 30 cells , 3 bio . repl . ; * , p < 0 . 05 using unpaired , two - tailed Student ' s T - test ) .

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D Chromatin expansion in Hela cells containing a doxocyclin (doxo.)-inducible macroH2A1.1 transgene. Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti-macroH2A1.1-immunoblotting. Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 using unpaired, two-tailed Student's T-test).


Quantified chromatin expansion in cells transfected with indicated histone constructs . Boxplots represent single cell measurement of chromatin expansion at 120 sec . post DNA damage ( n > 30 cells , 3 bio . repl . ) . The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test comparing to H2A ; n . s . , not significant ) .

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Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant).


A Western blot showing the expression of macroH2A proteins in control HepG2 cells , knock - down for macroH2A1 and macroH2A2 ( DKD ) and DKD with ectopic expression of the indicated YFP - tagged constructs . H3 is used as a loading control .

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A Western blot showing the expression of macroH2A proteins in control HepG2 cells, knock-down for macroH2A1 and macroH2A2 (DKD) and DKD with ectopic expression of the indicated YFP-tagged constructs. H3 is used as a loading control.


B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI . The indicated areas are zoomed - in for a better observation of the H3K9me3 signal profile . All images are maximum intensity Z - projections of confocal stacks .

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B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed-in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z-projections of confocal stacks.


Quantification of the nuclear area The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test ; n . s . , not significant ) .

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Quantification of the nuclear area The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).


Quantification of H3K9me3 foci per nucleus , The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test ; n . s . , not significant ) .

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Quantification of H3K9me3 foci per nucleus, The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).


( c - f ) The indicated marker genes were used to select clusters for subsetting into stromal cells ( c ) , epithelial cells ( d ) , endothelial cells ( e ) , and leukocytes ( f ) .

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(c-f) The indicated marker genes were used to select clusters for subsetting into stromal cells (c), epithelial cells (d), endothelial cells (e), and leukocytes (f).


( g - j ) The heatmaps show the relative gene expression levels for the indicated marker genes for the indicated stromal ( g ) , epithelial ( h ) , endothelial ( i ) , and leukocyte ( j ) cell types .

(g-j) The heatmaps show the relative gene expression levels for the indicated marker genes for the indicated stromal (g), epithelial (h), endothelial (i), and leukocyte (j) cell types.


( d ) Differential gene expression between end stage lung disease patients and controls across cohorts was compared for the indicated cell type identities . The color code demonstrates similarities of gene expression changes calculated by Pearson correlation of the t - value coefficient , which represents differences in the likelihood of detection for any gene between health and disease .

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(d) Differential gene expression between end stage lung disease patients and controls across cohorts was compared for the indicated cell type identities. The color code demonstrates similarities of gene expression changes calculated by Pearson correlation of the t-value coefficient, which represents differences in the likelihood of detection for any gene between health and disease.


( e ) The heatmap shows the top 79 genes differentially expressed in the indicated cell type identities .

(e) The heatmap shows the top 79 genes differentially expressed in the indicated cell type identities.


( f - h ) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in ( f ) alveolar epithelial cells , ( g ) fibroblasts , and ( h ) macrophages when compared to control tissue . The boxes represent the interquartile range , the horizontal line in the box is the median , and the whiskers represent 1 . 5 times the interquartile range ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(f-h) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (f) alveolar epithelial cells, (g) fibroblasts, and (h) macrophages when compared to control tissue. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e ) The heatmap shows changes of the indicated cell type signatures in published bulk RNA - seq data ( GEO GSE124685 ) across different histopathological stages that represent increasing extent of fibrosis from stage 1 - 3 , as determined by quantitative micro - CT imaging and tissue histology Samples used in this study were 10 IPF and 6 control patients .

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(e) The heatmap shows changes of the indicated cell type signatures in published bulk RNA-seq data (GEO GSE124685) across different histopathological stages that represent increasing extent of fibrosis from stage 1-3, as determined by quantitative micro-CT imaging and tissue histology Samples used in this study were 10 IPF and 6 control patients.


( f ) The heatmaps show z - scores for the individual marker genes of the indicated cell types across IPF stages and controls .

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(f) The heatmaps show z-scores for the individual marker genes of the indicated cell types across IPF stages and controls.


( b ) The boxplots show the number of proteins quantified ( y - axis ) across various diagnoses ( x - axis ) . The mean and 10 - 90 percentiles are shown ; the dotted line marks 1000 proteins ( IPF n = 16 , HP / EAA n = 8 , COP n = 11 , NSIP n = 10 , RB - IL , n = 3 , Sarcoidosis n = 22 , other ILDs n = 25 , non - IL , n = 29 ) .

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(b) The boxplots show the number of proteins quantified (y-axis) across various diagnoses (x-axis). The mean and 10-90 percentiles are shown; the dotted line marks 1000 proteins (IPF n=16, HP/EAA n=8, COP n=11, NSIP n=10, RB-IL, n=3, Sarcoidosis n=22, other ILDs n=25, non-IL, n=29).


( c Empirical cumulative density plots depict the distribution of correlation coefficients for ( c ) Myofibroblast markers ( red points ) with DLCO in comparison to all background proteins ( black line ) .

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(c Empirical cumulative density plots depict the distribution of correlation coefficients for (c) Myofibroblast markers (red points) with DLCO in comparison to all background proteins (black line).


d ) Empirical cumulative density plots depict the distribution of correlation coefficients for ( d ) Plasma cell markers ( red points ) with % alveolar macrophages in BAL in comparison to all background proteins ( black line ) .

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d) Empirical cumulative density plots depict the distribution of correlation coefficients for (d) Plasma cell markers (red points) with % alveolar macrophages in BAL in comparison to all background proteins (black line).


( e ) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level .

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(e) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level.


( f ) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types ( columns ) for selected BALF protein biomarkers ( rows ) . The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls ( GSE47460 ) . Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls .

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(f) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types (columns) for selected BALF protein biomarkers (rows). The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls (GSE47460). Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls.


( a , b ) Scatter plots stratify genes based on the protein lung function associations ( y - axis ) and cell type specific ILD associations ( x - axis ) . The size of the dots represents the detection level of each gene in the corresponding cell type . Colors highlight genes with marginal associations at the protein and RNA levels .

(a, b) Scatter plots stratify genes based on the protein lung function associations (y-axis) and cell type specific ILD associations (x-axis). The size of the dots represents the detection level of each gene in the corresponding cell type. Colors highlight genes with marginal associations at the protein and RNA levels.


( c , d ) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in ( c ) basal cells , and ( d ) alveolar epithelial cells when compared to controls . The boxes represent the interquartile range , the horizontal line in the box is the median , and the whiskers represent 1 . 5 times the interquartile range ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(c, d) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (c) basal cells, and (d) alveolar epithelial cells when compared to controls. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e , f ) The scatter plots show the positive correlation of the indicated proteins in BALF ( MS - intensity , x - axis ) with meta lung function ( y - axis ) .

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(e, f) The scatter plots show the positive correlation of the indicated proteins in BALF (MS-intensity, x-axis) with meta lung function (y-axis).


( g - j ) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF ( n = 3 ) and control samples ( n = 3 ) .

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(g-j) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF (n=3) and control samples (n=3).


( a ) The scatter plots show the positive correlation of CFHR1 in BALF ( MS - intensity , x - axis ) with meta lung function ( y - axis ) .

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(a) The scatter plots show the positive correlation of CFHR1 in BALF (MS-intensity, x-axis) with meta lung function (y-axis).


( c ) The volcano plot depicts the fold changes ( x - axis ) and the - log10 p - values ( y - axis ) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB + pericytes ( outlier values truncated ) .

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(c) The volcano plot depicts the fold changes (x-axis) and the -log10 p-values (y-axis) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB+ pericytes (outlier values truncated).


( e ) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes ( left color bar indicates significant correlation or anti - correlation ) .

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(e) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes (left color bar indicates significant correlation or anti-correlation).


( f ) Immunofluorescence analysis of SSTR2 , the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF ( n = 3 ) and control samples ( n = 3 ) .

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(f) Immunofluorescence analysis of SSTR2, the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF (n=3) and control samples (n=3).


( g ) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions ( n = 474 ) from ILD patients ( n = 53 ) and control patients ( n = 26 ) .

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(g) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions (n = 474) from ILD patients (n = 53) and control patients (n = 26).


( h ) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores .

(h) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores.


( i ) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category " transcription regulators " . The dotted line marks a correlation coefficient of zero .

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(i) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category "transcription regulators". The dotted line marks a correlation coefficient of zero.


( c ) Relative expression level of CRTAC1 across human organs .

(c) Relative expression level of CRTAC1 across human organs.


( d ) The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(d) The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e ) Relative gene expression levels of CRTAC1 in GSE47460 . Dots represent average expression in the tissue of individual patients . The line represents the mean , error bars show SD . CRTAC1 is significantly downregulated in ILD but not COPD

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(e) Relative gene expression levels of CRTAC1 in GSE47460. Dots represent average expression in the tissue of individual patients. The line represents the mean, error bars show SD. CRTAC1 is significantly downregulated in ILD but not COPD patients


( f ) For each single cell cohort , the gene - gene correlations with CRTAC1 within the SFTPC + AT - 2 cells were calculated . The indicated genes were selected based on their common direction of correlation across cohorts .

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(f) For each single cell cohort, the gene-gene correlations with CRTAC1 within the SFTPC+ AT-2 cells were calculated. The indicated genes were selected based on their common direction of correlation across cohorts.


( h ) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of " transcription regulators " . The dotted line marks a correlation coefficient of zero .

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(h) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of "transcription regulators". The dotted line marks a correlation coefficient of zero.


( n ) Immunofluorescence analysis of SPRR1A , KRT8 as well as SFTPC in IPF ( n = 3 ) and control samples ( n = 2 ) .

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(n) Immunofluorescence analysis of SPRR1A, KRT8 as well as SFTPC in IPF (n=3) and control samples (n=2).


( o ) A high throughput experimental workflow for plasma proteomics ( Niu allowed for profiling of two independent cohorts of ILD patients ( Munich , n = 30 and Hannover , n = 81 ; healthy age matched controls , n = 30 ) . All proteins quantified in plasma are shown , ranked by their abundance measured by mass spectrometry ( MS - intensity ) .

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(o) A high throughput experimental workflow for plasma proteomics(Niu allowed for profiling of two independent cohorts of ILD patients (Munich, n=30 and Hannover, n=81; healthy age matched controls, n=30). All proteins quantified in plasma are shown, ranked by their abundance measured by mass spectrometry (MS-intensity).


( p ) The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics .

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(p) The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics.


( q ) The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function ( forced vital capacity - FVC ) . Patients were split in two groups , one with a mild decline in lung function [ FVC 60 - 100 % ] and one with severe loss of lung function [ FVC 20 - 60 % ] and compared to healthy age matched controls .

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(q) The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function (forced vital capacity - FVC). Patients were split in two groups, one with a mild decline in lung function [FVC 60-100%] and one with severe loss of lung function [FVC 20-60%] and compared to healthy age matched controls.


CSTB gene expression analysis in hCOs , starting from day ( d ) 16 until d140 . For every time point , at least 3 different samples were analyzed ; each sample was made by a pool of 3 to 4 hCOs . Data are represented as mean ± SEM . Unpaired t - test ( * P < 0 . 05 , * * P < 0 . 01 ) .

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CSTB gene expression analysis in hCOs, starting from day (d) 16 until d140. For every time point, at least 3 different samples were analyzed; each sample was made by a pool of 3 to 4 hCOs. Data are represented as mean ± SEM. Unpaired t-test (*P < 0.05, **P < 0.01).


Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Nuclei ( blue ) are stained with DAPI .

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Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Nuclei (blue) are stained with DAPI.


Real time qPCR results of PAX6 , NEUN , NESTIN , CSTB gene expression levels in PAX6 - and NEUN - FACS sorted nuclei from f - CTRL d135 hCOs . Data are represented as mean ± SEM . Statistical significance was based on T - student test ( * P < 0 . 05 , * * P < 0 . 01 ) . For PAX6 , 10 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei and 5 for Neun + sorted Nuclei ; for NEUN , NESTIN and CSTB , 9 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei and 4 for Neun + sorted Nuclei .

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Real time qPCR results of PAX6, NEUN, NESTIN, CSTB gene expression levels in PAX6- and NEUN- FACS sorted nuclei from f-CTRL d135 hCOs. Data are represented as mean ± SEM. Statistical significance was based on T-student test (*P < 0.05, **P < 0.01). For PAX6, 10 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 5 for Neun+sorted Nuclei; for NEUN, NESTIN and CSTB, 9 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 4 for Neun+sorted Nuclei.


Western blot analysis and relative quantifications ( arbitrary units , a . u . ) on protein extracts from cortical ( Cx ) and ganglionic eminence ( GE ) tissues for Cstb using Actb as a loading control . Cx and GE tissues from mouse embryos at different time of development as indicated in figure .

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Western blot analysis and relative quantifications (arbitrary units, a.u.) on protein extracts from cortical (Cx) and ganglionic eminence (GE) tissues for Cstb using Actb as a loading control. Cx and GE tissues from mouse embryos at different time of development as indicated in figure.


Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin ( acTub ) as marker for stabilized microtubules . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Nuclei ( blue ) are stained with DAPI .

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Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin (acTub) as marker for stabilized microtubules. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Nuclei (blue) are stained with DAPI.


Micrograph sections of d40 hCOs electroporated with GFP - empty vector control or GFP - CSTB and analyzed 5 dpe . Sections were then immunostained for GFP and KI67 . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Quantification of the total number of proliferating KI67 + cells / area ( µm2 ) of V - like structures transfected with GFP - empty vector control or GFP - CSTB in ( A ) .

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Micrograph sections of d40 hCOs electroporated with GFP-empty vector control or GFP-CSTB and analyzed 5 dpe. Sections were then immunostained for GFP and KI67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area (µm2) of V-like structures transfected with GFP-empty vector control or GFP-CSTB in (A).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb and analyzed 2 dpe and immunostained with GFP , Ki67 and BrdU 30 minutes after BrdU injection . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) ( D ) and Ki67 + Brdu + cells / area ( µm2 ) ( E ) of ventricles transfected with GFP - empty vector or GFP - Cstb in ( C ) .

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Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb and analyzed 2 dpe and immunostained with GFP,Ki67 and BrdU 30 minutes after BrdU injection. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area(µm2)(D) and Ki67+Brdu+ cells/area(µm2)(E) of ventricles transfected with GFP-empty vector or GFP-Cstb in (C).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb , analyzed 2 dpe and immunostained with GFP and Phospho - Histone H3 ( PH3 ) ( F ) and with GFP , Pax6 and Tbr2 ( G ) . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles .

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Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3)(F) and with GFP, Pax6 and Tbr2 (G). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.


Distribution of proliferating Ki67 + / BrdU + cells ( H ) and mitotic progenitor PH3 + cells ( I ) in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3 + cells or Ki67 + / BrdU + cells calculated in each Bin .

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Distribution of proliferating Ki67+/BrdU+cells (H) and mitotic progenitor PH3+ cells (I) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells or Ki67+/BrdU+ cells calculated in each Bin.


Distribution of Tbr2 + intermediate progenitors ( J ) and Pax6 + Tbr2 + cells ( K ) in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2 + cells or Pax6 + Tbr2 + cells calculated in each Bin .

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Distribution of Tbr2+ intermediate progenitors (J) and Pax6+Tbr2+ cells (K) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2+ cells or Pax6+Tbr2+ cells calculated in each Bin.


Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos ( A ) , in the CM from E14 cortical cells in culture for 4

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Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos (A), in the CM from E14 cortical cells in culture for 4 days


Western blot analysis for CSTB in the CM from hCOs after 4 days in culture ( C ) . The CM was collected from hCOs at different time points in culture ( d19 , d38 , d60 , d80 ) . Protein extract ( PE ) from 60 days hCOs was used as a positive control . GAPDH immunostaining was used as a negative control on hCOs CM . Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM

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Western blot analysis for CSTB in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samples


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector and HA - empty vector or - Cstb , and analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65 - GFP transgenic mouse line . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of GFP + interneurons in the 5 equal Bins of the mouse developing cortex - Bin1 corresponded to apical side and Bin5 to pial side . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles .

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Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-empty vector or -Cstb, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex-Bin1 corresponded to apical side and Bin5 to pial side. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry and HA - Cstb expressing vector , analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67 - GFP transgenic mouse line . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of GFP + interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro - caudally .

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Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry and HA-Cstb expressing vector, analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro-caudally.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - miR - neg vector ( CTRL ) and GFP - miRNA - Cstb ( KD ) , analyzed 3 dpe and immunostained with GFP . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of electroporated GFP + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP + cells calculated in each Bin . Nuclei ( blue ) are stained with DAPI .

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Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-miR-neg vector (CTRL) and GFP-miRNA-Cstb (KD), analyzed 3 dpe and immunostained with GFP. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin. Nuclei (blue) are stained with DAPI.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - empty vector control and GFP - miRNA - Cstb , analyzed 3 dpe and immunostained with GFP and Ki67 . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) of ventricular structures transfected with GFP - miR - neg vector or GFP - miRNA - Cstb in ( C ) . Nuclei ( blue ) are stained with DAPI .

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Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-empty vector control and GFP-miRNA-Cstb, analyzed 3 dpe and immunostained with GFP and Ki67. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricular structures transfected with GFP-miR-neg vector or GFP-miRNA- Cstb in (C). Nuclei (blue) are stained with DAPI.


Micrograph sections of d40 hCOs electroporated with GFP - CSTB expressing vector or GFP - R68X mutant and analyzed 5 dpe . Sections were immunostained for GFP and Ki67 . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Quantification of the total number of proliferating KI67 + cells / area ( µm2 ) ( F ) and Ki67 + GFP + cells / area ( µm2 ) ( G ) of ventricles like structures transfected with GFP - empty vector control , GFP - CSTB or GFP - R68X mutant in ( E ) .

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Micrograph sections of d40 hCOs electroporated with GFP-CSTB expressing vector or GFP-R68X mutant and analyzed 5 dpe. Sections were immunostained for GFP and Ki67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area(µm2)(F)and Ki67+GFP+ cells/area(µm2)(G) of ventricles like structures transfected with GFP-empty vector control, GFP-CSTB or GFP-R68X mutant in (E).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X , analyzed 2 dpe and immunostained with GFP and Phospho - Histone H3 ( PH3 ) . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of electroporated GFP + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP + cells calculated in each Bin .

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Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin.


Distribution of mitotic progenitor PH3 + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3 + cells calculated in each Bin .

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Distribution of mitotic progenitor PH3+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells calculated in each Bin.


K . Quantification of the total number of proliferating PH3 + cells / area ( µm2 ) of ventricular structures transfected with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant in ( H ) .

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K.Quantification of the total number of proliferating PH3+ cells/area(µm2)of ventricular structures transfected with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant in (H).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant , analyzed 2 dpe and immunostained with GFP and Tbr2 . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of Tbr2 + cells / cortical area ( µm2 ) in ( L ) . Data shown as Z - scores relative to the mean of GFP control vector .

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

Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant, analyzed 2 dpe and immunostained with GFP and Tbr2. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of Tbr2+ cells/ cortical area (µm2) in (L). Data shown as Z-scores relative to the mean of GFP control vector.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector and HA - Cstb or R68X , and analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65 - GFP transgenic mouse line . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of GAD65 - GFP interneurons / cortical area ( µm2 ) in ( N ) . Data shown as Z - scores relative to the mean of GFP control vector . Nuclei ( blue ) are stained with DAPI .

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

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-Cstb or R68X, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of GAD65-GFP interneurons/ cortical area (µm2) in (N). Data shown as Z-scores relative to the mean of GFP control vector. Nuclei (blue) are stained with DAPI.


Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points . Right panel shows the difference of the areas of f - CTRL , b - CTRL , UL1 and UL4 hCOs at different time in culture . Data shown as Z - scores relative to the mean of b - CTRL .

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

Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points. Right panel shows the difference of the areas of f-CTRL, b-CTRL, UL1 and UL4 hCOs at different time in culture. Data shown as Z-scores relative to the mean of b-CTRL.


Micrograph sections of d30 b - CTRL , UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3 . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) of ventricles like structures and of the number of PH3 + cells facing the ventricle / apical surface length ( µm ) . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

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 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

Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricles like structures and of the number of PH3+ cells facing the ventricle/apical surface length (µm). Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d60 b - CTRL , UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3 . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the total number of proliferating KI67 + cells / area of ventricles like structures and of the number of PH3 + cells facing the ventricle / the apical surface length ( µm ) . Data shown as Z - scores relative to the mean of CTRL . Nuclei ( blue ) are stained with DAPI .

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 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

Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating KI67+ cells/ area of ventricles like structures and of the number of PH3+ cells facing the ventricle/the apical surface length (µm). Data shown as Z-scores relative to the mean of CTRL. Nuclei (blue) are stained with DAPI.


Scheme of the experiment with conditioned medium exchange between b - CTRL and UL4 hCOs and quantification of the Ki67 + cells by FACS analysis on 36 days old dissociated hCOs . Four samples were analyzed as biological replicates .

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

Scheme of the experiment with conditioned medium exchange between b-CTRL and UL4 hCOs and quantification of the Ki67+ cells by FACS analysis on 36 days old dissociated hCOs. Four samples were analyzed as biological replicates.


Micrograph sections of d30 b - CTRL , UL1 and UL4 hCOs immunostained for the neuronal marker DCX . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the DCX + area . The area of DCX + cells was calculated and related to the total area ( µm2 ) of the ventricular structure . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

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 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

Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for the neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area of DCX+ cells was calculated and related to the total area (µm2) of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d60 b - CTRL , UL1 and UL4 hCOs immunostained for neuronal marker DCX . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the DCX + area . The area ( µm2 ) of DCX + cells was calculated and related to the total area of the ventricular structure . Data shown as Z - scores relative to the mean of b - CTRL . Quantification of the number of neuronal DCX + cells by FACS sorting on 60 days old dissociated hCOs . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

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

Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area (µm2) of DCX+ cells was calculated and related to the total area of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Quantification of the number of neuronal DCX+ cells by FACS sorting on 60 days old dissociated hCOs. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d30 fused - organoid ; ventral GFP - f - CTRL and dorsal UL1 .

Micrograph sections of d30 fused-organoid; ventral GFP-f-CTRL and dorsal UL1.


Micrographs of sections from d30 fused organoids , showing ventral GFP + cells in the dorsal area . The migrating GFP + cells were counted starting from 100 µm far from the ventral / dorsal border . Quantification of ventrally - derived GFP + cells in dorsal / thickness of the counted area ( µm3 ) . The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig . EV5D . Every dot represents a different hCOs and different areas were counted for each hCO . Nuclei ( blue ) are stained with DAPI .

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

Micrographs of sections from d30 fused organoids, showing ventral GFP+ cells in the dorsal area. The migrating GFP+ cells were counted starting from 100 µm far from the ventral/dorsal border. Quantification of ventrally-derived GFP+ cells in dorsal/thickness of the counted area (µm3). The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig.EV5D. Every dot represents a different hCOs and different areas were counted for each hCO. Nuclei (blue) are stained with DAPI.


Volcano plot representing proteins identified by high - resolution nanoLC MS / MS analysis . Up - and down - regulated proteins ( - 0 . 3 ≥ log2FC ≥ 0 . 3 ) in human cerebral organoids ( UL1 vs b - CTRL ) are shown in red and green , respectively .

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

Volcano plot representing proteins identified by high-resolution nanoLC MS/MS analysis. Up- and down-regulated proteins (-0.3≥log2FC≥0.3) in human cerebral organoids (UL1 vs b-CTRL) are shown in red and green, respectively.


A - C Calcium flux measured by FACScan for splenic B cells isolated from B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( see Figure EV1 , 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . The addition of the stimuli to the cells is indicated by arrows .

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

A-C Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (see Figure EV1, 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows.


D Representative microscopy images showing Fab - PLA results measuring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . PLA signals are shown as red dots and the nuclei are visualized by DAPI staining . Scale bar : 5 μm . E The Fab - PLA results are quantified by Blobfinder software and presented as box plots . The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . Data from treated samples were compared to data from resting cells , p - values were calculated by Kruskal - Wallis one - way analysis of variance ( ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative microscopy images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots and the nuclei are visualized by DAPI staining. Scale bar: 5 μm.E The Fab-PLA results are quantified by Blobfinder software and presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from treated samples were compared to data from resting cells, p-values were calculated by Kruskal-Wallis one-way analysis of variance (ANOVA).


A - C Calcium flux measured by FACScan for splenic B cells isolated from Lyn - deficient B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


D Representative microscopy images showing Fab - PLA results monitoring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . Scale bar : 5 μm . E Quantified Fab - PLA results are presented as box plots , where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . p - values were calculated by Kruskal - Wallis one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative microscopy images showing Fab-PLA results monitoring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. Scale bar: 5 μm.E Quantified Fab-PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. p-values were calculated by Kruskal-Wallis one-way ANOVA.


A - D Proximity between IgM - BCR on the surface of 3046SM ( A , C ) or 3046M ( B , D ) cells before and after a 1 - min stimulation with the indicated reagents , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . Scale bar : 5 μm . In the box plots , the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA .

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

A-D Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SM ( E , F ) and 3046M ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the 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 O O O O O O O O O O O O O O

E-H Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


A - D Proximity between IgD - BCRs on the surface of 3046SD ( A , C ) or 3046D ( B , D ) cells before and after a 1 - min stimulation , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . PLA signals are shown as red dots and nuclei were visualized by DAPI staining . Scale bar : 5 μm . In the box plots , the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA .

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

A-D Proximity between IgD-BCRs on the surface of 3046SD (A, C) or 3046D (B, D) cells before and after a 1-min stimulation, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SD ( E , F ) and 3046D ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

E-H Calcium flux measured by FACScan for 3046SD (E, F) and 3046D (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


Calcium flux measured by FACScan for 3046M ( A , D ) , 3046SD ( B , E ) and 3046D ( C , F ) cells stimulated with anti - HC ( 2 μl / ml ) ( A , B , C ) , or anti - λ ( 2 μl / ml ) ( D , E , F ) antibodies with or without 1 min prestimulation with 1NIP - pep ( 80 nM ) . The addition of antibodies is indicated by black arrows . The 1NIP - pep was added immediately before recording . Data are representative of three independent experiments .

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

Calcium flux measured by FACScan for 3046M (A, D), 3046SD (B, E) and 3046D (C, F) cells stimulated with anti-HC (2 μl/ml) (A, B, C), or anti-λ (2 μl/ml) (D, E, F) antibodies with or without 1 min prestimulation with 1NIP-pep (80 nM). The addition of antibodies is indicated by black arrows. The 1NIP-pep was added immediately before recording. Data are representative of three independent experiments.


A - E B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD19 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD19 antibody . The calcium fluxes were measured by FACScan .

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

A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan.