Additional file 1: of M1-like tumor-associated macrophages activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma

Heat-map indication of the densitometry values detected using the PathScan Immune Cell Signaling Antibody Array Kit. Colors illustrate fold changes (see color scale). Red: up-regulation; green: down-regulation. (DOCX 276 kb

Additional file 2: of M1-like tumor-associated macrophages activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma

Validation of THBS1 knockdown in SCC25 and Cal27 cells. A. Relative mRNA expression of THBS1 in SCC25 and Cal27 after THBS1 knockdown (Scrambled as control), as determined by quantitative real-time PCR. Data are represented as the mean ± SD of three independent experiments, **p < 0.01. B. Relative protein expression of THBS1 in SCC25 and Cal27 after knockdown of THBS1 (Scrambled as control), as determined by Western blotting. Data are represented as the mean ± SD of three independent experiments, **p < 0.01. C. Expression level of THBS1 in CM of SCC25 and Cal27 after THBS1 knockdown (Scrambled as control), as determined by ELISA assays, **p < 0.01. D. Expression level of THBS1 in exosome supernatants of SCC25 and Cal27 cells after THBS1 knockdown (Scrambled as control), as determined by ELISA assays, **p < 0.01. (DOCX 199 kb

Additional file 3: of M1-like tumor-associated macrophages activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma

Negative control for tracing exosome uptake by macrophages. Cultured macrophages were fixed, permeabilized, and stained with Acti-stain™ 488-Phalloidin and DAPI. Then, these macrophages were examined under confocal microscope. No red signals were captured in macrophages with an excitation at 460 nm without the incubation of labelled exosomes. (DOCX 240 kb

Additional file 4: of M1-like tumor-associated macrophages activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma

Control images for the Chromogenic double staining with CD80 (pink)/CD68 (brown) in primary OSCC samples. Sections stained for hematoxylin were used as negative control. Sections stained with CD68 were used as single-positive control. (DOCX 557 kb

TCR induced CD25Foxp3T cells were immunosuppressive to CD4CD25T cell proliferation in vitro

<p><b>Copyright information:</b></p><p>Taken from "Endogenous TGF-β activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4CD25T cells"</p><p>http://www.retrovirology.com/content/4/1/57</p><p>Retrovirology 2007;4():57-57.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC2096626.</p><p></p> . CD4CD25T cells were cultured with anti-CD3 and anti-CD28 for 5 days. The converted CD4CD25Foxp3T cells were purified and washed extensively. The converted Tregs were then used at varying concentrations in a co-culture suppression assay along with CD4CD25(5 × 10) T cells pre-labeled with CFSE as responders and autologous monocytes (2 × 10) as accessory cells. Anti-CD3 antibody was added into the start of the co-culture suppression assay (0.5 μg/ml). CFSE dilution of responder cells was measured after 72 hrs using flow cytometry. . IFN-γ production of responder cells in the co-culture assay as detected by flow cytometry after 72 hrs. iTreg: induced Foxp3CD4CD25T cells. The experiment was repeated for three times with similar results

The cell-free supernatant from TCR stimulated CD4CD25T cell culture contained ROS

<p><b>Copyright information:</b></p><p>Taken from "Endogenous TGF-β activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4CD25T cells"</p><p>http://www.retrovirology.com/content/4/1/57</p><p>Retrovirology 2007;4():57-57.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC2096626.</p><p></p> CD4CD25T cells were cultured with anti-CD3 and anti-CD28 for the indicated time points and ROS in the culture supernatant was detected using DCFH-DA as described in the Method section. Oxidation of DCFH-DA was measured using a spectrofluorometer at wavelength 485/535 nm and is represented as fluorescent units. The experiment was repeated twice with similar results

Foxp3T cells exist in both non-proliferating (CFSE) and dividing (CFSE) TCR-stimulated CD4CD25T cells

<p><b>Copyright information:</b></p><p>Taken from "Endogenous TGF-β activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4CD25T cells"</p><p>http://www.retrovirology.com/content/4/1/57</p><p>Retrovirology 2007;4():57-57.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC2096626.</p><p></p> CD4CD25T cells were labeled with CFSE (2.5 μM) and cultured with anti-CD3 and anti-CD28 for 3 and 5 days. Cells were then counter-stained intracellularly with PE-conjugated anti-Foxp3 antibody. The cells were analyzed with FACS and a representative profile of CFSE vs. Foxp3 or its control antibody (mIgG2a) is displayed. The experiments were repeated three times with similar results. Data not shown here are the cultures with cells in medium alone. No CFSE dilution (CFSE) or Foxp3cells were observed

Neutralization of endogenous TGF-β abrogated TCR-induced Foxp3 expression

<p><b>Copyright information:</b></p><p>Taken from "Endogenous TGF-β activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4CD25T cells"</p><p>http://www.retrovirology.com/content/4/1/57</p><p>Retrovirology 2007;4():57-57.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC2096626.</p><p></p> . Western blot analysis of Foxp3 protein in cultured CD4CD25T cells with indicated reagents (72 hrs). . FACS analysis of intracellular Foxp3 protein cultured with TCR and CD28 in the presence of anti-TGF-β1,2,3 (αTGF-β) or control (mIgG1) antibodies (72 hr). The data are shown for a representative donor. The values are presented as the percentage of CD25Foxp3T cells. . CD25Foxp3T cells (%) in the TCR- and CD28-stimulated CD4CD25T cells in the absence (-) or presence of anti-TGF-β1,2,3 antibody (αTGF-β) at days 3 and 5. Each symbol represents one donor

A representative FACS profile of cell size ), DHE fluorescence () and apoptotic cells () between anti-CD3 plus anti-CD28-stimulated and medium-treated CD4CD25T cells is displayed

<p><b>Copyright information:</b></p><p>Taken from "Endogenous TGF-β activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4CD25T cells"</p><p>http://www.retrovirology.com/content/4/1/57</p><p>Retrovirology 2007;4():57-57.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC2096626.</p><p></p> . Profile of FSC vs SSC is displayed to show the cell size. The cells were electronically gated as two populations based on their size. R1 (red) represents live or early apoptotic cells (see C). R2 (green) represents dead and/or late apoptotic cells (see C). . Profile of DHE fluorescence (ROS) on FL-2 vs. FSC of R1 and R2 cells. The values are shown as the MFI of R1 and R2 cells (R1/R2). Data not shown here are the MFI of unlabeled cells (negative control for DHE staining) on FL2, which is usually < 10. . The profile of Annexin-V vs. 7-AAD staining of cultured cells compensating the R1 and R2 regions as gated in A. The quadrant gates were set according to the negative isotype control antibodies in the respective cells

HIV infection upregulated Foxp3 expression in TCR activated CD4CD25T cells

<p><b>Copyright information:</b></p><p>Taken from "Endogenous TGF-β activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4CD25T cells"</p><p>http://www.retrovirology.com/content/4/1/57</p><p>Retrovirology 2007;4():57-57.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC2096626.</p><p></p> . Purified CD4CD25T cells were infected with HIV1 (HIV NLA-3) and cultured with anti-CD3 and anti-CD28 antibodies in the absence (HIV-1) or presence of active TGF-β1 (2 ng/ml) (HIV-1+TGF-β) or anti-TGF-β1,2,3 antibody (HIV-1+anti-TGF-β) for 3 (data not shown) and 5 days. T cells were then stained for surface CD25 and intracellular Foxp3. A representative of two experiments is displayed. A parallel culture of TCR- and CD28-stimulated CD25T cells without HIV infection (uninfected) was used as control. . The supernatants from the same cultures in A were collected at the indicated time points and tested for HIV p24 with ELISA. The experiment was repeated twice with similar results