D - E . Immunohistochemistry of p62 mitochondrial mass marker , consecutive sections . Asterisks indicate p62 - positive corresponding affected ragged - red . F - G . p62 mitochondrial mass after mAD ; consecutive sections . Arrowheads indicate that show p62 positivity but have normal mitochondrial mass .

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D - E. Immunohistochemistry analysis of p62 and mitochondrial mass marker, consecutive sections. Asterisks indicate p62-positive fibers and corresponding affected ragged-red fibers.F - G. p62 and mitochondrial mass after mAD; consecutive sections. Arrowheads indicate fibers that show p62 positivity but have normal mitochondrial mass.


H . p62 LC3 positive ragged - red ( * ) in PEO ( P2 ) , normal diet . I . Muscle showing granular LC3 - positivity , PEO ( P1 ) , after mAD . Immunohistochemistry on frozen sections ; bars : 50 µm , except for H , 100 µm .

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H. p62 and LC3 positive ragged-red fibers (*) in PEO patient (P2), normal diet.I. Muscle fibers showing granular LC3-positivity, PEO patient (P1), after mAD. Immunohistochemistry on frozen sections; bars: 50 µm, except for H, 100 µm.


A - C . Muscle strength performance of PEO ( P1 - P4 ) on normal diet ( ND ) after modified Atkins diet ( mAD ) ( one six months 2 . 5 years after initiation of diet ) .

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A-C. Muscle strength and performance of PEO patients (P1-P4) on normal diet (ND) and after modified Atkins diet (mAD) (one and six months and 2.5 years after initiation of diet).


D - G . Spiroergometry results from PEO control subjects on normal diet ( ND ) , as well as one month six months after mAD initiation . PEO had higher serumlactate , attenuated respiratory response to exercise . These effects were further enhanced after mAD remained after 6 months of study . mAD increased ' serumammonium ( NH4 + ) . Data presented as mean SD .

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D-G. Spiroergometry results from PEO patients and control subjects on normal diet (ND), as well as one month and six months after mAD initiation. PEO patients had higher serumlactate levels, and attenuated respiratory response to exercise. These effects were further enhanced after mAD and remained after 6 months of the study. mAD increased patients' serumammonium (NH4+) levels. Data presented as mean and SD.


A . PEO , relative acid on normal diet ( PEO ) , immediately after finishing mAD ( PEO _ mAD ) , one month after mAD . B . Control subjects , relative acid on normal diet ( Control ) immediately after finishing mAD ( Control _ mAD ) .

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A. PEO patients, relative amino acid levels on normal diet (PEO), immediately after finishing mAD (PEO_mAD), and one month after mAD.B. Control subjects, relative amino acid levels on normal diet (Control) and immediately after finishing mAD (Control_mAD).


C . Relative values of plasma metabolites in creatine pathway , compared to controls on ND . Right : illustration of major metabolites pathway of creatine synthesis .

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C. Relative values of plasma metabolites in creatine pathway, compared to controls on ND. Right: illustration of major metabolites and pathway of creatine synthesis.


D . Relative values of plasma purine pyrimidine degradation pathway intermediates , compared to controls on ND . Right : illustration of major metabolites synthesis of pyrimidine purines .

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D. Relative values of plasma purine and pyrimidine degradation pathway intermediates, compared to controls on ND. Right: illustration of major metabolites and synthesis of pyrimidine and purines.


( B ) Box plots show MIG - 6 ( ERRFI1 ) gene expression in different breast subtypes in Servant dataset of 343 breast cancer carcinomas , analyzed using Illumina HumanWG - 6 _ v3 Arrays . gene expression are determined using R2 Genomics Visualization Platform ( http : / / r2 . amc . nl ) . In box plot , error bars are 95 % confidence interval , bottom top of box are 25th 75th percentiles , inside box is 50th percentile ( median ) , any outliers are shown as open circles . * * p < 0 . 01 , by Student ' s t - .

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(B) Box plots show the MIG-6 (ERRFI1) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas, analyzed using Illumina HumanWG-6_v3 Arrays. The gene expression levels are determined using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median), and any outliers are shown as open circles. **p < 0.01, by Student's t-test.


( C ) Immunoblotting for MIG - 6 expression in various human breast cancer lines .

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(C) Immunoblotting analysis for MIG-6 protein expression in various human breast cancer cell lines.


( D ) Representative of histological analyses of MIG - 6 in TNBC in low - grade ( stage I ) high - grade ( stage III ) tumors . Scale bar , 200 µm .

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(D) Representative images of histological analyses of MIG-6 in TNBC patients in low-grade (stage I) and high-grade (stage III) tumors. Scale bar, 200 µm.


( E ) of MIG - 6 expression in 85 cases of resected TNBCs . In box plot , error bars are 95 % confidence interval , bottom top of box are 25th 75th percentiles , inside box is 50th percentile ( median ) . p - value 0 . 011 is used for comparison of three groups , determined by Kruskal - Wallis H .

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(E) Quantification analysis of MIG-6 expression in 85 cases of resected TNBCs. In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median). The p-value 0.011 is used for the comparison of all three groups, determined by the Kruskal-Wallis H test.


( F ) Kaplan - Meier plot of disease - specific survival of 85 TNBC breast cancer with low or high expression of MIG - 6 .

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(F) Kaplan-Meier plot analysis of the disease-specific survival of 85 TNBC breast cancer patients with low or high expression of MIG-6.


( G ) Kaplan - Meier plot of metastasis - specific survival of 85 TNBC with low or high expression of MIG - 6 . MIG - 6 expression in 85 TNBC specimens was determined by H - score widely distributed . MIG - 6 expression greater than or equal to median is classified as " high , " while expression less than median is classified as " low . "

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(G) Kaplan-Meier plot analysis of the metastasis-specific survival of 85 TNBC patients with low or high expression of MIG-6. MIG-6 protein expression in the 85 TNBC specimens was determined by H-score and widely distributed. MIG-6 expression greater than or equal to the median is classified as "high," while expression less than the median is classified as "low."


( A ) proliferation immunoblotting assays in BT549 with Luciferase or MIG - 6 knockdown .

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(A) Cell proliferation and immunoblotting assays in BT549 cells with Luciferase or MIG-6 knockdown.


( B ) proliferation immunoblotting assays in MDA - MB - 231 with Luciferase or MIG - 6 knockdown .

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(B) Cell proliferation and immunoblotting assays in MDA-MB-231 cells with Luciferase or MIG-6 knockdown.


( C ) Representative results for a colony formation in BT549 with GFP or MIG - 6 knockdown .

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(C) Representative images and quantification results for a colony formation assay in BT549 cells with GFP or MIG-6 knockdown.


( D Immunoblotting for EGFR signaling in BT549 with GFP or MIG - 6 knockdown .

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(D Immunoblotting analysis for EGFR signaling in BT549 cells with GFP or MIG-6 knockdown.


E ) Immunoblotting for EGFR signaling in MDA - MB - 231 with GFP or MIG - 6 knockdown .

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E) Immunoblotting analysis for EGFR signaling in MDA-MB-231 cells with GFP or MIG-6 knockdown.


( F ) growth in BT549 with GFP or MIG - 6 knockdown in absence presence of gefitinib for 72 hours .

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(F) Cell growth assay in BT549 cells with GFP or MIG-6 knockdown in the absence and presence of gefitinib for 72 hours.


( A ) Immunoblotting for MIG - 6 expression in BT549 under normoxia hypoxia .

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(A) Immunoblotting analysis for MIG-6 expression in BT549 cells under normoxia and hypoxia.


( B ) Extracellular acidification rate ( ECAR ) in BT549 with GFP or MIG - 6 knockdown . ECAR flux profile of GFP MIG - 6 knockdown BT549 upon stimulation of glucose , oligomycin , 2 - DG is shown on left . Quantitative results for basal glycolysis maximal glycolysis capacities in GFP MIG - 6 knockdown BT549 are indicated by orange green boxes , graphed on right . Results are presented as mean ± ( n = 15 ; biological replicates ) .

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(B) Extracellular acidification rate (ECAR) in BT549 cells with GFP or MIG-6 knockdown. The ECAR flux profile of GFP and MIG-6 knockdown BT549 cells upon stimulation of glucose, oligomycin, and 2-DG is shown on the left. Quantitative results for the basal glycolysis and maximal glycolysis capacities in GFP and MIG-6 knockdown BT549 cells are indicated by orange and green boxes, graphed on the right. Results are presented as mean ± SEM (n=15; biological replicates).


( C ) A diagram shows expression profile of genes involved in glycolysis , TCA cycle , pentose phosphate pathway , pyruvate oxidation , gluconeogenesis , glycogen metabolism obtained from two sets of GFP MIG - 6 knockdown BT549 .

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(C) A heatmap diagram shows the expression profile of genes involved in glycolysis, the TCA cycle, the pentose phosphate pathway, pyruvate oxidation, gluconeogenesis, and glycogen metabolism obtained from two sets of GFP and MIG-6 knockdown BT549 cells.


Histograms show of gene dysregulation in glycolysis , TCA cycle , upon MIG - 6 knockdown in BT549 . For each gene , change was calculated on equation : ( shGFP - shMIG6 ) / shMIG - 6 . Results are presented as mean ± ( n = 3 ; biological replicates ) .

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Histograms show the percentage of gene dysregulation in glycolysis, TCA cycle, upon MIG-6 knockdown in BT549 cells. For each gene, the change was calculated based on the equation: (shGFP-shMIG6)/shMIG-6. Results are presented as mean ± SEM (n=3; biological replicates).


F ) Histograms show of pyruvate oxidation pathways upon MIG - 6 knockdown in BT549 . For each gene , change was calculated on equation : ( shGFP - shMIG6 ) / shMIG - 6 . Results are presented as mean ± ( n = 3 ; biological replicates ) .

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F) Histograms show the percentage of pyruvate oxidation pathways upon MIG-6 knockdown in BT549 cells. For each gene, the change was calculated based on the equation: (shGFP-shMIG6)/shMIG-6. Results are presented as mean ± SEM (n=3; biological replicates).


( H ) Lactate production in BT549 with GFP or MIG - 6 knockdown ( n = 3 ) . medium was collected to measure lactate using lactate strips an Accutrend Lactate analyzer . rate of lactate production was determined ( lactate production rate = lactate / / time ) normalized to rate detected in control group . quantified results are presented as mean ± ( n = 3 ; biological replicates ) .

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(H) Lactate production assay in BT549 cells with GFP or MIG-6 knockdown (n=3). The medium was collected to measure lactate concentration using lactate test strips and an Accutrend Lactate analyzer. The rate of lactate production was determined (lactate production rate = lactate concentration/cells/time) and normalized to the rate detected in the control group. The quantified results are presented as mean ± SEM (n=3; biological replicates).


( A ) Box plots show GLUT1 ( SLC2A1 ) gene expression in different breast subtypes in Servant dataset of 343 breast cancer carcinomas , analyzed using Illumina HumanWG - 6 _ v3 Arrays . gene expression are determined using R2 platform . In box plot , error bars are 95 % confidence interval , bottom top of box are 25th 75th percentiles , inside box is 50th percentile ( median ) , any outliers are shown as open circles . * * p < 0 . 01 , by Student ' s t - . .

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(A) Box plots show the GLUT1 (SLC2A1) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas, analyzed using Illumina HumanWG-6_v3 Arrays. The gene expression levels are determined using the R2 platform. In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median), and any outliers are shown as open circles. **p < 0.01, by Student's t-test..


( B ) Real - Time PCR for GLUT1 MIG - 6 mRNA expression in GFP MIG - 6 knockdown BT549 ( n = 3 ) .

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(B) Real-Time PCR analysis for GLUT1 and MIG-6 mRNA expression in GFP and MIG-6 knockdown BT549 cells (n=3).


( C ) Immunoblotting for GLUT1 expression in BT549 with GFP or MIG - 6 knockdown .

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(C) Immunoblotting analysis for GLUT1 protein expression in BT549 cells with GFP or MIG-6 knockdown.


( D ) Confocal image results for - bound GLUT1 in GFP MIG - 6 knockdown BT549 are shown in top panel . arrow indicates - localized GLUT1 . Scale bar , 25 μm . Quantitative results of of GFP MIG - 6 knockdown BT549 expressing - localized GLUT1 are shown in bottom panel ( n = 3 ) .

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(D) Confocal image analysis results for membrane-bound GLUT1 in GFP and MIG-6 knockdown BT549 cells are shown in the top panel. The arrow indicates membrane-localized GLUT1. Scale bar, 25 μm. Quantitative analysis results of the percentage of GFP and MIG-6 knockdown BT549 cells expressing membrane-localized GLUT1 are shown in the bottom panel (n=3).


( E ) Representative flow cytometry ( upper ) quantitative results ( lower ) of measuring glucose uptake in GFP MIG - 6 knockdown BT549 ( n = 3 ) . were grown in presence of analog 2 - NBDG for various time periods , glucose uptake was quantified using flow cytometric . red box indicates gating for 2 - NBDG + number indicates of 2 - NBDG + .

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(E) Representative flow cytometry images (upper) and quantitative results (lower) of measuring glucose uptake in GFP and MIG-6 knockdown BT549 cells (n=3). Cells were grown in the presence of the fluorescent analog 2-NBDG for various time periods, and glucose uptake was quantified using flow cytometric analysis. The red box indicates the gating for 2-NBDG+ cells and the number indicates the percentage of 2-NBDG+ cells.


( F ) Immunoblotting for GLUT1 expression in GFP MIG - 6 knockdown BT549 with or without MG132 treatment .

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(F) Immunoblotting analysis for GLUT1 protein expression in GFP and MIG-6 knockdown BT549 cells with or without MG132 treatment.


( B ) Immunoblotting for HIF1α expression in BT549 with GFP or MIG - 6 knockdown . ( C ) Immunoblotting for HIF1α expression in MDA - MB - 231 with GFP or MIG - 6 knockdown .

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(B) Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG-6 knockdown. (C) Immunoblotting analysis for HIF1α protein expression in MDA-MB-231 cells with GFP or MIG-6 knockdown.


( B ) Immunoblotting for HIF1α expression in BT549 with GFP or MIG - 6 knockdown .

(B) Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG-6 knockdown.


( D ) Real - Time PCR for HIF1α mRNA expression in BT549 with GFP or MIG - 6 knockdown . quantified results are presented as mean ± SD ( n = 4 ) . * * * p < 0 . 001 , by Student ' s t - .

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(D) Real-Time PCR analysis for HIF1α mRNA expression in BT549 cells with GFP or MIG-6 knockdown. The quantified results are presented as mean ± SD (n=4). ***p < 0.001, by Student's t-test.


( E ) Immunoblotting for HIF1α expression in GFP MIG - 6 knockdown BT549 in absence or presence of proteasome inhibitor MG132 .

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(E) Immunoblotting analysis for HIF1α protein expression in GFP and MIG-6 knockdown BT549 cells in the absence or presence of the proteasome inhibitor MG132.


( F ) GFP MIG - 6 knockdown BT549 transfected with indicated plasmids were treated with MG132 , subjected to immunoprecipitation ( IP ) with Flag - tag or HIF1α antibody , followed by immunoblotting . MG132 was used to rescue HIF1α degradation mediated by MIG - 6 knockdown , leading to similar HIF1α in control MIG - 6 knockdown BT549 , which were used as input to examine role of MIG - 6 in interaction between HIF1α HAUSP .

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(F) GFP and MIG-6 knockdown BT549 cells transfected with the indicated plasmids were treated with MG132, subjected to immunoprecipitation (IP) with Flag-tag or HIF1α antibody, followed by immunoblotting analysis. MG132 was used to rescue HIF1α protein degradation mediated by MIG-6 knockdown, leading to similar HIF1α protein levels in control and MIG-6 knockdown BT549 cells, which were used as the input to examine the role of MIG-6 in the interaction between HIF1α and HAUSP.


( G ) Luciferase MIG - 6 knockdown 293 were transfected with indicated plasmids subjected to MG132 treatment . Afterward , were harvested for IP with HA antibody , followed by immunoblotting to determine level of K48 - linked ubiquitination of HIF1α . MG132 was used to preserve degradative K48 - linked ubiquitination signals .

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(G) Luciferase and MIG-6 knockdown 293 cells were transfected with the indicated plasmids and subjected to MG132 treatment. Afterward, cells were harvested for IP with HA antibody, followed by immunoblotting analysis to determine the level of K48-linked ubiquitination of HIF1α. MG132 was used to preserve the degradative K48-linked ubiquitination signals.


( H ) Immunoblotting for MIG - 6 HIF1α expression in Luciferase MIG - 6 knockdown 293 without MG132 treatment .

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(H) Immunoblotting analysis for MIG-6 and HIF1α expression in Luciferase and MIG-6 knockdown 293 without MG132 treatment.


( I ) Immunoblotting for GLUT1 expression upon MIG - 6 overexpression in GFP HIF1α knockdown BT549 .

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(I) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HIF1α knockdown BT549 cells.


( J ) Immunoblotting for GLUT1 expression upon MIG - 6 overexpression in GFP HAUSP knockdown BT549 .

(J) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HAUSP knockdown BT549 cells.


( K ) Immunoblotting for HIF1α expression upon HAUSP overexpression in GFP MIG - 6 knockdown BT549 .

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(K) Immunoblotting analysis for HIF1α protein expression upon HAUSP overexpression in GFP and MIG-6 knockdown BT549 cells.


( A B ) Lactate production in BT549 ( A ) MDA - MB - 231 ( B ) with Luciferase or GLUT1 knockdown .

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(A and B) Lactate production assay in BT549 (A) and MDA-MB-231 (B) cells with Luciferase or GLUT1 knockdown.


( C ) proliferation in BT549 with Luciferase or GLUT1 knockdown .

(C) Cell proliferation assay in BT549 cells with Luciferase or GLUT1 knockdown.


( D ) proliferation in MDA - MB - 231 with GFP or GLUT1 knockdown .

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(D) Cell proliferation assay in MDA-MB-231 cells with GFP or GLUT1 knockdown.


( E F ) Immunoblotting for GLUT1 expression in control MIG - 6 knockdown BT549 ( E ) MDA - MB - 231 ( F ) with or without GLUT1 overexpression .

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(E and F) Immunoblotting analysis for GLUT1 protein expression in control and MIG-6 knockdown BT549 (E) and MDA-MB-231 (F) cells with or without GLUT1 overexpression.


( G Lactate production in control MIG - 6 knockdown BT549 with or without GLUT1 overexpression .

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(G Lactate production assay in control and MIG-6 knockdown BT549 cells with or without GLUT1 overexpression.


H ) Lactate production in control MIG - 6 knockdown MDA - MB - 231 with or without GLUT1 overexpression .

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H) Lactate production assay in control and MIG-6 knockdown MDA-MB-231 cells with or without GLUT1 overexpression.


( I J ) proliferation in BT549 ( I ) MDA - MB - 231 ( J ) with control knockdown , MIG - 6 knockdown , or MIG - 6 knockdown plus GLUT1 overexpression .

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(I and J) Cell proliferation assay in BT549 (I) and MDA-MB-231 (J) cells with control knockdown, MIG-6 knockdown, or MIG-6 knockdown plus GLUT1 overexpression.


( A B ) In vivo growth derived from BT549 with Luciferase or MIG - 6 knockdown ( six mice per group ) . were injected into mammary fat pads of nude mice , sizes were measured weekly by caliper . Kaplan - Meier plot is used to determine incidence of Luciferase or MIG - 6 knockdown BT549 - xenograft tumors ( A ) . Volumes of Luciferase or MIG - 6 knockdown BT549 tumors at week 10 are presented as mean ± ( B ) .

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(A and B) In vivo primary tumor growth derived from BT549 cells with Luciferase or MIG-6 knockdown (six mice per group). Cells were injected into the mammary fat pads of nude mice, and tumor sizes were measured weekly by caliper. Kaplan-Meier plot analysis is used to determine the incidence of Luciferase or MIG-6 knockdown BT549-xenograft tumors (A). Volumes of Luciferase or MIG-6 knockdown BT549 tumors at week 10 are presented as mean ± SEM (B).


( C In vivo growth derived from MDA - MB - 231 with Luciferase or MIG - 6 knockdown ( nine mice per group ) . Volumes of Luciferase or MIG - 6 knockdown MDA - MB - 231 tumors were measured weekly by caliper are presented as mean ±

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(C In vivo primary tumor growth derived from MDA-MB-231 cells with Luciferase or MIG-6 knockdown (nine mice per group). Volumes of Luciferase or MIG-6 knockdown MDA-MB-231 tumors were measured weekly by caliper are presented as mean ± SEM


D ) In vivo derived from MDA - MB - 231 with Luciferase or MIG - 6 knockdown ( nine mice per group ) . weights of MDA - MB - 231 - derived xenografts were measured at endpoint ( day 33 ) are presented as mean ± ( D ) .

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D) In vivo primary tumor derived from MDA-MB-231 cells with Luciferase or MIG-6 knockdown (nine mice per group). Tumor weights of MDA-MB-231-derived xenografts were measured at the endpoint (day 33) and are presented as mean ± SEM (D).


( E ) Immunoblotting for MIG - 6 expression in BT549 with MIG - 6 inducible knockdown ( iMIG - 6 - shRNA ) non - targeting shRNA control ( iNT - shRNA ) upon doxycycline treatment .

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(E) Immunoblotting analysis for MIG-6 expression in BT549 cells with MIG-6 inducible knockdown (iMIG-6-shRNA) and the non-targeting shRNA control (iNT-shRNA) upon doxycycline treatment.


( F ) In vivo growth derived from BT549 containing iNT - shRNA iMIG - 6 - shRNA ( n = 7 per group ) . arrow indicates time point when diet was given to induce MIG - 6 knockdown in vivo . Volumes of iNT - shRNA iMIG - 6 - shRNA BT549 tumors are presented as mean ± .

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(F) In vivo primary tumor growth derived from BT549 cells containing iNT-shRNA and iMIG-6-shRNA (n=7 per group). The arrow indicates the time point when the diet was given to induce MIG-6 knockdown in vivo. Volumes of iNT-shRNA and iMIG-6-shRNA BT549 tumors are presented as mean ± SEM.


( G ) Representative for growth quantitative results for at experimental endpoint ( seven mice per group ) . Results are presented as mean ± .

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(G) Representative images for tumor growth and quantitative results for tumor weight at the experimental endpoint (seven mice per group). Results are presented as mean ± SEM.


( A ) Representative of histological for GLUT1 expression in BT549 - derived xenograft tumors . Scale bar , 100 μm . ( B ) Representative of histological for GLUT1 expression in xenograft tumors derived from BT549 containing iNT - shRNA iMIG - 6 - shRNA . Scale bar , 100 μm .

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(A) Representative images of histological analysis for GLUT1 protein expression in BT549-derived xenograft tumors. Scale bar, 100 μm. (B) Representative images of histological analysis for GLUT1 protein expression in xenograft tumors derived from BT549 cells containing iNT-shRNA and iMIG-6-shRNA. Scale bar, 100 μm.


( A ) Representative of histological for GLUT1 expression in BT549 - derived xenograft tumors . Scale bar , 100 μm .

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(A) Representative images of histological analysis for GLUT1 protein expression in BT549-derived xenograft tumors. Scale bar, 100 μm.


( C ) Representative of histological analyses for GLUT1 MIG - 6 expressions in TNBC in low - grade ( stage I ) high - grade ( stage III ) tumors Scale bar , 200 µm .

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(C) Representative images of histological analyses for GLUT1 and MIG-6 expressions in TNBC patients in low-grade (stage I) and high-grade (stage III) tumors Scale bar, 200 µm.


( D ) Scatterplot for correlation between GLUT1 MIG - 6 expression ( n = 85 ) . r value was calculated by Spearman ' s rank correlation coefficient .

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(D) Scatterplot analysis for the correlation between GLUT1 and MIG-6 expression levels (n=85). The r value was calculated by Spearman's rank correlation coefficient analysis.


( E ) Representative of immunofluorescence multiplex for colocalization of MIG - 6 HIF1α expression in TNBC specimens . triangle indicates colocalization , star indicates colocalization .

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(E) Representative images of the immunofluorescence multiplex assay for colocalization of MIG-6 and HIF1α protein expression in TNBC tumor specimens. The triangle indicates nuclear colocalization, and the star indicates cytoplasmic colocalization.


B ) Bar chart indicating , localization ( according to UniProt ) in secretome of neurons , comparing previous SPECS ( blue ) to new hiSPECS method using DDA ( green ) or DIA ( green ) . were counted if quantified in at least 9 of 11 biological replicates of hiSPECS or 4 of 5 biological replicates category glycoprotein * includes UniProt annotations annotated as glycoproteins can have multiple UniProt annotations , e . g . for APP , TM , cytoplasm nucleus , because distinct proteolytic fragments are found in different organelles , so that some in categories cytoplasm nucleus may overlap with categories secreted TM + GPI . Data information : TM : single - pass transmembrane ; GPI : Glycosylphosphatidylinositol - anchored .

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

B) Bar chart indicating protein quantification, protein localization (according to UniProt) in the secretome of primary neurons, comparing the previous SPECS (blue) to the new hiSPECS method using DDA (light green) or DIA (dark green). Proteins were counted if quantified in at least 9 of the 11 biological replicates of hiSPECS or 4 of 5 biological replicates The category glycoprotein* includes UniProt annotations and proteins annotated as glycoproteins Proteins can have multiple UniProt annotations, e.g. for APP membrane, TM, cytoplasm and nucleus, because distinct proteolytic fragments are found in different organelles, so that some proteins in categories cytoplasm and nucleus may overlap with the categories secreted and TM+GPI. Data information: TM: single-pass transmembrane protein; GPI: Glycosylphosphatidylinositol-anchored membrane protein.


B ) Number of peptides ( left ) groups ( right ) quantified in secretome of investigated brain types with hiSPECS DIA method . quantified in at least 5 out of 6 biological replicates in at least one type are considered . In total 995 groups were detected .

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B) Number of peptides (left) and protein groups (right) quantified in the secretome of the investigated brain cell types with the hiSPECS DIA method. Proteins quantified in at least 5 out of 6 biological replicates in at least one cell type are considered. In total 995 protein groups were detected.


B , C ) Comparison of hiSPECS secretome resource lysate % enrichment is indicated normalized to average of most abundant type . B ) TREM2 CD200 are jointly enriched in both secretome lysate in microglia or neurons , respectively . In C ) two examples , of specifically enriched in secretome , but not in lysate are shown . ADIPOQ is specifically secreted from oligodendrocytes , but reveals highest expression in astrocytes . CACHD1 is specifically secreted from neurons , but high can be found in astrocytes , neurons oligodendrocytes . black indicates mean .

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B,C) Comparison of the hiSPECS secretome resource and lysate The % enrichment is indicated normalized to the average of the most abundant cell type. B) TREM2 and CD200 are jointly enriched in both secretome and lysate in microglia or neurons, respectively. In C) two examples, of proteins specifically enriched in the secretome, but not in the lysate are shown. ADIPOQ is specifically secreted from oligodendrocytes, but reveals highest expression in astrocytes. CACHD1 is specifically secreted from neurons, but high protein levels can be found in astrocytes, neurons and oligodendrocytes. The black line indicates the mean.


B ) Volcano plot showing changes in in secretome of neurons upon BACE1 inhibitor C3 treatment using hiSPECS DIA method . negative log10 p - values ( two sample t - ) of are plotted against their log2 fold changes ( C3 vs control ) ( N = 11 ) . grey hyperbolic curves depict a permutation false discovery rate estimation ( p = 0 . 05 ; s0 = 0 . 1 ) . Significantly regulated ( p < 0 . 05 ) are indicated with a blue dot known BACE1 substrates are indicated with blue letters . two newly validated BACE1 substrates CD200 ADAM22 are indicated in red .

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B) Volcano plot showing changes in protein levels in the secretome of primary neurons upon BACE1 inhibitor C3 treatment using the hiSPECS DIA method. The negative log10 p-values (two sample t-test) of all proteins are plotted against their log2 fold changes (C3 vs control) (N=11). The grey hyperbolic curves depict a permutation based false discovery rate estimation (p = 0.05; s0 = 0.1). Significantly regulated proteins (p<0.05) are indicated with a dark blue dot and known BACE1 substrates are indicated with blue letters. The two newly validated BACE1 substrates CD200 and ADAM22 are indicated in red.


C ) Independent validation of novel BACE1 substrate candidates CD200 ADAM22 by Western blotting in supernatants lysates of neurons incubated with or without BACE1 ( B1 ) inhibitor C3 for 48h . Full - ( fl ) ADAM22 ( mainly mature ADAM22 , lower band ) CD200 in neuronal lysate were mildly increased upon BACE1 inhibition , as expected due to reduced cleavage by BACE1 . Calnexin served as a loading control . soluble ectodomain of ADAM22 ( sADAM22 ) was strongly reduced in conditioned medium upon BACE1 inhibition . Ectodomain of known BACE1 substrate SEZ6 ( sSEZ6 ) were strongly reduced upon BACE1 inhibition served as positive control . Arrows indicate mature ( m ) immature ( im ) ( before prodomain cleavage ) form of ADAM22 .

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C) Independent validation of the novel BACE1 substrate candidates CD200 and ADAM22 by Western blotting in supernatants and lysates of primary neurons incubated with or without the BACE1 (B1) inhibitor C3 for 48h. Full-length (fl) ADAM22 (mainly mature ADAM22, lower band) and CD200 levels in the neuronal lysate and were mildly increased upon BACE1 inhibition, as expected due to reduced cleavage by BACE1. Calnexin served as a loading control. The soluble ectodomain of ADAM22 (sADAM22) was strongly reduced in the conditioned medium upon BACE1 inhibition. Ectodomain levels of the known BACE1 substrate SEZ6 (sSEZ6) were strongly reduced upon BACE1 inhibition and served as positive control. Arrows indicate the mature (m) and immature (im) (before prodomain cleavage) form of ADAM22.


D ) of Western blots in ( C ) ( N = 6 ) . Signals were normalized to calnexin quantified relative to control ( ctr ) condition . Statistical testing was performed with N = 6 biological replicates , using one sample t - with significance criteria of p < 0 . 05 . According to this criterion , ADAM22 CD200 were significantly increased in total lysates upon C3 treatment ( flADAM22 : * p - value 0 . 0251 , flCD200 : * * p - value 0 . 011 ) . Soluble ADAM22 was significantly reduced in supernatant upon C3 treatment ( * * * * p - value < 0 . 0001 ) . black central horizontal indicates mean error bars , mean ± SD .

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D) Quantification of the Western blots in (C) (N=6). Signals were normalized to calnexin levels and quantified relative to the control (ctr) condition. Statistical testing was performed with N=6 biological replicates, using the one sample t-test with the significance criteria of p < 0.05. According to this criterion, ADAM22 and CD200 were significantly increased in total lysates upon C3 treatment (flADAM22: *p-value 0.0251, flCD200: **p-value 0.011). Soluble ADAM22 was significantly reduced in the supernatant upon C3 treatment (****p-value <0.0001). The black central horizontal line indicates the mean and error bars, mean ± SD.


E ) reduction of soluble ectodomain of CD200 ( sCD200 ) was detected by ELISA ( one sample t - , * p - value 0 . 0116 ) , because available antibodies were not sensitive enough for Western blots of conditioned medium ( N = 4 ) . black central horizontal indicates mean error bars , mean ± SD .

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E) The reduction of the soluble ectodomain of CD200 (sCD200) was detected by ELISA (one sample t-test, *p-value 0.0116), because the available antibodies were not sensitive enough for Western blots of the conditioned medium (N=4). The black central horizontal line indicates the mean and error bars, mean ± SD.


A ) dynamic range plot of log10 transformed LFQ intensities of murine CSF quantified in at least 3 of 4 biological replicates measured with DIA . are split into quartiles according to their intensities , with 1st quartile representing 25 % most abundant . of annotated in UniProt with following subcellular locations / keywords are visualized for : , secreted , cytoplasm , glycoprotein . Examples of type specifically secreted are indicated with circles colored according to UniProt annotation ( purple : secreted , blue : ) . B ) dynamic range plot of log10 transformed LFQ intensities specifically of glycoproteins in murine CSF quantified in at least 3 of 4 biological replicates measured with DIA ( N = 4 ) . are split into quartiles according to their intensities . of identified in secretome of astrocytes , microglia , neurons or oligodendrocytes in at least 5 of 6 biological replicates is illustrated below . Selected specifically secreted from one type are indicated with color code of corresponding type .

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

A) Protein dynamic range plot of the log10 transformed LFQ intensities of the murine CSF proteins quantified in at least 3 of 4 biological replicates measured with DIA. The proteins are split into quartiles according to their intensities, with the 1st quartile representing the 25% most abundant proteins. The percentage of proteins annotated in UniProt with the following subcellular locations/keywords are visualized for: membrane, secreted, cytoplasm, and glycoprotein. Examples of cell type specifically secreted proteins are indicated with circles colored according to the UniProt annotation (purple: secreted, blue: membrane). B) Protein dynamic range plot of the log10 transformed LFQ intensities specifically of glycoproteins in the murine CSF quantified in at least 3 of 4 biological replicates measured with DIA (N=4). The proteins are split into quartiles according to their intensities. The percentage of proteins identified in the secretome of astrocytes, microglia, neurons or oligodendrocytes in at least 5 of 6 biological replicates is illustrated below. Selected proteins specifically secreted from one cell type are indicated with the color code of the corresponding cell type.


A ) hiSPECS DDA DIA of brain slice cultures in presence of 25 % serum . bar chart comparing hiSPECS DDA DIA method indicates number their localization according to UniProt identified in secretome of brain slices . quantified in at least 5 of 6 biological replicates are considered ( N = 6 ) .

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A) hiSPECS DDA and DIA analysis of brain slice cultures in the presence of 25% serum. The bar chart comparing the hiSPECS DDA and DIA method indicates the protein number and their localization according to UniProt identified in the secretome of brain slices. Proteins quantified in at least 5 of 6 biological replicates are considered (N=6).


B ) dynamic range plot of log10 LFQ intensities of secretome of brain slices in descending order . According to their , are grouped into quartiles of with following UniProt keywords is visualized : , secreted , cytoplasm . of identified in secretome of astrocytes , microglia , neurons or oligodendrocytes in at least 5 of 6 biological replicates is illustrated . Selected specifically secreted from one type are indicated with color code of corresponding type . circle of APOE indicates it as a soluble secreted .

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B) Protein dynamic range plot of the log10 LFQ intensities of secretome proteins of brain slices in descending order. According to their intensity, proteins are grouped into quartiles and the percentage of proteins with the following UniProt keywords is visualized: membrane, secreted, cytoplasm. The percentage of proteins identified in the secretome of astrocytes, microglia, neurons or oligodendrocytes in at least 5 of 6 biological replicates is illustrated. Selected proteins specifically secreted from one cell type are indicated with the color code of the corresponding cell type. The circle of APOE indicates it as a soluble secreted protein.


D ) Volcano plot showing changes in in secretome of cultured brain slices upon 6 h LPS treatment in a 24 h collection window using hiSPECS DIA method . negative log10 p - values ( two sample t - ) of are plotted against their log2 fold changes ( LPS vs control ) ( N = 6 ) . grey hyperbolic curves depict a permutation false discovery rate estimation ( p = 0 . 05 ; s0 = 0 . 1 ) . Significantly regulated ( p < 0 . 05 ) are indicated with a blue dot . highlighted in red indicate known to increase upon LPS treatment , whereas labeled in blue are not upregulated in this

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D) Volcano plot showing changes in protein levels in the secretome of primary cultured brain slices upon 6 h LPS treatment in a 24 h collection window using the hiSPECS DIA method. The negative log10 p-values (two sample t-test) of all proteins are plotted against their log2 fold changes (LPS vs control) (N=6). The grey hyperbolic curves depict a permutation based false discovery rate estimation (p = 0.05; s0 = 0.1). Significantly regulated proteins (p<0.05) are indicated with a dark blue dot. Proteins highlighted in red indicate proteins known to increase upon LPS treatment, whereas proteins labeled in blue are not upregulated in this study


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . Representative confocal microscopy of CMAC - stained conjugates formed between B lymphocytes ( isolated from DICER - KO mouse splenocytes pre - activated with a mixture of LPS plus IL - 4 ) CD4 + T ( from OVA - specific OT - II transgenic mice ) ; conjugates were formed either in presence or in absence of OVA . A representative experiment of at least 3 independent experiments is shown .

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Representative confocal microscopy images of CMAC-stained conjugates formed between B lymphocytes (isolated from DICER-KO mouse splenocytes and pre-activated with a mixture of LPS plus IL-4) and CD4+ T cells (from OVA-specific OT-II transgenic mice); conjugates were formed either in the presence or in the absence of OVA. A representative experiment of at least 3 independent experiments is shown.


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . showing of B forming

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. showing the percentage of B cells forming conjugates


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . accumulation at IS of TCR Conjugates were assessed using Image J plug - in for IS

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. the accumulation at the IS of the TCR Conjugates were assessed using the Image J plug-in for IS analysis


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . accumulation of actin at IS Conjugates were assessed using Image J plug - in for IS

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. the accumulation of the actin at the IS Conjugates were assessed using the Image J plug-in for IS analysis


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . from CD4 + T MTOC to contact site with B MTOC - B - was measured with Imaris software .

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. the distance from the CD4+ T cell MTOC to the contact site with the B cell MTOC-B-cell distance was measured with Imaris analysis software.


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . Flow cytometry of B lymphocytes co - cultured with OT - II isolated T in presence or absence of OVA , showing IgG1 expression after 24h of co - culture ,

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA, showing IgG1 expression after 24h of co-culture,


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . Flow cytometry of B lymphocytes co - cultured with OT - II isolated T in presence or absence of OVA B proliferation after 24h , 48h 72h co - culture ( Ki67 expression in live B violet tracer dilution , respectively )

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA B cell proliferation after 24h, 48h and 72h co-culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively)


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . B proliferation after 24h , 48h 72h co - culture ( Ki67 expression in live B violet tracer dilution , respectively ) ,

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. B cell proliferation after 24h, 48h and 72h co-culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively),


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . Flow cytometry of B lymphocytes co - cultured with OT - II isolated T in presence or absence of OVA survival ( with live marker DAPI )

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA survival (staining with the live marker DAPI)


DICER - KO B were co - cultured for 24 hours with OTII - derived T in presence or absence of OVA . Flow cytometry of B lymphocytes co - cultured with OT - II isolated T in presence or absence of OVA apoptosis ( Caspase - 3 expression ) 24h , 48h 72h after IS formation .

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DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA apoptosis (cleaved Caspase-3 expression) 24h, 48h and 72h after IS formation.


CD4 + T DICER - KO B were co - cultured for 24 hours with or without OVA , flow - cytometry sorted B were analyzed by small next generation sequencing ( NGS ) . Heat map showing microRNAs significantly upregulated in B after co - culture with T in presence of OVA . Data are from 3 independent experiments ( samples 1 - 3 ) , only microRNAs with an adjusted P - value < 0 . 02 are shown . Fold increase in B content of upregulated microRNAs in presence of OVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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CD4+ T and DICER-KO B cells were co-cultured for 24 hours with or without OVA, and flow-cytometry sorted B cells were analyzed by small RNA next generation sequencing (NGS). Heat map showing microRNAs significantly upregulated in B cells after co-culture with T cells in the presence of OVA. Data are from 3 independent experiments (samples 1-3), and only microRNAs with an adjusted P-value < 0.02 are shown. Fold increase in the B cell content of upregulated microRNAs in the presence of OVA; *P<0.05, **P<0.01, ***P<0.001.


Heat map showing NGS of microRNAs differentially expressed in activated WT C57BL / 6 CD4 + T their secreted exosomes . Data are from 3 independent experiments ( samples a - c ) , only microRNAs with an adjusted P - value < 0 . 02 are shown .

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Heat map showing NGS analysis of microRNAs differentially expressed in activated WT C57BL/6 CD4+ T cells and their secreted exosomes. Data are from 3 independent experiments (samples a-c), and only microRNAs with an adjusted P-value < 0.02 are shown.


Mean tags per million ( TPMs ) for differentially expressed microRNAs under study in exosomes from activated CD4 + T . Bars represent mean ± .

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Mean tags per million (TPMs) for the differentially expressed microRNAs under study in exosomes from activated CD4+ T cells. Bars represent the mean ± SEM.


Quantitative real - time PCR of upregulated microRNAs after co - culture of OT - II derived CD4 + T with DICER - KO B activated with LPS + IL - 4 Bars represent mean ± of at least three independent experiments data were obtained by 2 - ∆ ∆ Ct method using Biogazelle software . Results are expressed as a proportion of microRNA expression in non - OVA containing co - cultures , with normalization to small nucleolar RNAs RNU1A1 RNU5G .

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Quantitative real-time PCR of upregulated microRNAs after co-culture of OT-II derived CD4+ T cells with DICER-KO B cells activated with LPS+IL-4 Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2-∆∆Ct method using Biogazelle software. Results are expressed as a proportion of the microRNA expression in non-OVA containing co-cultures, with normalization to the small nucleolar RNAs RNU1A1 and RNU5G.


Quantitative real - time PCR of upregulated microRNAs after co - culture of OT - II derived CD4 + T with DICER - KO B activated with CD40 + IgM Bars represent mean ± of at least three independent experiments data were obtained by 2 - ∆ ∆ Ct method using Biogazelle software . Results are expressed as a proportion of microRNA expression in non - OVA containing co - cultures , with normalization to small nucleolar RNAs RNU1A1 RNU5G .

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Quantitative real-time PCR of upregulated microRNAs after co-culture of OT-II derived CD4+ T cells with DICER-KO B cells activated with CD40+IgM Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2-∆∆Ct method using Biogazelle software. Results are expressed as a proportion of the microRNA expression in non-OVA containing co-cultures, with normalization to the small nucleolar RNAs RNU1A1 and RNU5G.


Quantitative real - time PCR showing expression of down - modulated mRNA targets upon IS formation between OT - II CD4 + T DICER - KO B pre - activated with LPS + IL - 4 Results are means ± of at least four independent experiments are expressed as a proportion of mRNA expression in non - OVA condition , with normalization to GAPDH .

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Quantitative real-time PCR showing the expression of down-modulated mRNA targets upon IS formation between OT-II CD4+ T cells and DICER-KO B cells pre-activated with LPS+IL-4 Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non-OVA condition, with normalization to GAPDH.


Quantitative real - time PCR showing expression of down - modulated mRNA targets upon IS formation between OT - II CD4 + T DICER - KO B pre - activated with CD40 + IgM Results are means ± of at least four independent experiments are expressed as a proportion of mRNA expression in non - OVA condition , with normalization to GAPDH .

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Quantitative real-time PCR showing the expression of down-modulated mRNA targets upon IS formation between OT-II CD4+ T cells and DICER-KO B cells pre-activated with CD40+IgM Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non-OVA condition, with normalization to GAPDH.


Fluorescently tagged mimics of microRNAs mmu - miR - 20a - 5p , mmu - miR - 25 - 3p mmu - miR - 155 - 3p were nucleofected into activated B lymphocytes from C57 / BL6 mice . Quantitative real - time PCR showing expression of nucleofected microRNA mimics in FACS - sorted FAM + B with normalization to RNU1A1

Fluorescently tagged mimics of the microRNAs mmu-miR-20a-5p, mmu-miR-25-3p and mmu-miR-155-3p were nucleofected into activated B lymphocytes from C57/BL6 mice. Quantitative real-time PCR showing expression of nucleofected microRNA mimics in FACS-sorted FAM+ B cells with normalization to RNU1A1 and RNU5G


Fluorescently tagged mimics of microRNAs mmu - miR - 20a - 5p , mmu - miR - 25 - 3p mmu - miR - 155 - 3p were nucleofected into activated B lymphocytes from C57 / BL6 mice . Quantitative real - time PCR showing expression of Bcl2l11 Pten mRNA targets in mimic - nucleofected B with normalization to

Fluorescently tagged mimics of the microRNAs mmu-miR-20a-5p, mmu-miR-25-3p and mmu-miR-155-3p were nucleofected into activated B lymphocytes from C57/BL6 mice. Quantitative real-time PCR showing expression of Bcl2l11 and Pten mRNA targets in mimic-nucleofected B cells with normalization to GAPDH


Flow cytometry of mimic - nucleofected B lymphocytes . Dot - plots show B220 + IgG1 + B 48h after mimic nucleofection from at least 4 independent experiments bar charts show mean values ± of 4 independent experiments .

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Flow cytometry analysis of mimic-nucleofected B lymphocytes. Dot-plots show B220+ IgG1+ B cells 48h after mimic nucleofection from at least 4 independent experiments and bar charts show mean values ± SEM of 4 independent experiments.


Quantitative real - time PCR ( RNU1A1 RNU5G - normalized ) showing blockade of microRNA transfer from CD4 + T pre - treated with siRNA targeting exosome secretion mediator Rab27 . Bar charts represent mean values ± of at least 4 independent experiments .

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Quantitative real-time PCR (RNU1A1 and RNU5G- normalized) showing blockade of microRNA transfer from CD4+ T cells pre-treated with siRNA targeting the exosome secretion mediator Rab27. Bar charts represent the mean values ± SEM of at least 4 independent experiments.


Quantitative real - time PCR showing mRNA target down - modulation after IS formation between DICER - KO B CD4 + T nucleofected with either siRab27 or scrambled control siRNA , with normalization to GAPDH . Bar charts represent mean values ± of at least 4 independent experiments .

Quantitative real-time PCR showing mRNA target down-modulation after IS formation between DICER-KO B cells and CD4+ T cells nucleofected with either siRab27 or scrambled control siRNA, with normalization to GAPDH. Bar charts represent the mean values ± SEM of at least 4 independent experiments.


Representative dot - plots , showing IgG1 expression of gated B220 + B after IS formation as in ( G ) ; bar chart shows mean ± of 3 independent experiments is shown .

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Representative dot-plots, showing IgG1 expression of gated B220+ B cells after IS formation as in (G); the bar chart shows the mean fluorescence Intensity ± SEM and quantification of 3 independent experiments is shown.


Representative plot ( left ) ( right ) of reconstitution of B T compartments after adoptive transfer . graph shows mean of CD19 CD4 positive from n ≥ 5 reconstituted mice ± .

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Representative plot (left) and quantification (right) of the reconstitution of the B cell and T cell compartments after adoptive transfer. The graph shows the mean percentage of CD19 and CD4 positive cells from n ≥ 5 reconstituted mice ± SEM.


Representative flow cytometry plot ( left ) ( right ) of GL7 CD95 / Fas expression in B . graph shows mean of GL7 + Fas + from n ≥ 5 reconstituted mice ± .

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Representative flow cytometry plot (left) and quantification (right) of GL7 and CD95/Fas expression in B cells. The graph shows the mean percentage of GL7+Fas+ cells from n ≥ 5 reconstituted mice ± SEM.


Representative dot - plot showing CXCR4 CD86 expression on B for zone ( DZ ) zone ( LZ ) ( gated on GC GL7 + Fas + B ) . graph shows mean of DZ ( CXCR4 + CD86 - ) LZ ( CXCR4 - CD86 + ) populations from n ≥ 5 reconstituted mice ± .

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Representative dot-plot analysis showing CXCR4 and CD86 expression on B cells for Dark zone (DZ) and Light zone (LZ) analysis (gated on GC GL7+ Fas+ B cells). The graph shows the mean percentage of DZ (CXCR4+CD86-) and LZ (CXCR4-CD86+) populations from n ≥ 5 reconstituted mice ± SEM.


ELISA of IgM ( H ) IgG ( I ) in serum from immunized adoptively transferred mice . graph shows mean serum of IgM IgG from n ≥ 5 reconstituted mice , respectively ± .

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ELISA quantification of IgM (H) and IgG (I) in serum from immunized adoptively transferred mice. The graph shows the mean serum concentration of IgM and IgG from n ≥ 5 reconstituted mice, respectively ± SEM.


72h after stimulation with anti - CD3 anti - CD28 , were re - stimulated with P + I for 2h , mRNA was subjected to qRT - PCR using indicated primers for lineage specifying transcription factors ( B ) or cytokines ( C ) . graphs represent an average of at least 4 independent experiments . In each graph , expression level in control was set as 100 % . P - value was calculated by t - . * p < 0 . 05 , * * * p < 0 .

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72h after stimulation with anti-CD3 and anti-CD28, the cells were re-stimulated with P+I for 2h, and the mRNA was subjected to qRT-PCR using the indicated primers for the lineage specifying transcription factors (B) or cytokines (C). The graphs represent an average of at least 4 independent experiments. In each graph, the expression level in the control was set as 100%. P-value was calculated by t-test. * p<0.05, ***p<0.005


EVs were extracted from schistosomal - growing medium were subjected to cryo - : were taken as described previously [ 90 ] . black arrows indicate EVs at of ~ 100nm . EVs were extracted from schistosomal - growing medium were subjected AFM : were taken from schistosomal - growing medium ( I ) or from 1 / 10 dilution of this medium ( II ) . small magnify arrow - indicated EVs . ( III ) Image of extract deriving from unused schistosomal - medium . EVs were extracted from schistosomal - growing medium were subjected NTA fractionation of particles ; graph represents average of 5 different measurements .

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EVs were extracted from the schistosomal-growing medium and were subjected to cryo-TEM analysis: images were taken as described previously [90]. The black arrows indicate EVs at the size of ~100nm. EVs were extracted from the schistosomal-growing medium and were subjected AFM analysis: images were taken from the schistosomal-growing medium (I) or from 1/10 dilution of this medium (II). The small images magnify the arrow-indicated EVs. (III) Image of the extract deriving from the unused schistosomal-medium. EVs were extracted from the schistosomal-growing medium and were subjected NTA size fractionation of particles analysis; the graph represents average of 5 different measurements.


EVs were purified from either schistosomal - growing medium ( supernatant ) or unused schistosomal - medium as control . Both pellets were stained using Thiazole Orange . Purified labeled ~ 5 × 106 EVs were incubated with 5 × 105 freshly purified Th that were stimulated with anti - CD3 anti - CD28 antibodies for 10 minutes . from same slide were taken by inverted confocal microscopy at indicated time points . arrows in enlarged point toward labeled with EVs . scale bars in represent 20μm . of labeled was calculated by dividing number of labeled by total numbers of in same image . mean + / - was calculated from 3 independent

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EVs were purified from either schistosomal-growing medium (supernatant) or unused schistosomal-medium as control. Both pellets were stained using Thiazole Orange. Purified labeled ~5 × 106 EVs were incubated with 5 × 105 freshly purified Th cells that were stimulated with anti-CD3 and anti-CD28 antibodies for 10 minutes. Images from the same slide were taken by inverted confocal microscopy at the indicated time points. The arrows in the enlarged images point toward cells labeled with EVs. The scale bars in all images represent 20μm. The percentage of labeled cells was calculated by dividing the number of labeled cells by the total numbers of cells in the same image. The mean +/- SEM was calculated from 3 independent images


Schistosomal - miRNA was extracted from either EVs that were isolated from schistosomal - growing medium or control pellets that were isolated from unused schistosomal - medium as described in Materials Methods ( Schistosomal - EVs purification section ) . Equal aliquots were subjected to qRT - PCR with specific primers to schistosomal - miR - 10 , Bantam miR - 125 ; expression is presented as ∆ Ct from background Ct that was set as 40 cycles .

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Schistosomal- miRNA was extracted from either EVs that were isolated from schistosomal-growing medium or control pellets that were isolated from unused schistosomal-medium as described in the Materials and Methods (Schistosomal-EVs purification section). Equal aliquots were subjected to qRT-PCR with specific primers to the schistosomal-miR-10, Bantam and miR-125; the expression is presented as ∆Ct from background Ct that was set as 40 cycles.


trans - well system was performed as in Fig . 1 . Schistosome - exposed Th or control Th were subjected to qRT - PCR with specific schistosomal - Taqman primers to ( A ) miR - 10 - 5p , ( B ) Bantam , ( C ) miR - 125b .

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The trans-well system was performed as in Fig. 1. Schistosome-exposed Th cells or control Th cells were subjected to qRT-PCR with specific schistosomal-Taqman primers to (A) miR-10-5p, (B) Bantam, and (C) miR-125b.


trans - well system was performed as in Fig . 1 . Control wells contained only unused schistosomal - medium ; Supernatant wells contained 0 . 1 μm of filtered schistosomal - growing medium ; Th wells contained sorted CD4 + CD11c - from spleens ; Th + APCs contained sorted CD4 + CD11c - CD4 - CD11c + in 5 : 1 , respectively . In Data information : In ( A - E ) expression is presented as arbitrary units calculated as 2 ∆ ∆ CT of miRNA relative to snRNA U6 in ( F ) mRNA relative to β2 microglobulin . mean + / - was calculated from at least 3 independent experiments ( except in F of 2 independent experiments ) .

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The trans-well system was performed as in Fig. 1. The Control wells contained only unused schistosomal-medium; Supernatant wells contained 0.1 μm of the filtered schistosomal-growing medium; Th wells contained sorted CD4+CD11c- from spleens; Th+APCs contained sorted CD4+CD11c- and CD4-CD11c+ in 5:1 ratio, respectively. In Data information: In (A-E) the expression is presented as arbitrary units calculated as 2∆∆CT of miRNA relative to snRNA U6 and in (F) mRNA relative to β2 microglobulin. The mean +/- SEM was calculated from at least 3 independent experiments (except in F of 2 independent experiments).


Image of a schistosome - infected mouse . lymph nodes , from which Th were isolated , are indicated .

Image of a schistosome-infected mouse. The lymph nodes, from which the Th cells were isolated, are indicated.


Th were isolated from indicated lymph nodes spleen . In case of uninfected mice , Th from different lymph nodes spleen were mixed . qRT - PCR was performed with specific Taqman primers to either schistosomal - miR - 10 or - Bantam . presence of miRNAs is presented as arbitrary units calculated as 2 ∆ ∆ CT of miRNA divided by snRNA U6 . mean + / - was calculated from 4 independent experiments .

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Th cells were isolated from the indicated lymph nodes and spleen. In the case of uninfected mice, the Th cells from different lymph nodes and the spleen were mixed. qRT-PCR was performed with specific Taqman primers to either schistosomal-miR-10 or -Bantam. The presence of the miRNAs is presented as arbitrary units calculated as 2∆∆CT of the miRNA divided by snRNA U6. The mean +/- SEM was calculated from 4 independent experiments.


HEK - 293 were transfected with a plasmid expressing schistosomal - miR - 10 at indicated together with either psiCHECK - II vector , psiCHECK - WT - MAP3K7 - 3 ' UTR or psiCHECK - mut - MAP3K7 . After 24h , lysates were subjected to luciferase . results are presented as of renilla / luciferase expression that was normalized relative to transfected only with psiCHECK - II . Values are expressed as mean + / - SD of at least 3 independent experiments .

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HEK-293 cells were transfected with a plasmid expressing schistosomal-miR-10 at the indicated concentration together with either psiCHECK-II vector, psiCHECK-WT-MAP3K7-3'UTR or psiCHECK-mut-MAP3K7. After 24h, the cells lysates were subjected to luciferase assay. The results are presented as the ratio of renilla/luciferase expression that was normalized relative to cells transfected only with psiCHECK-II. Values are expressed as the mean+/-SD of at least 3 independent experiments.


A representative experiment in which extracted from Jurkat - T stably over expressing stably control vector or miR - 10 expressing vector were subjected to WB with anti - MAP3K7 or anti - GAPDH antibodies . graph presents densitometry of 3 WBs analyses performed as in ( C ) . mean + / - SD was calculated from 4 independent experiments . Statistics were performed using t - ( * p < 0 . 05 ) .

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A representative experiment in which protein extracted from Jurkat-T cells stably over expressing stably control vector or miR-10 expressing vector were subjected to WB analysis with anti-MAP3K7 or anti-GAPDH antibodies. The graph presents densitometry analysis of 3 WBs analyses performed as in (C). The mean +/- SD was calculated from 4 independent experiments. Statistics were performed using t-test (*p<0.05).


Jurkat - T stably expressing either control vector or a vector containing schistosomal - miR - 10 were co - transfected with a control Luciferase plasmid or NF - κB luciferase reporter vector . In transfections , a plasmid expressing Renilla vector as an internal control was added . 48h post - transfection , were harvested were analyzed by luciferase reporter . Values were normalized to Renilla activity . Data are represented as mean + / - from 5 independent experiments . Statistics were performed using t - , * * p < 0 . 01 .

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Jurkat-T cells stably expressing either control vector or a vector containing schistosomal-miR-10 were co-transfected with a control Luciferase plasmid or NF-κB luciferase reporter vector. In all transfections, a plasmid expressing Renilla vector as an internal control was added. 48h post-transfection, cells were harvested and were analyzed by luciferase reporter assay. Values were normalized to the Renilla activity. Data are represented as mean +/-SEM from 5 independent experiments. Statistics were performed using t-test, **p<0.01.


Representative WB experiment demonstrating that MAP3K7 is downregulated in schistosomal - exposed Th F ) or to Jurkat ( H ) in comparison to control unexposed . ( G , I ) graphs present densitometry of 3 WB analyses performed as in ( F H ) , respectively . mean + / - SD was calculated from 3 independent experiments . Statistics were performed using paired one - t - ( * p < 0 . 05 ) .

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Representative WB experiment demonstrating that MAP3K7 is downregulated in schistosomal-exposed Th cells F) or to Jurkat (H) in comparison to the control unexposed cells. (G, I) The graphs present densitometry analysis of 3 WB analyses performed as in (F and H), respectively. The mean +/- SD was calculated from 3 independent experiments. Statistics were performed using paired one-tailed t-test (*p<0.05).


A . qRT - PCR of samples from normal human skin ( NS ) AK for activin βA ( INHBA ) , normalized to hypoxanthine phosphoribosyltransferase ( HPRT ) . Expression in one of normal skin samples were set to 1 . Scatter plots are shown . NS : N = 4 ; AK : N = 21 . ns = non - significant ( Mann - Whitney ) .

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A. qRT-PCR analysis of RNA samples from normal human skin (NS) and AK for activin βA (INHBA), normalized to hypoxanthine phosphoribosyltransferase (HPRT). Expression levels in one of the normal skin samples were set to 1. Scatter plots are shown. NS: N=4; AK: N=21. ns = non-significant (Mann-Whitney test).


B . qRT - PCR of samples from normal back skin of wild - type mice ( N = 4 , n = 4 ) , non - tumorigenic back skin from HPV8 / wt mice ( N = 7 , n = 9 ) papillomas from HPV8 / wt transgenic mice ( N = 8 , n = 11 ) for Inhba , normalized to glyceraldehyde 3 - phosphate dehydrogenase ( Gapdh ) . Expression in one of wild - type back skin samples were set to 1 . Statistical significance was determined using 1 - way ANOVA Bonferroni ` s Multiple Comparisons of log - transformed data . N , number of mice ; n , number of biopsies .

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B. qRT-PCR analysis of RNA samples from normal back skin of wild-type mice (N=4, n=4), non-tumorigenic back skin from HPV8/wt mice (N=7, n=9) and papillomas from HPV8/wt transgenic mice (N=8, n=11) for Inhba, normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Expression levels in one of the wild-type back skin samples were set to 1. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test of log-transformed data. N, number of mice; n, number of biopsies.


C - E . Sections from a collected from a 64 week - old HPV8 / wt mouse ( C , D ) or from non - tumorigenic skin of 5 week - old HPV8 / wt mice ( E ) stained with an anti - activin A antibody counterstained with hematoxylin . E : Epidermis , D : Dermis , HF : Hair follicle . ( D ) Shows a higher magnification of boxed in ( C ) . Scale bars : 200μm ( C ) 100μm ( D , E ) .

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C-E. Sections from a tumor collected from a 64 week-old HPV8/wt mouse (C,D) or from non-tumorigenic skin of 5 week-old HPV8/wt mice (E) stained with an anti-activin A antibody and counterstained with hematoxylin. E: Epidermis, D: Dermis, HF: Hair follicle. (D) Shows a higher magnification of the boxed area in (C). Scale bars: 200μm (C) and 100μm (D,E).


F . Kinetics of incidence in HPV8 / wt HPV8 / Act mice ; N = 20 mice per genotype . Statistical significance was determined using Log - rank ( Mantel - Cox ) .

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F. Kinetics of tumor incidence in HPV8/wt and HPV8/Act mice; N=20 mice per genotype. Statistical significance was determined using Log-rank (Mantel-Cox) test.


G . of mice when first tumors appeared on certain sites . Each dot represents one .

G. Age of the mice when the first tumors appeared on certain body sites. Each dot represents one tumor.


H . Representative macroscopic pictures of ear tumors ( left panel ) H & amp ; amp ; E - stained sections of ear acanthopapillomas ( upper middle panel ) or an ear acanthopapilloma with trichoepitheliomatous differentiation ( lower panel ) . Boxed areas are shown at higher magnification in subsequent pictures to right in each horizontal picture panel . E : epidermis ; D : dermis ; C : cartilage . Scale bars : 200μm ( left panel ) , 100μm ( middle panel ) or 50μm ( right panel ) .

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H. Representative macroscopic pictures of ear tumors (left panel) and H&amp;E-stained sections of ear acanthopapillomas (upper and middle panel) or an ear acanthopapilloma with trichoepitheliomatous differentiation (lower panel). Boxed areas are shown at higher magnification in subsequent pictures to the right in each horizontal picture panel. E: epidermis; D: dermis; C: cartilage. Scale bars: 200μm (left panel), 100μm (middle panel) or 50μm (right panel).


A , B . Representative pictures of ear sections from 10 week - old mice stained for δTcR ( green ) ; counterstained with Hoechst ( blue ) ( A ) of δTcR + in ear epidermis ( B ) . Dotted lines indicate epidermal - dermal border . Scale bar : 20μm . N = 6 wt / wt mice , N = 7 HPV8 / wt mice , N = 6 wt / Act mice ( N = 8 ) HPV8 / Act mice .

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A,B. Representative pictures of ear sections from 10 week-old mice stained for δTcR (green); counterstained with Hoechst (blue) (A) and quantification of δTcR+ cells in the ear epidermis (B). Dotted lines indicate the epidermal-dermal border. Scale bar: 20μm. N=6 wt/wt mice, N=7 HPV8/wt mice, N=6 wt/Act mice and (N=8) HPV8/Act mice.


C . of βTcR + in ear skin cryosections from 10 week - old wt / wt ( N = 5 ) ; HPV8 / wt ( N = 5 ) ; wt / Act ( N = 8 ) or HPV8 / Act ( N = 8 ) mice .

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C. Quantification of βTcR+ cells in ear skin cryosections from 10 week-old wt/wt (N=5); HPV8/wt (N=5); wt/Act (N=8) or HPV8/Act (N=8) mice.


D - F . of viable CD4 + βTcR + ( D ) , CD8 + βTcR + ( E ) or CD4 + CD25 + ( F ) among epidermal analyzed by flow cytometry . Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown . Representative flow cytometry scatter plots are shown in Appendix Fig S1A - C .

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D-F. Percentage of viable CD4+ βTcR+ (D), CD8+ βTcR+ (E) or CD4+CD25+ (F) cells among all epidermal cells analyzed by flow cytometry. Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown. Representative flow cytometry scatter plots are shown in Appendix Fig S1A-C.


G . Representative pictures of ear cryosections from 10 week - old mice stained with antibodies against Foxp3 ( red ) counterstained with Hoechst ( blue ) . Insert shows higher magnification of Foxp3 - positive in wt / Act skin . Dotted lines indicate epidermal - dermal border . Scale bar : 50μm .

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G. Representative pictures of ear cryosections from 10 week-old mice stained with antibodies against Foxp3 (red) and counterstained with Hoechst (blue). Insert shows higher magnification of the Foxp3-positive cells in wt/Act skin. Dotted lines indicate the epidermal-dermal border. Scale bar: 50μm.


H . Kinetics of incidence in HPV8 / Act / wt ( N = 29 ) , HPV8 / Act / CD4KO ( N = 10 ) , HPV8 / wt / wt ( N = 23 ) HPV8 / wt / CD4KO ( N = 22 ) mice per genotype . Statistical significance was determined using Log - rank ( Mantel - Cox ) ; ns = non - significant .

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H. Kinetics of tumor incidence in HPV8/Act/wt (N=29), HPV8/Act/CD4KO (N=10), HPV8/wt/wt (N=23) and HPV8/wt/CD4KO (N=22) mice per genotype. Statistical significance was determined using the Log-rank (Mantel-Cox) test; ns = non-significant.


A , B . Representative pictures of ear skin cryosections from 10 - 12 week - old mice stained with anti - CD206 antibodies ( green ) ; counterstained with Hoechst ( blue ) ( A ) , of CD206 + as a of total dermal ( B ) in wt / wt ( N = 3 ) , HPV8 / wt ( N = 7 ) , wt / Act ( N = 2 ) or HPV8 / Act ( N = 8 ) mice . basement is indicated with a dotted .

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A,B. Representative pictures of ear skin cryosections from 10-12 week-old mice stained with anti-CD206 antibodies (green); counterstained with Hoechst (blue) (A), and quantification of CD206+ area as a percentage of total dermal area (B) in wt/wt (N=3), HPV8/wt (N=7), wt/Act (N=2) or HPV8/Act (N=8) mice. The basement membrane is indicated with a white dotted line.


C . Abundance of MerTK + CD64 + macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12 - 16 week - old mice . N = 5 - 6 . Representative flow cytometry scatter plots are shown in Appendix Fig S1D .

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C. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12-16 week-old mice. N = 5-6. Representative flow cytometry scatter plots are shown in Appendix Fig S1D.


D . of CD206 + as a of total dermal in wt ( N = 7 ) Act ( N = 6 ) mice in back skin treated 8x with TPA ( Antsiferova et al , 2011 ) .

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D. Quantification of CD206+ area as a percentage of total dermal area in wt (N=7) and Act (N=6) mice in back skin treated 8x with TPA (Antsiferova et al, 2011).


E . Abundance of MerTK + CD64 + macrophages analyzed by flow cytometry of pre - tumorigenic ear skin from 12 week - old mice expressing CCR2 - CFP - DTR transgene ( wt / CCR2 ) or double - transgenic ( Act / CCR2 ) , untreated or treated with diphtheria toxin ( + DT ) 3 times every 48h analyzed 24h after last injection . of CD45 + CD11b + MerTK + CD64 + macrophages among live is shown . N = 3 control wt / CCR2 Act / CCR2 mice ; N = 4 DT - injected wt / CCR2 Act / CCR2 mice . Representative flow cytometry scatter plots are shown in Appendix Fig S1E .

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E. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of pre-tumorigenic ear skin from 12 week-old mice expressing the CCR2-CFP-DTR transgene (wt/CCR2) or double-transgenic animals (Act/CCR2), untreated or treated with diphtheria toxin (+DT) 3 times every 48h and analyzed 24h after the last injection. Percentage of CD45+CD11b+MerTK+CD64+ macrophages among live cells is shown. N=3 control wt/CCR2 and Act/CCR2 mice; N=4 DT-injected wt/CCR2 and Act/CCR2 mice. Representative flow cytometry scatter plots are shown in Appendix Fig S1E.


F , G . Inflammatory thioglycollate - elicited peritoneal macrophages ( F ) or mouse peripheral blood monocytes ( G ) were isolated from wt mice allowed to migrate for 24h in a Transwell . Activin A 0 . 1ng / ml ( A0 . 1 ) , 1ng / ml ( A1 ) , or 10ng / ml ( A10 ) was added to lower well . Alternatively , 10ng / ml was added to lower well insert ( A10 / A10 ) . Random migration to medium ( ctrl ) was set to 1 for each mouse ; fold over control is shown . Log - transformed data were used for statistical . Results in ( F ) are representative of 4 independent experiments performed with 8 mice in total ( results obtained in one experiment performed with 3 mice are shown ) . Results in ( G ) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice .

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F, G. Inflammatory thioglycollate-elicited peritoneal macrophages (F) or mouse peripheral blood monocytes (G) were isolated from wt mice and allowed to migrate for 24h in a Transwell assay. Activin A 0.1ng/ml (A0.1), 1ng/ml (A1), or 10ng/ml (A10) was added to the lower well. Alternatively, 10ng/ml was added to the lower well and the insert (A10/A10). Random migration to medium (ctrl) was set to 1 for each mouse; fold over control is shown. Log-transformed data were used for statistical analysis. Results in (F) are representative of 4 independent experiments performed with 8 mice in total (results obtained in one experiment performed with 3 mice are shown). Results in (G) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice.


B . of FACS data of sorted macrophages shows relative of CD45 + CD11b + F4 / 80 + in each genotype absolute number of sorted used for sequencing . Representative flow cytometry gating strategy for sorting is shown in Fig EV3B . N = 5 - 9 . Statistical significance was determined using 1 - way ANOVA Bonferroni ` s Multiple Comparisons .

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B. Analysis of FACS data of sorted macrophages shows the relative percentage of CD45+CD11b+F4/80+ cells in each genotype and the absolute number of sorted cells used for RNA sequencing. Representative flow cytometry gating strategy for sorting is shown in Fig EV3B. N=5-9. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test.


C . Differential expression of genes was analyzed using edgeR software package . IPA was performed with filtered lists of up - downregulated genes that exhibited an FDR - value of < 0 . 1 in different genotype comparisons ( wt / Act vs wt / wt ; HPV8 / Act vs HPV8 / wt ; HPV8 / Act vs wt / wt ) . number of differentially expressed genes used for is indicated . Data are presented as pseudo - with magnitudes of Activation Z - scores color - coded as indicated in legend ; non - grey comparisons are - log10 ( p - value ) > 2 . 5 unless otherwise indicated .

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C. Differential expression of genes was analyzed using the edgeR software package. IPA was performed with filtered lists of up- and downregulated genes that exhibited an FDR-value of <0.1 in different genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt). The number of differentially expressed genes used for analysis is indicated. Data are presented as pseudo-heatmap with magnitudes of Activation Z-scores color-coded as indicated in the legend; all non-grey comparisons are -log10(p-value)>2.5 unless otherwise indicated.


D . Gene Set Enrichment ( GSEA ) was performed to compare transcriptomes of genotype comparisons ( wt / Act vs wt / wt ; HPV8 / Act vs HPV8 / wt ; HPV8 / Act vs wt / wt , [ ] / Act vs [ ] / wt ) to 3 published datasets of - Associated Macrophages ( TAM ) ( Franklin et al . 2014 ; Ojalvo et al , 2009 ; Galletti et al , 2016 ) . Gene sets are composed of top genes upregulated ( TAM UP ) or downregulated ( TAM DOWN ) in TAM vs normal tissue macrophages . Positive Normalized Enrichment Score ( NES ) corresponds to enrichment of gene set in activin - upregulated genes ; negative NES corresponds to enrichment in activin - downregulated genes . Data are presented as pseudo - with magnitudes of NES color - coded as indicated in legend ; non - grey comparisons are FDR < 0 . 10 .

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D. Gene Set Enrichment Analysis (GSEA) was performed to compare transcriptomes of genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt, [all]/Act vs [all]/wt) to 3 published datasets of Tumor-Associated Macrophages (TAM) (Franklin et al. 2014; Ojalvo et al, 2009; Galletti et al, 2016). Gene sets are composed of top genes upregulated (TAM UP) or downregulated (TAM DOWN) in TAM vs normal tissue macrophages. Positive Normalized Enrichment Score (NES) corresponds to enrichment of gene set in activin-upregulated genes; negative NES corresponds to enrichment in activin-downregulated genes. Data are presented as pseudo-heatmap with magnitudes of NES color-coded as indicated in the legend; all non-grey comparisons are FDR<0.10.