Journal: Frontiers in Oncology

Article Title: TIPE2 Suppresses Malignancy of Pancreatic Cancer Through Inhibiting TGFβ1 Mediated Signaling Pathway

doi: 10.3389/fonc.2021.680985

Figure Lengend Snippet: TIPE2 inhibited PI3K/AKT and Raf/MEK/ERK signaling pathways triggered by TGFβ1. (A) The total proteins extracted from cells were analyzed for the expression of AKT, ERK and Bax using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK, anti-Bax and anti-GAPDH. (B) ELISA analysis of TGFβ1 secretion from AsPC-1/vector and AsPC-1/TIPE2 cells. (C) Immunohistochemistry analysis of the TGFβ1 expression in AsPC-1/vector and AsPC-1/TIPE2 tumor tissues. (D) Western blot analysis of the expression of p-TGFBR1 and total TGFBR1 in AsPC-1/vector and AsPC-1/TIPE2 cells. (E) AsPC-1 cells were seeded in 6-well plate and added with or without anti-TGFβ1 antibody or rhTGFβ1 protein. After 48 h incubation, the total proteins extracted from the cultured AsPC-1 cells were analyzed for the expression of AKT and ERK using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK and anti-GAPDH. Data shown were representative of three independent experiments. Values are presented as means ± SD. ***p < 0.001.

Article Snippet: In AsPC-1 cells, the neutralizing anti-TGFβ1 monoclonal antibody (1 μg/ml, ProteinTech) or 5 ng/ml human recombinant TGFβ1 (ProteinTech) was added for 48 h incubation, respectively.

Techniques: Protein-Protein interactions, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Immunohistochemistry, Incubation, Cell Culture

Journal: Frontiers in Oncology

Article Title: TIPE2 Suppresses Malignancy of Pancreatic Cancer Through Inhibiting TGFβ1 Mediated Signaling Pathway

doi: 10.3389/fonc.2021.680985

Figure Lengend Snippet: TIPE2 suppressed the growth of pancreatic cancer through inhibiting TGFβ1 expression in subcutaneous tumor model. (A) The tumor volume was measured weekly one week after tumor inoculation and the tumor growth curve was calculated. (B, C) The tumors were isolated, weighted and photographed five weeks after tumor cells inoculation. (D) ELISA analysis of TGFβ1 secretion from Panc02/vector and Panc02/TIPE2 cells. (E) Immunohistochemistry analysis of the TGFβ1 expression in Panc02/vector and Panc02/TIPE2 tumor tissues. (F) Western blot analysis of the expression of p-TGFBR1 and total TGFBR1 in Panc02/vector and Panc02/TIPE2 cells. The data shown are the representative of three experiments. Values are presented as means ± SD. *p < 0.05, ***p < 0.001.

Article Snippet: In AsPC-1 cells, the neutralizing anti-TGFβ1 monoclonal antibody (1 μg/ml, ProteinTech) or 5 ng/ml human recombinant TGFβ1 (ProteinTech) was added for 48 h incubation, respectively.

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Immunohistochemistry, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo

doi: 10.1080/20013078.2020.1795362

Figure Lengend Snippet: RegDCs EXO protect encapsulated immunoregulatory cargo (TGFB1 and IL10) from proteolytic degradation . (A) Nano-tracking analysis to determine EXO number and size distribution in nm. (B) Transmission electron microscopy (TEM) to visualize EXO shape and immuno-gold TEM to detect EXO marker tetraspanin CD63, showing unstained (left) and positive staining (right)(arrows). (C) Western blotting to detect other EXO-related markers including CD81, TSG101, GRP94 and B-actin in donor DCs and EXO (left) and anti/pro-inflammatory cytokines including TGFB1, IL10, IL6, IL1B and TNF and the costimulatory molecule CD86 as well as EXO associated proteins ALIX and TSG101 in DCs EXO subsets (right). (D) TGFB1 and IL10 content of lysed EXO and in regDCs EXO supernatant by ELISA. (E) TGFB1 and IL10 content of non-lysed EXO (transmembrane domain) by ELISA. (F) Immunogold TEM to detect luminal and transmembrane TGFB1 in regDCs EXO. (G) regDCs EXO (left) or equivalent concentration of free TGFB1 and IL10 (right) were treated with trypsin (upper panel) or proteinase-K (lower panel) and incubated in control buffer (1hour at 37°C) and analysed by western blotting to detect the levels of TGFB1, IL10 and exosomal markers TSG101 (upper panel) and CD81 (lower panel).

Article Snippet: Analyses were performed in the presence or absence of DC stimulators/inhibitors including LPS and neutralizing antibodies against TGFB1 (MAB1835, R and D) and IL10 (JES5-2A5, Thermo fisher scientific).

Techniques: Transmission Assay, Electron Microscopy, Marker, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation

Journal: Journal of Extracellular Vesicles

Article Title: Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo

doi: 10.1080/20013078.2020.1795362

Figure Lengend Snippet: LPS/inflammation induced miRNAs and mRNA are down regulated in regDCs EXO in vitro . (A) Colocalization of TGFB1 (back arrow) and EXO (red arrow) in multivesicular bodies in regDCs. (B) miRNA analysis of LPS/inflammation induced miRNAs in EXO. (C) IL6mRNA expression in EXO by PCR.

Article Snippet: Analyses were performed in the presence or absence of DC stimulators/inhibitors including LPS and neutralizing antibodies against TGFB1 (MAB1835, R and D) and IL10 (JES5-2A5, Thermo fisher scientific).

Techniques: In Vitro, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo

doi: 10.1080/20013078.2020.1795362

Figure Lengend Snippet: DC EXO uptake by acceptor DCs, with EXO subtype determining maturation and cytokine profile of acceptor DCs in vitro . (A) Uptake of Dil labelled EXO (red) by bone marrow derived DCs, counterstained with nuclear stain DAPI (blue), phalloidin (green) for cell membrane and visualized under confocal microscopy. (B) Flow cytometry scattergrams showing % of (top panel) CD11c MHCII and (bottom panel) CD11c CD86 double positive cells in DCs treated or not treated with EXO (C) Bar graphs showing % of (left panel) CD11c MHCII and (right panel) CD11c CD86 double positive cells in DCs treated or not treated with each EXO subtype. (D) mRNA expression in DCs treated or not treated with EXO of IL6 (left panel), IL12 (middle panel) and IL23 (right panel). (E) Western blotting analysis of levels of phosphorylation of TGFB1and IL10 transcription factors, SMAD2/3 and STAT3 respectively and MHCII in DCs treated with or without EXO. (B) Flow cytometry scattergrams (top panels) showing % of double positive CD11c MHCII cells in DCs treated with or without regDCs EXO in presence or absence of neutralizing antibodies against TGFB1 and or IL10 and (bottom panel) representative bar graph. Results shown are representative of three independent experiments (* P<0.05 by one-way ANOVA followed by Tukeys multiple comparisons).

Article Snippet: Analyses were performed in the presence or absence of DC stimulators/inhibitors including LPS and neutralizing antibodies against TGFB1 (MAB1835, R and D) and IL10 (JES5-2A5, Thermo fisher scientific).

Techniques: In Vitro, Derivative Assay, Staining, Confocal Microscopy, Flow Cytometry, Expressing, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo

doi: 10.1080/20013078.2020.1795362

Figure Lengend Snippet: RegDCs EXO uptake by CD4T-cells, promoting TGFB1 dependent Tregs induction while stim DCs EXO induce T-helper17 response in vitro . (A) Uptake of Dil labelled EXO (red) by splenic CD4T-cells, showing DAPI-labelled nuclei, and phalloidin (green) labelled cell membrane, as visualized under confocal microscopy. (B) Flowcytometry scattergrams showing in vitro influence of EXO on induction of T-regulatory cells, as measured by % of double positive CD25 and Foxp3 cells in gated CD4T-cell population, stimulated with antiCD3/CD28 antibodies. (C) Summary bar graph of flow cytometry data. (D) Western blotting analysis of Foxp3 expression in acceptor CD4T-cells. (E) Flow cytometry analysis showing proliferation % of CFSE-labelled splenic CD4+T-cells, as expressed by histograms (left panel) and bar graphs (right panel), after stimulation with anti-CD3/CD28 in presence or absence of EXO subtypes. (F) Flow cytometry analysis of CD4+IL-17+T cells %, as expressed by histograms (left) and bar graph (right). (G) ELISA of IL17 expression in supernatant of T cells treated with EXO subtypes. Results shown are representative of three independent experiments (* P<0.05 by one-way ANOVA followed by Tukeys multiple comparisons).

Article Snippet: Analyses were performed in the presence or absence of DC stimulators/inhibitors including LPS and neutralizing antibodies against TGFB1 (MAB1835, R and D) and IL10 (JES5-2A5, Thermo fisher scientific).

Techniques: In Vitro, Confocal Microscopy, Flow Cytometry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Extracellular Vesicles

Article Title: Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo

doi: 10.1080/20013078.2020.1795362

Figure Lengend Snippet: Cytokine and DCs maturation marker CD86 profile in DCs derived exosomes subsets.

Article Snippet: Analyses were performed in the presence or absence of DC stimulators/inhibitors including LPS and neutralizing antibodies against TGFB1 (MAB1835, R and D) and IL10 (JES5-2A5, Thermo fisher scientific).

Techniques: Marker, Derivative Assay

Journal: Cornea

Article Title: Evaluation of the Transforming Growth Factor-β Activity in Normal and Dry Eye Human Tears by CCL-185 Cell Bioassay

doi: 10.1097/ico.0b013e3181cf98ff

Figure Lengend Snippet: FIGURE 3. Neutralization of the growth inhibitory effect of TGF-b1 (0.3 ng/mL) in CCL-185 by anti-human TGF-b1 IgG1 (concentrations ranging from 0 to 100 mg/mL) measured with the WST-1 assay.

Article Snippet: Cells were cultured in Dulbecco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 50 mg/mL of gentamicin, 1.25mg/mL amphotericin B and 2mM l-glutamine in a 100 cm2 plate at 37 C in 5%CO2 using a previously published method.15 To perform the assay, wells were rinsed with DMEM, then 50 mL of each sample including tears, recombinant human TGFb1 or TGF-b2, anti-human TGF-b1 immunoglobulin G 1 (IgG1) (MAB 240) or anti-human TGF-b1,2,3 (all from R & D Systems, Minneapolis, MN) were added to each well of a 96- well cell culture plate.

Techniques: Neutralization, WST-1 Assay