Journal: Molecular and Cellular Biology

Article Title: G Protein-Coupled Receptor 83 Is Dispensable for the Development and Function of Regulatory T Cells

doi: 10.1128/mcb.01075-07

Figure Lengend Snippet: FIG. 5. Th17 differentiation in vitro is unaffected by GPR83 deficiency. (A) Purified naı¨ve CD25 CD62Lhi CD4 T cells were stimulated with soluble CD3 antibodies and Ly5.1-marked T-cell-depleted splenocytes in the presence or absence of recombinant TGF-1 and IL-6 for 4 days. During the last 6 h, cells were cultured with phorbol myristate acetate-ionomycin and IL-17 secretion by Ly5.1 CD4 T cells was measured using intracellular flow cytometric analysis. (B) Percentages of IL-17-expressing T cells among G protein-coupled receptor-deficient and nondeficient CD4 T cells activated under Th17 polarizing conditions. Each square represents an average number of IL-17 cells in an individual experiment with two to three mice per group.

Article Snippet: For Foxp3 induction, 2 105 purified CD4 CD25 CD62Lhi naı̈ve T cells were cocultured with 2 106 irradiated T-cell-depleted splenocytes isolated from Ly5.1 C56BL/6 mice in the presence of CD3 (1- g/ml) antibodies and 1-ng/ml human recombinant TGF- (R&D Systems) in the wells of 24-well plates.

Techniques: In Vitro, Recombinant, Cell Culture, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Altered proTGFα/cleaved TGFα ratios offer new therapeutic strategies in renal carcinoma

doi: 10.1186/s13046-021-02051-0

Figure Lengend Snippet: TGFα protein levels in renal cancer patients. A . (Left) Schematic representation of proTGFα structure, indicating domains and the proteolytic cleavage sites that lead to the membrane anchored proTGFα or tail. The antibody used for western blotting detection is showed as well. (Right) Western blot showing the different forms of TGFα detected in a renal cell carcinoma sample. B . Protein levels of different molecular forms of TGFα in normal and tumor kidney tissues from 24 patients. The same antibody was used for both the immunoprecipitation and the western blot. C . Ratio of proTGFα and tail fragment between tumor and normal tissue, calculated as the mean pixel intensity of the upper band (17 KDa) to the lower band (15 KDa) of the above WB scans. p -value was calculated by the Mann-Whitney U test. D . Pie chart showing the percentage of paired samples where levels of TGFα were higher (pink), lower (green) or equal (yellow) in the tumor samples than in the healthy tissue counterpart

Article Snippet: The neutralizing anti-human TGFα was from R&D Systems (Minneapolis, MN, USA).

Techniques: Membrane, Western Blot, Immunoprecipitation, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Altered proTGFα/cleaved TGFα ratios offer new therapeutic strategies in renal carcinoma

doi: 10.1186/s13046-021-02051-0

Figure Lengend Snippet: Expression of EGFR and TGFα in kidney cancer cell lines. A . Differential expression of EGFR and B . TGFA in different cancer cell lines. RNA-Seq data were obtained from the Cancer Cell Line Encyclopedia. The number of different cell lines for each cancer subtype is indicated in brackets and the data corresponding to the expression of both genes in kidney are highlighted with a red dashed square. C . Phosphorylation level of different RTKs, calculated as the mean pixel intensity of dot duplicates from the antibody array relative to the reference dots (in a.u.). D . Protein levels of phosphorylated and total EGFR, as well as two ligands (TGFα and amphiregullin) in kidney cancer cell lines. E . Level of expression (measured by quantitative PCR) of the seven EGFR ligands in the cell lines. Data are represented as 2^-delta Ct, in arbitrary units (a.u.). U_AE, upper aerodigestive; ESO, esophagus; KID, kidney; U_TR, urinary tract; THY, thyroid; PANC, pancreas; BD, bile duct; L_NSC, lung non-small cell carcinoma; LIV, liver; PROS, prostate; GLI, glioma; STO, stomach; OV, ovary; END, endometrium; CR, colorectal; OST, osteosarcoma; BRE, breast; NEU, neuroblastoma; S_TIS, soft tissue; MEL, melanoma; LSC, lung small cells; HL, Hodgkin lymphoma; E_SAR, Ewing sarcoma; BL, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; T_ALL, T-cell acute lymphoid leukemia; B_ALL, B-cell acute lymphoid leukemia; MM, multiple myeloma; AML, acute myeloid leukemia; CML, chronic myeloid leukemia

Article Snippet: The neutralizing anti-human TGFα was from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative Proteomics, RNA Sequencing, Phospho-proteomics, Ab Array, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Altered proTGFα/cleaved TGFα ratios offer new therapeutic strategies in renal carcinoma

doi: 10.1186/s13046-021-02051-0

Figure Lengend Snippet: Role of EGFR and TGFα in the proliferation of kidney cancer cell lines. A . Knockdown of EGFR and D . TGFα in renal cancer cell lines. Cells were infected with control vector (pLKO) and viruses including two different short hairpin sequences targeting EGFR ( A ) and TGFA ( D ). Cell extracts were obtained and the receptor expression was measured by immunoprecipitation followed by western blot with the specific antibody. B . EGFR or E . TGFα knockdown effect on the proliferation of renal cancer cells. Cells were infected with the indicated shRNAs. After selection, cells were then plated and counted after 5 days. Results are plotted as the mean ± s.d. of triplicates with respect to the proliferation of cultures infected with the control vector. C . Effect of cetuximab on the proliferation of renal cancer cells. Cells were treated with 10 nM of cetuximab or anti-human IgG antibody (hIgG) for 5 days and then counted. Graph bars represent the mean ± s.d. of triplicates normalized to cells treated with hIgG as a control. F . Effect of a neutralizing anti-TGFα antibody on the proliferation of renal cancer cells. Cells were treated as in C . and counted

Article Snippet: The neutralizing anti-human TGFα was from R&D Systems (Minneapolis, MN, USA).

Techniques: Knockdown, Infection, Control, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Selection

Journal: Pharmacology Research & Perspectives

Article Title: Autocrine EGF and TGF ‐α promote primary and acquired resistance to ALK / c‐Met kinase inhibitors in non‐small‐cell lung cancer

doi: 10.1002/prp2.1047

Figure Lengend Snippet: Transient exposure to EGF and TGF‐α promoted primary resistance to ALK/c‐Met TKI in H3122 and EBC‐1 cells. (A) Summary of results from H3122 and EBC‐1 cells treated with eight widespread growth factors in the presence of TAE684 (1 μM) or SGX‐523 (1 μM). CR, complete resistance; PR, partial resistance; NR, no rescue. (B, C) H3122 cells (B) or EBC‐1 cells (C) were treated with growth factors in the presence of TAE684 (1 μM) or SGX‐523 (1 μM) for 72 h and cell viability was assessed by MTT assay. Data are shown as mean ± SD from three independent experiments. ** p < .01, *** p < .001. (D, E) H3122 cells (D) or EBC‐1 cells (E) were treated with TAE684 (1 μM) or SGX‐523 (1 μM) for 12 h and incubated with growth factors for additional 30 min followed by western blotting analysis with indicated antibodies.

Article Snippet: DNA plasmids EGF cDNA (SC127840) and TGF‐α cDNA (SC311226) were obtained from Origene (Rockville, MD).

Techniques: MTT Assay, Incubation, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Autocrine EGF and TGF ‐α promote primary and acquired resistance to ALK / c‐Met kinase inhibitors in non‐small‐cell lung cancer

doi: 10.1002/prp2.1047

Figure Lengend Snippet: Increased autocrine EGF and TGF‐α activated EGFR pathway in acquired resistant H3122/TR and EBC‐1/SR cells. (A, B) Sensitive (H3122, EBC‐1) and resistant (H3122/TR, EBC‐1/SR) cells were treated with different concentrations of TAE684 (A) or SGX‐523 (B) for 72 h and cell viability was assessed by MTT assay. Data are shown as mean ± SD from three independent experiments. (C, D) Sensitive and resistant cells were treated with TAE684 (1 μM) (C) or SGX‐523 (1 μM) (D) for 3 h and the whole cell lysates was subjected to western blotting with indicated antibodies. (E, F) ELISA of HGF, EGF and TGF‐α secretion by sensitive or resistant cells. Data are shown as mean ± SD from three independent experiments. * p < .05, ** p < .01, *** p < .001. (G, H) Western blotting analysis of protein phosphorylation in sensitive and resistant cells with indicated antibodies. Results of three independent experiments are shown.

Article Snippet: DNA plasmids EGF cDNA (SC127840) and TGF‐α cDNA (SC311226) were obtained from Origene (Rockville, MD).

Techniques: MTT Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: Pharmacology Research & Perspectives

Article Title: Autocrine EGF and TGF ‐α promote primary and acquired resistance to ALK / c‐Met kinase inhibitors in non‐small‐cell lung cancer

doi: 10.1002/prp2.1047

Figure Lengend Snippet: High expression of EGF and TGF‐α promoted ALK/c‐Met TKI resistance in vitro and in clinical samples. (A) ELISA of EGF and TGF‐α secretion by NSCLC cells transfected with EGF (H3122/EGF, EBC‐1/EGF), TGF‐α (H3122/TGF‐α, EBC‐1/TGF‐α) or an empty vector (H3122/Vec, EBC‐1/Vec). Data are means ± SD from three independent experiments. * p < .05, ** p < .01. (B, C) H3122 cells (B) or EBC‐1 cells (C) transfected with EGF or TGF‐α were treated with different concentrations of TAE684 or SGX‐523 for 72 h and cell viability was assessed by MTT assay. Data are means ± SD from three independent experiments. (D) Characteristics of NSCLC patients and their response to crizotinib treatment. (E) Immunohistochemistry analysis of EGF and TGF‐α expression in specimens taken from NSCLC patients before crizotinib treatment. Images taken at 20 × magnification (scale bar −50 μm).

Article Snippet: DNA plasmids EGF cDNA (SC127840) and TGF‐α cDNA (SC311226) were obtained from Origene (Rockville, MD).

Techniques: Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, MTT Assay, Immunohistochemistry

Journal: Pharmacology Research & Perspectives

Article Title: Autocrine EGF and TGF ‐α promote primary and acquired resistance to ALK / c‐Met kinase inhibitors in non‐small‐cell lung cancer

doi: 10.1002/prp2.1047

Figure Lengend Snippet: Combination of ALK/c‐Met inhibitor with EGFR inhibitor circumvented EGF‐ and TGF‐α‐driven primary resistance. (A, B) H3122 cells treated with EGF (100 ng/ml) (A) or TGF‐α (100 ng/ml) (B) were incubated with different concentrations of TAE684 alone or in combination with gefitinib (1 μM) for 72 h and cell proliferation was detected by MTT assay. Data are shown as mean ± SD from three independent experiments. (C, D) EBC‐1 cells treated with EGF (100 ng/ml) (C) or TGF‐α (100 ng/ml) (D) were incubated with different concentrations of SGX‐523 alone or in combination with gefitinib (1 μM) for 72 h and cell proliferation was detected by MTT assay. Data are shown as mean ± SD from three independent experiments. (E, F) H3122 cells (E) or EBC‐1 cells (F) were treated with TKIs (1 μM) alone or in combination for 12 h and incubated with EGF (100 ng/ml) or TGF‐α (100 ng/ml) for additional 30 min followed by western blotting analysis with indicated antibodies. Results of three independent experiments are shown.

Article Snippet: DNA plasmids EGF cDNA (SC127840) and TGF‐α cDNA (SC311226) were obtained from Origene (Rockville, MD).

Techniques: Incubation, MTT Assay, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Autocrine EGF and TGF ‐α promote primary and acquired resistance to ALK / c‐Met kinase inhibitors in non‐small‐cell lung cancer

doi: 10.1002/prp2.1047

Figure Lengend Snippet: Combination treatment of c‐Met/ALK inhibitor and EGFR inhibitor circumvented EGF‐ and TGF‐α‐mediated acquired resistance. (A, B) H3122/TR cells (A) or EBC‐1/SR cells (B) were treated with TKIs (1 μM) alone or in combination for 72 h and cell proliferation was detected by MTT assay. Data are shown as mean ± SD from three independent experiments. ** p < .01. (C, D) H3122/TR cells (C) or EBC‐1/SR cells (D) were treated with TKIs (1 μM) alone or in combination for 3 h and the whole cell lysates was detected by western blotting with indicated antibodies. Results of three independent experiments are shown. (E) Graphical summary of autocrine EGF and TGF‐α promoting primary and acquired resistance to ALK−/c‐Met‐targeted TKI by activation of EGFR signal pathway in non‐small cell lung cancer.

Article Snippet: DNA plasmids EGF cDNA (SC127840) and TGF‐α cDNA (SC311226) were obtained from Origene (Rockville, MD).

Techniques: MTT Assay, Western Blot, Activation Assay