Journal: Biomedicines

Article Title: Effect of the Enrichment in c-Kit Stem Cell Potential of Foetal Human Amniotic Fluid Cells: Characterization from Single Cell Analysis to the Secretome Content

doi: 10.3390/biomedicines11020430

Figure Lengend Snippet: Comparison of the extracellular vesicles produced by amniotic fluid cells selected or not for c-Kit. ( A ) Representative TEM images of negative staining of vesicles obtained from two types of amniotic fluid cells. ( B ) Representative nanoparticle tracking analysis (NTA) performed on EV suspension with ZetaView. ( C ) Representative images of Western blot analysis of EV samples, revealed with anti-CD9, anti-CD81, anti-Rab5, anti-lamin A/C, and anti-actin as a loading control. ( D ) PBMCs, treated with PHA, were exposed for four days to EVs. The graphs show the cytofluorimetric analysis of the percentage of PBMC positive for CD4 and CD8 surface markers. ** p < 0.01 = samples significantly different. ( E ) ELISA analysis of TGFβ1, IDO, HGF, and IL-6 on the two lysates of isolated EVs. ** p < 0.01; * p < 0.05 = samples significantly different. ( F ) Representative images of Western blot analysis of EV samples, revealed with anti-HGF, anti-IDO, anti-TGFβ, anti-IL-6, and anti-actin, as a loading con.trol.

Article Snippet: Primary antibodies were raised against the following molecules: HIF-1α, GAPDH, actin, HGF, IDO, lamin A/C (Santa Cruz Biotechnology, CA, USA), phospho-c-Kit Tyr719 (Cell Signaling Technology, MA, USA), TGFβ (Novus, CO, USA), Rab5 (Lonza, SC, USA), CD9, and CD81 (Life Technologies, CA, USA).

Techniques: Comparison, Produced, Negative Staining, Suspension, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Isolation

Journal: International journal of molecular sciences

Article Title: Preconditioned Chorionic Villus Mesenchymal Stem/Stromal Cells (CVMSCs) Minimize the Invasive Phenotypes of Breast Cancer Cell Line MDA231 In Vitro.

doi: 10.3390/ijms24119569

Figure Lengend Snippet: Figure 6. Modulation in expression of tumor suppressor proteins and oncogenes in MDA231 cells treated with preconditioned CVMSCs: flow cytometry analysis for the expression of tumor suppressor proteins modulated in MDA231 cells after treatment with preconditioned CVMSCs showed significant increase in the expression levels for CDH1 and IFN-gamma in both IC and CM settings as compared to untreated control (A(i)). Expression levels of oncogenes such as IDO, IL6, MMP7, and TGF-β1 reduced significantly in MDA231 cells in both IC and SF setting as compared to untreated control (B(i)). Data obtained by FACS analysis from three independent experiments were quantified and are shown in bar graphs as Mean Florescence Index (MFI) pertaining to CDH1 and IFN-gamma (A(ii)), and IDO, IL6, MMP7, and TGF-β1 (B(ii)), respectively. Bars represent standard errors. * p < 0.05.

Article Snippet: Fluorescent-labeled antibodies for flow cytometry experiments, including IFN-γ (Human IFN-gamma PE-conjugated Antibody) cat# IC285P; CDH1 (Human E-Cadherin PE-conjugated Antibody) cat# FAB18381P; IDO (Human Indoleamine 2,3-dioxygenase/IDO PE-conjugated Antibody) cat# IC6030P; IL6 (Human IL-6 PE-conjugated Antibody)cat# IC206P; MMP7 (Human MMP-7 PE-conjugated Antibody) cat# IC9071P; and TGF-β1 (Human TGF-beta 1 Alexa Fluor® 488-conjugated Antibody) cat# IC10502G were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Cytometry, Control

Journal: Cells

Article Title: IDO-Mediated Immune and Metabolic Dysregulation in Schwann Cells Exposed to Mycobacterium leprae

doi: 10.3390/cells14191550

Figure Lengend Snippet: M. leprae differentially modulates IDO expression and Schwann cell metabolism. ( A ) Heatmap represents the IDO and AADAT genes expression in primary Schwann cells infected (in green) or not (in purple) with M. leprae (for 24 h (in pink) and 48 h (in blue). ( B ) Gate strategy in the cell’s population through the FSC-H and SSC-H and percentage of IDO1 positive cells (%). The FACScalibur flow cytometer was used for the acquisition and FlowJo software was used for the analysis. Statistical analysis of n = 6 independent experiment was performed by using the unpaired t -test. The data are represented as mean ± SD. ( C ) The figure represents the HPLC for IDO1 activity of the culture supernatant by kyn/tryp ratio. Statistical analysis of n = 5 independent experiments were performed using the unpaired t -test ( p = 0.0253). The data was represented as a mean with SEM. ( D ) The metabolic activity of Schwann cells incubated with irradiated M. leprae . Statistical analysis of n = 5 independent experiments were performed by Kruskal–Wallis non-parametric followed by Dunn’s multiple comparisons test ( p ≤ 0.0001). The data was represented as mean ± SD. Where **** means p < 0.0001, ** means p < 0.005 and * means p < 0.05.

Article Snippet: Intracellular staining was conducted using a PE-conjugated anti-IDO1 antibody (R&D Systems, Minneapolis, MN, USA; 1:50 dilution, cat. IC6030P) for 30 min at room temperature in the dark.

Techniques: Expressing, Infection, Flow Cytometry, Software, Activity Assay, Incubation, Irradiation

Journal: Neuro-Oncology Advances

Article Title: Superinduction of immunosuppressive glioblastoma extracellular vesicles by IFN-γ through PD-L1 and IDO1

doi: 10.1093/noajnl/vdac017

Figure Lengend Snippet: IFN-γ increases PD-L1 and IDO1 expression in human glioblastoma cells and extracellular vesicles. (A) Glioblastoma cell lines dBT114 or dBT116 ± 100 ng/mL IFN-γ for 24 h and expression of indicated proteins in whole-cell lysate (WCL) and extracellular vesicles (EVs) was assessed by western blot. (B) Immunoblots from (A) were normalized to loading control (HSP90) by densitometry. Relative intensity compared to baseline (WCL without IFN-γ exposure) is shown (mean ± SEM; n = 3). (C) dBT114 or dBT116 cells ± 10 or 100 ng/mL IFN-γ for 24 h and analyzed by western blot for IDO1, PD-L1, and GAPDH. Bar graphs for PD-L1 show median fluorescence intensity on flow cytometry relative to EVs without IFN-γ exposure (median ± standard deviation, n = 3) while those for IDO1 show relative densitometry intensity compared to baseline (no IFN-γ) after normalization to GAPDH (mean ± standard deviation; n = 3). (D) Nanoparticle tracker analysis histograms and photomicrographs showing the size distribution and frequency of dBT114 and dBT116 EVs ± 100 ng/mL IFN-γ. * P < .05, ** P < 0.01,*** P < .001, **** P < .0001. IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-gamma; PD-L1, programmed cell death ligand 1.

Article Snippet: Short interfering RNA (siRNA) sequences targeting PD-L1 (sc-39699), IDO1 (sc-45939), or a nontargeting siRNA control (sc-37007) were acquired (Santa Cruz Inc).

Techniques: Expressing, Western Blot, Control, Fluorescence, Flow Cytometry, Standard Deviation

Journal: Neuro-Oncology Advances

Article Title: Superinduction of immunosuppressive glioblastoma extracellular vesicles by IFN-γ through PD-L1 and IDO1

doi: 10.1093/noajnl/vdac017

Figure Lengend Snippet: PD-L1 and IDO1 expression are both required for MDSC and NCM superinduction in response to IFN-γ-exposed GBM EVs. (A) dBT114, dBT116, dBT120, and dBT165 cells were transfected with either nontargeting (sicon), PD-L1 (80 nmol/L) or IDO1 (80 nmol/L). After 72 h, the transient transfectants were stimulated in the absence (−) or presence (+) of 100 ng/mL IFN-γ for 24 h and western blotted for PD-L1, IDO1, and HSP90 as a loading control. (B) Immunoblots from (A) were normalized to loading control (HSP90) and evaluated. Bar graphs showing mean frequency of MDSC (C) or NCM (D) induction in serum-free media, EVs, EVs from dBT cell lines, PD-L1 knockdown dBT cells or IDO1 knockdown dBT cells treated with (+) or without (−) IFN-γ (100 ng/mL) for 3 days. Note that both PD-L1 and IDO1 knockdown markedly reduced the superinduction of MDSC and NCM in response to IFN-γ. No synergy or additive effects are seen. * P < .05, ** P <0.01, *** P < .001, **** P < .0001. EVs, extracellular vesicles; GBM, glioblastoma; IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-gamma; MDSC, myeloid-derived suppressor cell; NCM, nonclassical monocyte; PD-L1, programmed cell death ligand 1.

Article Snippet: Short interfering RNA (siRNA) sequences targeting PD-L1 (sc-39699), IDO1 (sc-45939), or a nontargeting siRNA control (sc-37007) were acquired (Santa Cruz Inc).

Techniques: Expressing, Transfection, Western Blot, Control, Knockdown, Derivative Assay

Journal: Neuro-Oncology Advances

Article Title: Superinduction of immunosuppressive glioblastoma extracellular vesicles by IFN-γ through PD-L1 and IDO1

doi: 10.1093/noajnl/vdac017

Figure Lengend Snippet: Superinduction of T cell inhibition by monocytes cultured with IFN-γ-exposed GBM EV depends on GBM PD-L1 and IDO1 expression. (A) Representative histograms showing proliferation of CFSE-stained T cells stimulated with or without anti-CD3/anti-CD28 in serum-free media alone, with naive GBM EV-treated monocytes, or with IFN-γ-treated GBM EVs. (B) Bar graphs showing mean T cell proliferation (from 4 donors) in response to anti-CD3/anti-CD28 antibodies in the conditions outlined. (C) Bar graphs showing mean T cell proliferation in response to anti-CD3/anti-CD28 antibodies in serum-free media alone or in the presence of monocytes exposed to GBM EVs, GBM EVs with PD-L1 knockdown, or GBM EVs with IDO1 knockdown. GBM cells treated with (+) or without (−) IFN-γ (100 ng/mL) for 3 days prior to EV harvest. * P < .05, ** P <0.01, *** P < .001, **** P < .0001. EV, extracellular vesicle; GBM, glioblastoma; IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-gamma; PD-L1, programmed cell death ligand 1.

Article Snippet: Short interfering RNA (siRNA) sequences targeting PD-L1 (sc-39699), IDO1 (sc-45939), or a nontargeting siRNA control (sc-37007) were acquired (Santa Cruz Inc).

Techniques: Inhibition, Cell Culture, Expressing, Staining, Knockdown