Journal: PLoS ONE

Article Title: Epidermal Growth Factor Protects Squamous Cell Carcinoma against Cisplatin-Induced Cytotoxicity through Increased Interleukin-1β Expression

doi: 10.1371/journal.pone.0055795

Figure Lengend Snippet: ( A ) A431 cells were treated with various concentration of LY294002 as indicated for 1 h and followed by treated with 50 ng/ml EGF for 10 min (lysates) or 3 h (RNA). Lysates and RNA were prepared for examining the phosphorylation of Akt on serine 473 and IL-1βmRNA, respectively by using Western blot analysis and RT-PCR. ( B and D ) A431 cells were treated with various concentrations of LY294002 or parthenolide as indicated for 1 h, and followed by treated with 50 ng/ml EGF for 3 h. The expression of IL-1β was analyzed by Real-time PCR. The induction fold of EGF-induced IL-1β expression was normalized by GAPDH. ( C ) A431 cells were treated with various concentrations of parthenolide as indicated for 1 h and followed by treated with 50 ng/ml EGF for 1 h (nuclear extracts) or 3 h (RNA). Nuclear extracts and RNA were prepared for examining the nuclear translocation of NF-κB and IL-1βmRNA, respectively by using Western blot analysis and RT-PCR. Values represent means ± S.E. of three determinations. n.s.: no significant difference. ***: P<0.005; **: P< 0.01; *: P<0.05.

Article Snippet: Antibodies against Akt, Phospho-Akt Ser473 (Cell Signaling techology®), NF-κB p65 C-20 (sc-372) and Ku70 (sc-12729) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), were used as the primary antibodies.

Techniques: Concentration Assay, Phospho-proteomics, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Translocation Assay

Journal: PLoS ONE

Article Title: Epidermal Growth Factor Protects Squamous Cell Carcinoma against Cisplatin-Induced Cytotoxicity through Increased Interleukin-1β Expression

doi: 10.1371/journal.pone.0055795

Figure Lengend Snippet: ( A ) The construct of pTK promoter with 5 repeated NF-κB biding sites bearing luciferase gene was presented. ( B and C ) SCC4 and SCC-25 cells were transfected with 0.5 µg pTK-NFκB promoter, 1 µg DN-IκB expression vector and 1 µg control vector by lipofection. The luciferase activities and protein concentrations were then determined and normalized. ( D and E ) SCC4 and SCC-25 cells were transfected with 1 µg dominant negative IκB (DN-IκB) expression vector or 1 µg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h before the extraction of RNA. The expression of IL-1β, IκB and GAPDH mRNA was analyzed by RT-PCR and examined in 2% agarose gel.

Article Snippet: Antibodies against Akt, Phospho-Akt Ser473 (Cell Signaling techology®), NF-κB p65 C-20 (sc-372) and Ku70 (sc-12729) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), were used as the primary antibodies.

Techniques: Construct, Luciferase, Transfection, Expressing, Plasmid Preparation, Control, Dominant Negative Mutation, Extraction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: PLoS ONE

Article Title: Epidermal Growth Factor Protects Squamous Cell Carcinoma against Cisplatin-Induced Cytotoxicity through Increased Interleukin-1β Expression

doi: 10.1371/journal.pone.0055795

Figure Lengend Snippet: A431 cells were treated with 20 µM LY294002 and 20 µM parthenolide for 1 h and followed by treating with 50 ng/ml EGF for 1 h. Anti-p65 antibodies and DAPI were used for staining the nuclear translocation of NF-κB (p65) and DNA, respectively in immunofluorescence analysis.

Article Snippet: Antibodies against Akt, Phospho-Akt Ser473 (Cell Signaling techology®), NF-κB p65 C-20 (sc-372) and Ku70 (sc-12729) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), were used as the primary antibodies.

Techniques: Staining, Translocation Assay, Immunofluorescence

Journal: PLoS ONE

Article Title: Epidermal Growth Factor Protects Squamous Cell Carcinoma against Cisplatin-Induced Cytotoxicity through Increased Interleukin-1β Expression

doi: 10.1371/journal.pone.0055795

Figure Lengend Snippet: ( A ) Constructs of IL-1β promoter or mutation sequence of NF-κB binding site bearing luciferase gene were presented. ( B and C ) Nuclear extracts (NE) from A431 cells were treated with 20 µM LY294002 (LY) and 20 µM parthenolide (PTL) for 1 h, and followed by treating with 50 ng/ml EGF for 1 h. DNA affinity precipitation assay was performed as described under “ ”. Binding of NF-κB p65 to wild-type probes, wt was analyzed by Western blot. The mutant sequence, NF-κB mut was used to serve as a negative control for wild-type sequence. ( D ) Cells were transfected with 0.5 µg IL-1β promoter construct by lipofection and then treated with 20 µM LY294002 (LY) and 20 µM parthenolide (PTL) for 1 h, and followed by treating with 50 ng/ml EGF for 6 h. The luciferase activities and protein concentrations were then determined and normalized. ( E ) Cells were transfected with 0.5 µg wild type (wt) or mutant (NF-κB mut) IL-1β promoter construct by lipofection and then treated with 50 ng/ml EGF for 6 h. The luciferase activities and protein concentrations were then determined and normalized. Values of luciferase activities are means ± S.E. of three determinations. ***: P<0.005; n.s.: no significant difference; Ctrl: control.

Article Snippet: Antibodies against Akt, Phospho-Akt Ser473 (Cell Signaling techology®), NF-κB p65 C-20 (sc-372) and Ku70 (sc-12729) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), were used as the primary antibodies.

Techniques: Construct, Mutagenesis, Sequencing, Binding Assay, Luciferase, Affinity Precipitation, Western Blot, Negative Control, Transfection, Control

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: A). The percentage of CD4 + /CD25 + /FoxP3 + cells in PHA-activated PBMCs after stimulation with increasing concentrations of NGAL: Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml), raised in a dose dependent manner. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of CD25 + /FoxP3 + cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4 + /CD25 + /FoxP3 + cells. Means ± SD; n = 8. *p<0.05.

Article Snippet: After permeabilization, cells were incubated for 30 minutes in the dark in the refrigerator (2–8°C) with an anti-FoxP3-PE antibody (Miltenyi, Bergish Gladbach, Germany).

Techniques:

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of CD25 + /FoxP3 + expression on CD4 + cells in PHA-activated PBMCs. Iron treatment (without NGAL) did not increase the percentage of CD25 + /FoxP3 + cells. Treatment with NGAL:enterobactin complex (without iron) reduced the percentage of CD25 + /FoxP3 + cells in contrast to stimulation with NGAL:Enterobactin:Iron. Incubation with anti-NGAL antibody reduced the percentage of CD25 + /FoxP3 + cells. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of CD25+foxP3 cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4+/CD25+/FoxP3+ cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: After permeabilization, cells were incubated for 30 minutes in the dark in the refrigerator (2–8°C) with an anti-FoxP3-PE antibody (Miltenyi, Bergish Gladbach, Germany).

Techniques: Flow Cytometry, Expressing, Incubation

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of HLA-G + /FoxP3 + expression on CD4 + cell PHA-activated PBMCs. PBMCs were treated with increasing concentrations of NGAL:Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml). A dose-dependent raise in the percentage of HLA-G + /FoxP3 + cells was evident. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of HLA-G + /FoxP3 + cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4 + /HLA-G + /FoxP3 + cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: After permeabilization, cells were incubated for 30 minutes in the dark in the refrigerator (2–8°C) with an anti-FoxP3-PE antibody (Miltenyi, Bergish Gladbach, Germany).

Techniques: Flow Cytometry, Expressing