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Additional file 1. Table S1. Identification of proteins with unique peptides in Helicobacter pylori (Hp) 60190 OMVs. The results of the LC–MS/MS analysis and the search using the Hp UniProtKB database (UP000000429.fasta) allowed to identify the total protein content of OMVs and the parental bacteria Hp 60190. The table shows the protein ID and the average number of unique peptides of proteins found in OMVs. Figure S1. Evaluation of the dose-response of the reactivity markers in DITNC1 ATCC astrocytes treated with different concentrations of OMVs from Helicobacter pylori (Hp) 60190. DITNC1 ATCC cells were incubated in the absence or presence of OMVs (1.25 to 20 µg/ml) from Hp 60190, at 37 °C for 24 h. As a positive reactivity control, the astrocytes were incubated with TNF (10 ng/ml, 48 h). After treatment, whole cell lysates were evaluated by immunoblot analysis of total connexin 43 (A), β3 integrin (B), GFAP (C), and vimentin (D), normalized to β-actin. Values in the graphs were obtained by averaging the immune-specific band intensity normalized to β-actin from 3 independent experiments (mean ± S.E.M). *p < 0.05 and **p < 0.01, compared to controls (Ctrl). Figure S2. Dose-response of the reactivity markers in DITNC1 ATCC astrocytes treated with 2.5 µg/ml of Helicobacter pylori (Hp) OMVs at different times. DITNC1 ATCC cells were incubated in the absence or presence of 2.5 µg/ml of Hp 60190 OMVs at 37 °C, from 3 to 72 h. As a positive reactivity control, the astrocytes were incubated with TNF (10 ng/ml, 48 h). After treatment, whole cell lysates were evaluated by immunoblot analysis of total connexin 43 (A), β3 integrin (B), GFAP (C), and vimentin (D), normalized to β-actin. Values in the graphs were obtained by averaging the immune-specific band intensity, normalized to β-actin, from 3 independent experiments (mean ± S.E.M). *p < 0,05, **p < 0.01, and ***p < 0.001, compared to controls (Ctrl). Figure S3. Effect of Helicobacter pylori (Hp) OMVs on astrocyte cell viability. Primary astrocytes (post 17 days in vitro) were incubated in the absence or presence of 2.5 µg/ml of OMVs for 12 h. Cell viability was evaluated with the Trypan blue assay in a Neubauer chamber. Values in the graph represent the average of the percentage of negative trypan blue cells from 3 independent experiments (mean ± S.E.M.). Figure S4. Effect of Helicobacter pylori (Hp) OMVs on p65 NF-ĸB subunit protein levels in DITNC1 ATCC astrocytes, following different times of exposure. DITNC1 ATCC cells were incubated in the absence or presence of 2.5 µg/ml of OMVs from 3 to 72 h, at 37 °C. As a positive reactivity control, the astrocytes were incubated with TNF (10 ng/ml, 48 h). After treatment, whole cell lysates were evaluated by immunoblot analysis of p65 NF-ĸB total levels. Values in the graph were obtained by averaging the immune-specific band intensity, normalized to β-actin, from 3 independent experiments (mean ± S.E.M). *p < 0.05, **p < 0.01, and ***p < 0.001, compared to controls (Ctrl). Table S2. Research Resource IdentifiersList of reagents, antibodies, inhibitors, kits, experimental models used in this study. Tee catalog number is included when available.

Additional file 1. Precipitation of Syndecan-4 binding Proteins for MS. (A) Syndecan-4 cytoplasmic tail contains three different regions named C1-V-C2. The peptides used to precipitate Syndecan-4 binding proteins contain the last 10 amino acids indicated (SDC4). In control peptides (CTRL) the last 4 amino acids, EFYA have been replaced to four glycine. (B) Protein extracts from DI TNC1 cells, stimulated or not (NS) with fetal bovine serum (FBS), were precipitated with either biotinylated peptides of the Syndecan-4 (SDC4)-C-terminal sequence, which includes the C2 sequence, or the control peptide (CTRL). Precipitates were size-fractionated by 4–12% SDS-PAGE and silver stained. Bands differentially precipitated with SDC4 peptides were cut from the gel and analyzed by MS as described in Material and Methods. Relative molecular weight (Mr) is indicated to the right in kDa. The symbols (*) and (*’) indicate the bands identified as PAR-3 and Syntenin, respectively.

Additional file 1. Cell culture condition. Tables S1,S2, Figures S1–S3. Data summarizing characterization of AuNP, AuNP-PEG, AuNP-PEG-FA, B16F10 EVs and EV-AuNP by DLS, Laser Doppler Anemometry, TEM, Cryo-TEM and western blot. Uptake of AuNP-PEG and AuNP-PEG-FA in B16F10 cells. Distribution of control EVs, AuNP-PEG-FA and EV-AuNP in mice without tumors by fluorescence imaging.

Supplemental material, Garrido_et_al._Supl_Material for Metformin prevents nerve growth factor-dependent proliferative and proangiogenic effects in epithelial ovarian cancer cells and endothelial cells by Maritza P. Garrido, Carolina Vera, Margarita Vega, Andrew F.G. Quest and Carmen Romero in Therapeutic Advances in Medical Oncology

A) Representative western blot of astrocyte reactivity markers GFAP and iNOS from wild-type rat astrocytes treated or not with TNF. B) Representative images of GFAP immunofluorescence from rat primary astrocytes treated or not with TNF. Magnification bar = 50 μm. C) Quantification of rat primary astrocytes harboring FAs in control cells or cells treated with IL-1β, IL-6, or TNF, and stimulated or not with Thy-1-Fc or FBS. Values shown are the means ± s.e.m. from three independent experiments. ***p

Representative western blot of β3 Integrin from rat astrocytes treated with Il-6, IL+db cAMP, IL-1β, IL-1β+db cAMP, or IFN-γ. (PDF 77 kb)

Representative western blot of β3 Integrin from rat astrocytes treated with Il-6, IL+db cAMP, IL-1β, IL-1β+db cAMP, or IFN-γ. (PDF 77 kb)

Representative western blot of the markers of astrocyte reactivity GFAP and iNOS from non-transgenic and hSOD1G93A-derived astrocytes. β-actin is a loading control. (PDF 166 kb)

A) Representative western blot of astrocyte reactivity markers GFAP and iNOS from wild-type rat astrocytes treated or not with TNF. B) Representative images of GFAP immunofluorescence from rat primary astrocytes treated or not with TNF. Magnification bar = 50 μm. C) Quantification of rat primary astrocytes harboring FAs in control cells or cells treated with IL-1β, IL-6, or TNF, and stimulated or not with Thy-1-Fc or FBS. Values shown are the means ± s.e.m. from three independent experiments. ***p

Representative western blot of the markers of astrocyte reactivity GFAP and iNOS from non-transgenic and hSOD1G93A-derived astrocytes. β-actin is a loading control. (PDF 166 kb)

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