Journal: The Journal of Experimental Medicine

Article Title: Phenotype, Localization, and Mechanism of Suppression of CD4 + CD25 + Human Thymocytes

doi: 10.1084/jem.20020110

Figure Lengend Snippet: Cell contact–dependent suppressive activity of CD4 + CD25 + human thymocytes is abrogated by the combined action of anti–CTLA-4 and anti-TGFβ1 mAbs. (A) CD4 + CD25 − thymocytes were stimulated with irradiated T cell–depleted allogeneic PBMNCs in the lower chamber of a transwell plate in the absence or presence of CD4 + CD25 + thymocytes, placed in the same or in the top chamber. On day 5, cells present in the bottom chamber were harvested and their proliferation assessed by measuring 3 [H]TdR uptake. (B) CD4 + CD25 − thymocytes were stimulated with allogeneic irradiated T cell–depleted PBMNCs in the presence of different numbers of autologous purified CD4 + CD25 + thymocytes without (black columns) or with isotype control (IgG1+IgG2a) (gray columns), anti-CTLA-4 (hatched columns), or anti–TGF-β1 mAb (dotted columns), or mixtures of them (white columns). Cell proliferation was measured by assessment of 3 [H]TdR uptake. Mean values (±SD) obtained in four separate experiments are reported.

Article Snippet: The PE-conjugated anti-TNFR2 (22235.311, mouse IgG2a), the purified anti–TGF-β1 (9016.2, mouse IgG1) neutralizing mAb, TGF-βR2 (goat IgG) neutralizing Ab, and biotin-conjugated anti–TGF-β1 (chicken IgY) and anti-LAP (TGF-β1) (goat IgG) Abs were purchased from R&D Systems.

Techniques: Activity Assay, Irradiation, Purification, Control

Journal: The Journal of Experimental Medicine

Article Title: Phenotype, Localization, and Mechanism of Suppression of CD4 + CD25 + Human Thymocytes

doi: 10.1084/jem.20020110

Figure Lengend Snippet: CTLA-4 and TGF-β1–mediated suppression is due to the inhibition of IL-2R α-chain expression and is overcome by IL-15. (A) CFSE-labeled CD4 + CD25 − thymocytes were stimulated with irradiated allogeneic PB non-T cells in absence or presence of autologous CD4 + CD25 + thymocytes with anti–CTLA-4, anti–TGF-β1, anti–CTLA-4 plus anti–TGF-β1 or isotype-matched (IgG1 plus IgG2a) mAbs, and assessed by flow cytometry for proliferation and surface CD25 expression. One representative of three separate experiments is shown. (B) CD25 (left) and CD69 (right) expression by CD4 + CD25 − thymocytes cultured under the experimental conditions mentioned in (A). CD4 + CD25 − thymocytes without mAb added (black columns), or containing isotype control (gray columns), anti–TGF-β1 (dotted columns), anti–CTLA-4 (hatched columns), or anti–CTLA-4 plus anti–TGF-β1 (white columns) mAbs. Columns represent mean values (±SD) found in three separate experiments. (C) Effect of the addition of exogenous IL-2 or IL-15 on the proliferative response of CD4 + CD25 − thymocytes stimulated with irradiated allogeneic non-T cells in absence or presence of CD4 + CD25 + autologous thymocytes. Columns represent mean values (±SD) obtained in three separate experiments.

Article Snippet: The PE-conjugated anti-TNFR2 (22235.311, mouse IgG2a), the purified anti–TGF-β1 (9016.2, mouse IgG1) neutralizing mAb, TGF-βR2 (goat IgG) neutralizing Ab, and biotin-conjugated anti–TGF-β1 (chicken IgY) and anti-LAP (TGF-β1) (goat IgG) Abs were purchased from R&D Systems.

Techniques: Inhibition, Expressing, Labeling, Irradiation, Flow Cytometry, Cell Culture, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: Analysis of ROS intracellular amounts and FHC antioxidant protein levels in OVCAR3 and OVCAR8 cells. (a) Immunofluorescence analysis of ROS levels in OVCAR3 and OVCAR8 cells by staining with CellROX® Green Reagent (green). Nuclei were stained with DAPI (blue). Analysis was performed in duplicate and representative images are reported. (b) Immunofluorescence analysis of superoxide radical levels in OVCAR3 and OVCAR8 cells by staining with MitoSOX™ Red Indicator (red). Nuclei were stained with DAPI (blue). Analysis was performed in duplicate and representative images are reported. (c) Representative western blot of antioxidant protein FHC in OVCAR3 and OVCAR8 cells. γ -Tubulin was used as internal control. WB has been quantified by using ImageJ software and optical densitometry is reported. WB analysis was performed three times and results were reproducible.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Western Blot, Control, Software

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: OVCAR3 cells exhibit increased growth potential compared to OVCAR8 cells. (a) Cell cycle FACS analysis of OVCAR3 and OVCAR8 cells stained with PI. The experiments were performed in triplicate. Representative plots of a single experiment (left); histograms showing the mean ± SD of three independent experiments (right). ∗ p value < 0.05, OVCAR3 vs . OVCAR8. (b) MTT analysis of OVCAR3 and OVCAR8 cell growth at 12 h, 24 h, 48 h, and 72 h. Data are reported as absorbance measured at 595 nm and shown as mean ± SD of three independent replicates ( ∗ p < 0.05, OVCAR3 vs . OVCAR8); (° p < 0.01, OVCAR3 vs . OVCAR8); N.S.: not significant. (c) Representative WB of c-Myc, cyclin E1 (CCNE1), pChk2 (Thr68), pERK1/2, and pAKT in OVCAR3 and OVCAR8 cells. γ -Tubulin was used as internal control. WB analysis was performed three times and results were reproducible.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Staining, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: OVCAR8 cells are characterized by ROS accumulation and FHC downregulation upon 6 μ M cisplatin treatment. (a) Cell viability assay performed by MTT analysis in OVCAR3 and OVCAR8 cells treated with 6 μ M, 12 μ M, 24 μ M and 48 μ M of cisplatin for 24 h. Cisplatin concentrations are reported as log [cisplatin ( μ M)]. Cell viability is expressed as percentage (%). Treatments were performed at least three times on independent biological replicates and the mean concentration of the drug that gives half-maximal response (log EC 50 ) was used to compare cytotoxicity. (b) Quantification of ROS amounts through DCFDA staining in OVCAR3 and OVCAR8 untreated (NT) and upon treatment with 6 μ M, 12 μ M, 24 μ M and 48 μ M cisplatin for 24 h. Data represent the mean ± SD of three biological replicates. ∗ p value < 0.01 OVCAR3 NT vs . OVCAR8 NT; ° p value < 0.01 OVCAR3 6 μ M cisplatin vs . OVCAR8 6 μ M cisplatin; ∗∗ p value < 0.05 OVCAR3 12 μ M cisplatin vs . OVCAR8 12 μ M cisplatin; °° p value < 0.05 OVCAR3 24 μ M cisplatin vs . OVCAR8 24 μ M cisplatin; N.S.: not significant: OVCAR3 48 μ M cisplatin vs . OVCAR8 48 μ M cisplatin. (c) Representative western blot of FHC, cleaved caspase 3, and caspase 3 in OVCAR3 and OVCAR8 untreated (NT) and upon treatment with 6 μ M cisplatin for 24h. γ -Tub was used as internal control. WB analysis was performed three times and results were reproducible. (d) Immunofluorescence analysis of ROS levels in untreated (NT) and treated with 6 μ M cisplatin OVCAR3 and OVCAR8 cells by staining with CellROX® Green Reagent (green). Nuclei were stained with DAPI (blue). Analysis was performed in duplicate and representative images are reported. (e) Immunofluorescence analysis of superoxide radical levels untreated (NT) and treated with 6 μ M cisplatin OVCAR3 and OVCAR8 cells by staining with MitoSOX™ Red Indicator (red). Nuclei were stained with DAPI (blue). Analysis was performed in duplicate and representative images are reported.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Viability Assay, Concentration Assay, Staining, Western Blot, Control, Immunofluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: FHC knockdown improves OVCAR3 response to 6 μ M cisplatin by increasing ROS production. (a) Representative plots of Annexin V/7-AAD apoptosis assays in OVCAR3 Neg Control , OVCAR3 Neg Control (6 μ M) cisplatin, OVCAR3 Neg Control 10 mM NAC, OVCAR3 Neg Control (6 μ M) cisplatin/10 mM NAC, OVCAR3 siFHC , and OVCAR3 siFHC (6 μ M) cisplatin. Cisplatin treatment was performed for 24 h while NAC treatment was performed for 2 h. FACS plots are representative of single experiments. Values are expressed as mean ± SD of three biological replicates. (b) Immunofluorescence analysis of ROS levels in OVCAR3 Neg Control , OVCAR3 Neg Control (6 μ M) cisplatin, OVCAR3 Neg Control 10 mM NAC OVCAR3 Neg Control (6 μ M) cisplatin/10 mM NAC, OVCAR3 siFHC , and OVCAR3 siFHC (6 μ M) cisplatin, by staining with CellROX® Green Reagent (green). Nuclei were stained with DAPI (blue). (c) Representative western blot of FHC in OVCAR3 Neg Control , OVCAR3 Neg Control (6 μ M) cisplatin, OVCAR3 Neg Control 10 mM NAC, OVCAR3 Neg Control (6 μ M) cisplatin/10 mM NAC, OVCAR3 siFHC , and OVCAR3 siFHC (6 μ M) cisplatin. γ -Tubulin was used as internal control. WB analysis was performed three times and results were reproducible.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Knockdown, Control, Immunofluorescence, Staining, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: FHC overexpression or NAC treatment reduces OVCAR8 response to cisplatin. (a) Representative plots of Annexin V/7-AAD apoptosis assays in OVCAR8 pc3DNA , OVCAR8 pc3DNA (6 μ M) cisplatin, OVCAR8 pc3DNA (6 μ M) cisplatin/10 mM NAC, and OVCAR8 pc3FHC (6 μ M) cisplatin. Cisplatin treatment was performed for 24 h while NAC treatment was performed for 2 h. FACS plots are representative of single experiments. Values are expressed as mean ± SD of three biological replicates. (b) Immunofluorescence analysis of ROS levels in OVCAR8 pc3DNA , OVCAR8 pc3DNA (6 μ M) cisplatin, OVCAR8 pc3DNA (6 μ M) cisplatin/10 mM NAC, and OVCAR8 pc3FHC (6 μ M) cisplatin, by staining with CellROX® Green Reagent (green). Nuclei were stained with DAPI (blue). (c) Representative western blot of FHC in OVCAR8 pc3DNA , OVCAR8 pc3DNA (6 μ M) cisplatin, OVCAR8 pc3DNA (6 μ M) cisplatin/10 mM NAC, and OVCAR8 pc3FHC (6 μ M) cisplatin. γ -Tubulin was used as internal control. WB analysis was performed three times and results were reproducible.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Over Expression, Immunofluorescence, Staining, Western Blot, Control

Journal: Scientific Reports

Article Title: Roles of GalNAc-disialyl Lactotetraosyl Antigens in Renal Cancer Cells

doi: 10.1038/s41598-018-25521-6

Figure Lengend Snippet: Integrin-ILK-Akt signaling was enhanced in GalNAc-DSLc4-expressing cells. ( A ) Phosphorylation of Akt during treatment with FCS in control cells and GalNAc-DSLc4 expressing cells was examined. Cells were prepared as described in “Materials and Methods”, and cell suspension (4 × 10 5 cells) were added to plates, and incubated for 0, 10, 30, 60, or 120 min. After incubation, cells were lysed and used for immunoblotting using anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), or anti-total Akt antibodies. Bands in autofluorograms ( a ) were quantified by a scanner, and the relative intensities of the bands were plotted after correction with total Akt bands ( b and c ). ( B ) Phosphorylation of Akt during adhesion to LN in GalNAc-DSLc4-expressing cells was examined. Cells (4 × 10 5 ) were added to pre-coated plates with LN, and incubated for 0, 15, 30, 60, or 120 min. After incubation, cells were lysed and used for immunoblotting using anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), or anti-total Akt antibodies. Bands in autofluorograms ( a ) were quantified by a scanner, and the relative intensities of the bands were plotted after correction with total Akt bands ( b and c ). Bars indicate mean ± S.D. (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.005. All cropped blots were run under the same experimental condition. The full-length blots are included in Supplemental Figure respectively.

Article Snippet: Goat polyclonal anti-integrin β1 antibody and mouse monoclonal anti-integrin α3 were purchased from Santa cruz biotechnology, Inc.

Techniques: Expressing, Phospho-proteomics, Control, Suspension, Incubation, Western Blot

Journal: Scientific Reports

Article Title: Roles of GalNAc-disialyl Lactotetraosyl Antigens in Renal Cancer Cells

doi: 10.1038/s41598-018-25521-6

Figure Lengend Snippet: Intracellular localization of integrin β1 and caveolin-1 during FCS stimulation. ( A ) Integrin β1 was found in GEM/rafts during adhesion to LN-coated surface in the transfectants. Cells were lysed after incubation for 15 min at 37 °C, and separated by sucrose density gradient ultracentrifugation, and used for immunoblotting using anti-integrin β1, anti-EGFR, anti-cMet, or anti-caveolin-1 antibodies. Fractions 1–4 contained low density fractions and fractions 6–10 corresponded to high density fractions. These were representative results among experiments repeated at least 3 times with similar results. All cropped blots were run under the same experimental condition. The full-length blots are presented in Supplemental Figure . ( B ) The clones were immunocytostained with anti-integrin β1, anti-caveolin-1, and RM2 antibodies during FCS stimulation and adhesion onto LN. Cells were rotated under serum-free conditions for 30 min, and stimulated with 10% FCS. After incubation for 0, 5, 15, 60 min, cells were fixed in 4% paraformaldehyde for 10 min. Then, cells were stained for integrin β1 (ITGB1, green ) and caveolin-1 ( red ), and their images were observed using a confocal microscope (Fluoview FV10i-DOC). DIC , image of differential interference contrast microscope. Scale bar indicate 10 μm. ( C ) Ratio of co-localization of integrin β1/caveolin-1. The number of dots ( red ) stained with anti-caveolin-1 antibody and the number of merged dots ( yellow ) stained with anti-integrin β1/caveolin-1 antibodies were counted, and percentage was obtained. The column represent mean ± S.D. ( n = 10). * P < 0.01. ( D ) Mean of diameter of co-localized integrin β1/caveolin-1 dot areas. The diameters of merged dots ( yellow ) were measured, and the average was obtained. Values are means ± S.D. ( n = 10). * P < 0.05, **** P < 0.0005. ( E ) Size distribution of lipid/rafts in control cells ( a ) and transfectant cells ( b ). Size distribution of merged dot area in control cells ( c ) and transfectant cells ( d ). The over 250 vesicular profiles were measured. Blue : < 0.025 μm, red : 0.26–0.50 μm, green : 0.51–0.75 μm, purple : 0.76–1.0 μm, light blue : 1.1–2.5 μm. In all experiments, it was confirmed that there was no cross-reaction between the individual antigens and non-relevant second reagents.

Article Snippet: Goat polyclonal anti-integrin β1 antibody and mouse monoclonal anti-integrin α3 were purchased from Santa cruz biotechnology, Inc.

Techniques: Incubation, Western Blot, Clone Assay, Staining, Microscopy, Control, Transfection

Journal: Scientific Reports

Article Title: Roles of GalNAc-disialyl Lactotetraosyl Antigens in Renal Cancer Cells

doi: 10.1038/s41598-018-25521-6

Figure Lengend Snippet: mAb RM2 suppressed malignant properties of the transfectant cells. ( A ) To investigate the effects of mAb RM2 on the cell growth of the transfectant cells and control cells, cells (2.5 × 10 3 cells/well) were prepared in 48-well plates in serum-containing D-MEM and cultured for 5 days with mAb RM2-containing D-MEM. MTT assay was performed as described in “Materials and Methods” ( a ). The absorbance (590 nm) was measured on day 5. Data are means of three independent experiments. In a same manner, the effects of mAb RM2 on 4 RCC lines (OS-RC-2, RCC10RGB, VMRC-RCW, and SK-RC-29) were examined ( b ). Surface expression of GalNAc-DSLc4 on these cell lines was analyzed by flow cytometry using mAb RM2, and MTT assay with mAb RM2-containing D-MEM was performed as described above. Black profiles mean GalNAc-DSLc4 expressing cells, and light gray profiles mean negative control cells in flow cytometric assay. Data of MTT assay are means of three independent experiments. ( B ) The effect of mAb RM2 on invasiveness was investigated the transfectant cells or control cells using in vitro invasion assay ( a ). In same manner, effects of mAb RM2 on 2 RCC lines (OS-RC-2 and SK-RC-29) were examined ( b ). Bars indicate mean ± S.D. (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.005. ( C ) RT-CES of cells treated with antibodies. Suppression of adhesion to LN-coated plates by anti-integrin Abs or mAb RM2. Transfectant cells and control cells were seeded in the wells of 96-well e-plates at 2.5 × 10 4 cells/well with FCS. RT-CES was performed as described in Fig. . Red and yellow lines mean transfectant cells, and green and blue lines mean control cells.

Article Snippet: Goat polyclonal anti-integrin β1 antibody and mouse monoclonal anti-integrin α3 were purchased from Santa cruz biotechnology, Inc.

Techniques: Transfection, Control, Cell Culture, MTT Assay, Expressing, Flow Cytometry, Negative Control, In Vitro, Invasion Assay