EMBO/SourceData huggingface dataset:
Figure 1B) 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.
Figure 2B) 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.
Figure 4B,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.
Figure 5B) 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.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.
Figure 6A) 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.
Figure 7A) 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.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
Figure 1DICER-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 conjugatesDICER-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 analysisDICER-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 analysisDICER-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,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),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.
Figure 3Quantitative 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.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.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.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.
Figure 172h 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
Figure 2EVs 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.
Figure 3EVs 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
Figure 4Schistosomal- miRNA was extracted from either EVs that were isolated from schistosomal-growing medium or control pellets that were isolated from unused schistosomal-medium as described in the Materials and Methods (Schistosomal-EVs purification section). Equal aliquots were subjected to qRT-PCR with specific primers to the schistosomal-miR-10, Bantam and miR-125; the expression is presented as ∆Ct from background Ct that was set as 40 cycles.
Figure 5The trans-well system was performed as in Fig. 1. Schistosome-exposed Th cells or control Th cells were subjected to qRT-PCR with specific schistosomal-Taqman primers to (A) miR-10-5p, (B) Bantam, and (C) miR-125b.The trans-well system was performed as in Fig. 1. The Control wells contained only unused schistosomal-medium; Supernatant wells contained 0.1 μm of the filtered schistosomal-growing medium; Th wells contained sorted CD4+CD11c- from spleens; Th+APCs contained sorted CD4+CD11c- and CD4-CD11c+ in 5:1 ratio, respectively. In Data information: In (A-E) the expression is presented as arbitrary units calculated as 2∆∆CT of miRNA relative to snRNA U6 and in (F) mRNA relative to β2 microglobulin. The mean +/- SEM was calculated from at least 3 independent experiments (except in F of 2 independent experiments).
Figure 6Image of a schistosome-infected mouse. The lymph nodes, from which the Th cells were isolated, are indicated.Th cells were isolated from the indicated lymph nodes and spleen. In the case of uninfected mice, the Th cells from different lymph nodes and the spleen were mixed. qRT-PCR was performed with specific Taqman primers to either schistosomal-miR-10 or -Bantam. The presence of the miRNAs is presented as arbitrary units calculated as 2∆∆CT of the miRNA divided by snRNA U6. The mean +/- SEM was calculated from 4 independent experiments.
Figure 7HEK-293 cells were transfected with a plasmid expressing schistosomal-miR-10 at the indicated concentration together with either psiCHECK-II vector, psiCHECK-WT-MAP3K7-3'UTR or psiCHECK-mut-MAP3K7. After 24h, the cells lysates were subjected to luciferase assay. The results are presented as the ratio of renilla/luciferase expression that was normalized relative to cells transfected only with psiCHECK-II. Values are expressed as the mean+/-SD of at least 3 independent experiments.A representative experiment in which protein extracted from Jurkat-T cells stably over expressing stably control vector or miR-10 expressing vector were subjected to WB analysis with anti-MAP3K7 or anti-GAPDH antibodies. The graph presents densitometry analysis of 3 WBs analyses performed as in (C). The mean +/- SD was calculated from 4 independent experiments. Statistics were performed using t-test (*p<0.05).Jurkat-T cells stably expressing either control vector or a vector containing schistosomal-miR-10 were co-transfected with a control Luciferase plasmid or NF-κB luciferase reporter vector. In all transfections, a plasmid expressing Renilla vector as an internal control was added. 48h post-transfection, cells were harvested and were analyzed by luciferase reporter assay. Values were normalized to the Renilla activity. Data are represented as mean +/-SEM from 5 independent experiments. Statistics were performed using t-test, **p<0.01.Representative WB experiment demonstrating that MAP3K7 is downregulated in schistosomal-exposed Th cells F) or to Jurkat (H) in comparison to the control unexposed cells. (G, I) The graphs present densitometry analysis of 3 WB analyses performed as in (F and H), respectively. The mean +/- SD was calculated from 3 independent experiments. Statistics were performed using paired one-tailed t-test (*p<0.05).
Figure 1A. qRT-PCR analysis of RNA samples from normal human skin (NS) and AK for activin βA (INHBA), normalized to hypoxanthine phosphoribosyltransferase (HPRT). Expression levels in one of the normal skin samples were set to 1. Scatter plots are shown. NS: N=4; AK: N=21. ns = non-significant (Mann-Whitney test).B. qRT-PCR analysis of RNA samples from normal back skin of wild-type mice (N=4, n=4), non-tumorigenic back skin from HPV8/wt mice (N=7, n=9) and papillomas from HPV8/wt transgenic mice (N=8, n=11) for Inhba, normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Expression levels in one of the wild-type back skin samples were set to 1. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test of log-transformed data. N, number of mice; n, number of biopsies.C-E. Sections from a tumor collected from a 64 week-old HPV8/wt mouse (C,D) or from non-tumorigenic skin of 5 week-old HPV8/wt mice (E) stained with an anti-activin A antibody and counterstained with hematoxylin. E: Epidermis, D: Dermis, HF: Hair follicle. (D) Shows a higher magnification of the boxed area in (C). Scale bars: 200μm (C) and 100μm (D,E).F. Kinetics of tumor incidence in HPV8/wt and HPV8/Act mice; N=20 mice per genotype. Statistical significance was determined using Log-rank (Mantel-Cox) test.G. Age of the mice when the first tumors appeared on certain body sites. Each dot represents one tumor.H. Representative macroscopic pictures of ear tumors (left panel) and H&amp;amp;E-stained sections of ear acanthopapillomas (upper and middle panel) or an ear acanthopapilloma with trichoepitheliomatous differentiation (lower panel). Boxed areas are shown at higher magnification in subsequent pictures to the right in each horizontal picture panel. E: epidermis; D: dermis; C: cartilage. Scale bars: 200μm (left panel), 100μm (middle panel) or 50μm (right panel).
Figure 2A,B. Representative pictures of ear sections from 10 week-old mice stained for δTcR (green); counterstained with Hoechst (blue) (A) and quantification of δTcR+ cells in the ear epidermis (B). Dotted lines indicate the epidermal-dermal border. Scale bar: 20μm. N=6 wt/wt mice, N=7 HPV8/wt mice, N=6 wt/Act mice and (N=8) HPV8/Act mice.C. Quantification of βTcR+ cells in ear skin cryosections from 10 week-old wt/wt (N=5); HPV8/wt (N=5); wt/Act (N=8) or HPV8/Act (N=8) mice.D-F. Percentage of viable CD4+ βTcR+ (D), CD8+ βTcR+ (E) or CD4+CD25+ (F) cells among all epidermal cells analyzed by flow cytometry. Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown. Representative flow cytometry scatter plots are shown in Appendix Fig S1A-C.G. Representative pictures of ear cryosections from 10 week-old mice stained with antibodies against Foxp3 (red) and counterstained with Hoechst (blue). Insert shows higher magnification of the Foxp3-positive cells in wt/Act skin. Dotted lines indicate the epidermal-dermal border. Scale bar: 50μm.H. Kinetics of tumor incidence in HPV8/Act/wt (N=29), HPV8/Act/CD4KO (N=10), HPV8/wt/wt (N=23) and HPV8/wt/CD4KO (N=22) mice per genotype. Statistical significance was determined using the Log-rank (Mantel-Cox) test; ns = non-significant.
Figure 3A,B. Representative pictures of ear skin cryosections from 10-12 week-old mice stained with anti-CD206 antibodies (green); counterstained with Hoechst (blue) (A), and quantification of CD206+ area as a percentage of total dermal area (B) in wt/wt (N=3), HPV8/wt (N=7), wt/Act (N=2) or HPV8/Act (N=8) mice. The basement membrane is indicated with a white dotted line.C. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12-16 week-old mice. N = 5-6. Representative flow cytometry scatter plots are shown in Appendix Fig S1D.D. Quantification of CD206+ area as a percentage of total dermal area in wt (N=7) and Act (N=6) mice in back skin treated 8x with TPA (Antsiferova et al, 2011).E. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of pre-tumorigenic ear skin from 12 week-old mice expressing the CCR2-CFP-DTR transgene (wt/CCR2) or double-transgenic animals (Act/CCR2), untreated or treated with diphtheria toxin (+DT) 3 times every 48h and analyzed 24h after the last injection. Percentage of CD45+CD11b+MerTK+CD64+ macrophages among live cells is shown. N=3 control wt/CCR2 and Act/CCR2 mice; N=4 DT-injected wt/CCR2 and Act/CCR2 mice. Representative flow cytometry scatter plots are shown in Appendix Fig S1E.F, G. Inflammatory thioglycollate-elicited peritoneal macrophages (F) or mouse peripheral blood monocytes (G) were isolated from wt mice and allowed to migrate for 24h in a Transwell assay. Activin A 0.1ng/ml (A0.1), 1ng/ml (A1), or 10ng/ml (A10) was added to the lower well. Alternatively, 10ng/ml was added to the lower well and the insert (A10/A10). Random migration to medium (ctrl) was set to 1 for each mouse; fold over control is shown. Log-transformed data were used for statistical analysis. Results in (F) are representative of 4 independent experiments performed with 8 mice in total (results obtained in one experiment performed with 3 mice are shown). Results in (G) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice.