E , F ) Representative Western blots ( E ) and quantification ( F ) of PARIS at disease end stage . Protein levels are normalized by β - actin and results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) . No statistically significant differences were found between G93A and PKO / G93A by paired Wilcoxon ' s test ( p = 0 . 99 ) . PARIS levels are unchanged in SOD1 - G93A mice relative to Non Tg at disease end stage .

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

E, F) Representative Western blots (E) and quantification (F) of PARIS at disease end stage. Protein levels are normalized by β-actin and results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.99). PARIS levels are unchanged in SOD1-G93A mice relative to Non Tg at disease end stage.


A , B ) Western blots ( A ) and quantification ( B ) of Miro1 in spinal cord homogenates at disease end stage . β - actin is used as loading reference . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group ; * p = 0 . 011 by paired Student ' s t test ( for comparison G93A vs . PKO / G93A ) and * * p = 0 . 003 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A ) . Parkin knockout increases the levels of Miro1 in SOD1 - G93A mice at disease end stage .

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

A, B) Western blots (A) and quantification (B) of Miro1 in spinal cord homogenates at disease end stage. β-actin is used as loading reference. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; *p= 0.011 by paired Student's t test (for comparison G93A vs. PKO/G93A) and **p= 0.003 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A). Parkin knockout increases the levels of Miro1 in SOD1-G93A mice at disease end stage.


C , D ) Western blots ( C ) and quantification ( D ) of Mfn2 in spinal cord homogenates at disease end stage . Protein levels are normalized by β - actin . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group ; No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 078 by paired Wilcoxon ' s test ) ; * * * p = 0 . 0007 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A ) and * p = 0 . 037 by paired Friedman ' s test with Dunn ' s correction ( for PKO vs . PKO / G93A ) . Parkin knockout increases the levels of Mfn2 in SOD1 - G93A mice at disease end stage .

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

C, D) Western blots (C) and quantification (D) of Mfn2 in spinal cord homogenates at disease end stage. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.078 by paired Wilcoxon's test); ***p= 0.0007 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and *p= 0.037 by paired Friedman's test with Dunn's correction (for PKO vs. PKO/G93A). Parkin knockout increases the levels of Mfn2 in SOD1-G93A mice at disease end stage.


E ) Western blots of spinal cord mitochondria at disease end stage , probed for lysine 48 ( left panel ) and lysine 63 ( right panel ) ubiquitin chains . Citrate synthase is used as loading control . Asterisks indicate ubiquitinated proteins with different abundance in SOD1 - G93A samples .

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

E) Western blots of spinal cord mitochondria at disease end stage, probed for lysine 48 (left panel) and lysine 63 (right panel) ubiquitin chains. Citrate synthase is used as loading control. Asterisks indicate ubiquitinated proteins with different abundance in SOD1-G93A samples.


F , G ) Representative Western blot ( F ) and quantification ( G ) of LC3 II in spinal cord mitochondria at end stage . Protein levels are normalized by Complex V . LC3 II is increased in both G93A and PKO / G93A mitochondria and Parkin knockout does not affect LC3 II levels . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group ; No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 504 by paired Student ' s t test ) . * p = 0 . 035 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A ) and * * p = 0 . 0014 by paired one - way ANOVA with Tukey ' s correction ( for PKO vs . PKO / G93A )

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

F, G) Representative Western blot (F) and quantification (G) of LC3 II in spinal cord mitochondria at end stage. Protein levels are normalized by Complex V. LC3 II is increased in both G93A and PKO/G93A mitochondria and Parkin knockout does not affect LC3 II levels. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.504 by paired Student's t test). *p= 0.035 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and **p= 0.0014 by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A)


H ) Representative Western blot of March5 and Mul1 in spinal cord mitochondria at end stage .

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H) Representative Western blot of March5 and Mul1 in spinal cord mitochondria at end stage.


I ) Quantification of March5 at disease end stage . Results are expressed as mean ± SEM and percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group . No statistically significant differences were found between G93A and PKO / G93A by paired Wilcoxon ' s test ( p = 0 . 742 ) . Protein levels are normalized by Complex V . March5 levels are unaffected by Parkin knockout .

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

I) Quantification of March5 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.742). Protein levels are normalized by Complex V. March5 levels are unaffected by Parkin knockout.


J ) Quantification of Mul1 at disease end stage . Results are expressed as mean ± SEM and percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group . No statistically significant differences were found between G93A and PKO / G93A by paired Wilcoxon ' s test ( p = 0 . 94 ) . Protein levels are normalized by Complex V . Mul1 levels are unaffected by Parkin knockout .

J) Quantification of Mul1 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.94). Protein levels are normalized by Complex V. Mul1 levels are unaffected by Parkin knockout.


B . Beta - hydroxybutyrate ( ketone body ) concentrations , plasma .

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B. Beta-hydroxybutyrate (ketone body) concentrations, plasma.


C . Creatine kinase ( CK ) concentrations , plasma .

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C. Creatine kinase (CK) concentrations, plasma.


D . Myoglobin concentrations , plasma . Dashed line : upper limit of control range . The individual patients are shown separately , and controls as mean with standard deviation ( n = 10 ) . Arrow : mAD endpoint in PEO patients ; lines under graphs : duration of mAD .

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D. Myoglobin concentrations, plasma. Dashed line: upper limit of control range. The individual patients are shown separately, and controls as mean with standard deviation (n=10). Arrow: mAD endpoint in PEO patients; lines under graphs: duration of mAD.


E - G . PEO patient ( P1 ) muscle before diet : E . Ultrathin section of abnormal muscle fiber ( arrowhead ) with subsarcolemmal accumulations of mitochondria . Scale bar 50 µmF . Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria ( same cell as in E ) . G . Enlargement of the area marked in F ; mitochondria with paracrystalline " parking lot " inclusions ( arrow ) and concentric cristae ( asterisk ) . Scale bar 1µmH - J . PEO patient ( P1 ) muscle after mAD ( H - J ) : H . Ultrathin section , lytic muscle fiber with centrally located organellar debris ( arrow ) . Scale bar 50 µmI . Electron micrograph of a degrading muscle fiber with an invaded macrophage ( arrow ) ( same cell as in H ) . J . Enlargement of the fiber in I : paracrystalline inclusions within mitochondria ( asterisk ) . Scale bar 1µmK . Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections . n . a . , not available .

O O O O O B-ORGANISM O O O B-TISSUE O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-ORGANISM O O O B-TISSUE O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O

E-G. PEO patient (P1) muscle before diet:E. Ultrathin section of abnormal muscle fiber (arrowhead) with subsarcolemmal accumulations of mitochondria. Scale bar 50 µmF. Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria (same cell as in E).G. Enlargement of the area marked in F; mitochondria with paracrystalline "parking lot" inclusions (arrow) and concentric cristae (asterisk). Scale bar 1µmH-J. PEO patient (P1) muscle after mAD (H-J):H. Ultrathin section, lytic muscle fiber with centrally located organellar debris (arrow). Scale bar 50 µmI. Electron micrograph of a degrading muscle fiber with an invaded macrophage (arrow) (same cell as in H).J. Enlargement of the fiber in I: paracrystalline inclusions within mitochondria (asterisk). Scale bar 1µmK. Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections. n.a., not available.


A - B . Cleaved caspase - 3 positive muscle fibers ( * ) . A . PEO patient ( P3 ) on normal diet ( inset : caspase staining in healthy control individual ) B . PEO patient ( P3 ) after modified Atkins diet . Arrowhead : caspase negative necrotic fiber .

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A-B. Cleaved caspase-3 positive muscle fibers (*).A. PEO patient (P3) on normal diet (inset: caspase staining in healthy control individual)B. PEO patient (P3) after modified Atkins diet. Arrowhead: caspase negative necrotic fiber.


C . Western blot analysis of autophagy receptor p62 and pax7 marking muscle satellite cell activation ; left , blot ; right , signal quantification in each patient .

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

C. Western blot analysis of autophagy receptor p62 and pax7 marking muscle satellite cell activation; left, blot; right, signal quantification in each patient.


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 .

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

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 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 .

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

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 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 ) .

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

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 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 .

O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-TISSUE O O O O O O O O O O O O O

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 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 ) .

O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 and pathway of creatine synthesis .

O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 analysis for MIG - 6 protein expression in various human breast cancer cell lines .

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

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


( 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 .

O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-DISEASE O 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 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 ) 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 .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 analysis of the disease - specific survival of 85 TNBC breast cancer patients with low or high expression of MIG - 6 .

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

(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 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 . "

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

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

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

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


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

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


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

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

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


( D Immunoblotting analysis for EGFR signaling in BT549 cells 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 analysis for EGFR signaling in MDA - MB - 231 cells 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 ) Cell growth assay in BT549 cells with GFP or MIG - 6 knockdown in the absence and presence of gefitinib for 72 hours .

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

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

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

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

(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 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 ) .

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

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 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 ) .

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

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 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 ) .

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

(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 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 . .

O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 analysis for GLUT1 and MIG - 6 mRNA expression in GFP and MIG - 6 knockdown BT549 cells ( n = 3 ) .

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

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


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

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

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


( 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 ) .

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

(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 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 .

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

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

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

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


( 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 .

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

(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 analysis for HIF1α protein expression in BT549 cells 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 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 .

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

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

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

(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 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 .

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

(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 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 .

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

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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


( 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 .

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

(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 assay in control and MIG - 6 knockdown BT549 cells 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 assay in control and MIG - 6 knockdown MDA - MB - 231 cells with or without GLUT1 overexpression .

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

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


( 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 .

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

(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 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 ) .

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

(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 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

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

(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


( 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 ±

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


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 ) .

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

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 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 .

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

(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 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 .

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

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

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

(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 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 .

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

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

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

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


( 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 .

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

(C) 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 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 .

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

(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 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 .

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

(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 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 ) 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 ) 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 .

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

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 ) 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 .

O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) 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 .

O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 ) 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 .

O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

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 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

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

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 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 .

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

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 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

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

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 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

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

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 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

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

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 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 .

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

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 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 ,

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

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 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 )

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

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 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 ) ,

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

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 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 )

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

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 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 .

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

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 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 .

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

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 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 .

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

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 the differentially expressed microRNAs under study in exosomes from activated CD4 + T cells . Bars represent the mean ± SEM .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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

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

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 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

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

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 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 .

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

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 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 .

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

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 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 .

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

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 cells after IS formation as in ( G ) ; the bar chart shows the mean fluorescence Intensity ± SEM and quantification of 3 independent experiments is shown .

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

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 ) 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 .

O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O

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 ) 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 .

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

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 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 .

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

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 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 .

B-EXP_ASSAY O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-TISSUE O O O O B-ORGANISM O O O O O O B-TISSUE O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-ORGANISM O O O O O

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 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 .

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

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 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 .

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

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 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

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

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