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: Scientific reports

Article Title: Polyphenols journey through blood-brain barrier towards neuronal protection.

doi: 10.1038/s41598-017-11512-6

Figure Lengend Snippet: Figure 4. Effects on neuroinflammation by Cat-sulf and Pyr-sulf. Pro-inflammatory markers were evaluated, namely (a) TNF-α release, (b) intracellular superoxide production, (c) nitric oxide, and (d) CD40 quantified in N9 microglial cells. Cells were pre-incubated for 6 h with each of the bioavailable (poly)phenol metabolite and then challenged with 300ng/mL of LPS. Statistical differences are denoted as ***p < 0.001, **p < 0.01 and *p < 0.05 relatively to lesion (LPS). (e) Microglial NF-κB p65 translocation into the nucleus after 60 minutes of LPS stimulation. Cells were pre-treated with Cat-sulf or Pyr-sulf for 6 h before LPS-stimulation. NF-κB (red); Nuclei (blue) stained with DAPI. Each capture is representative of at least 3 independent biological replicates. Scale bar: 10 µm. (f–i) Microglial NF-κB p65 phosphorylation ratio and IκBα fold change in protein levels. (f) IkBα protein levels along time after LPS stimulation and (g) after 60 min of LPS stimulation with representative western blots. (h) NF-κB activation profile along time after LPS stimulation looking at NF-κB p65 phosphorylation (ser536) ratio along time after LPS stimulation and (i)after 60 min of LPS stimulation with representative western blots. Cells were pre-treated either with Pyr-sulf or Cat-sulf before LPS stimulation. Control cells (white triangles, solid line), LPS-stimulated cells (black triangles, solid line), cells treated with Cat-sulf prior to LPS stimulation (black circles, dashed line), cells treated with Pyr-sulf prior to LPS stimulation (black squares, dotted line). Statistical differences are denoted as *p < 0.05 and **p < 0.01 relatively to lesion (LPS). Western blots were analyzed under the same experimental conditions. Data are presented as the means ± SD, n = 3.

Article Snippet: Briefly, HBMEC coverslips were incubated overnight at 4 °C with primary antibodies anti-P-gp (1:50, Calbiochem), anti-MRP1 (1:100, Millipore) and anti-BCRP (1:100, Millipore) and N9 cells coverslips were incubated overnight at 4 °C with rabbit polyclonal anti-NF-κB p65 (C-20) (1:200, Santa Cruz Biotechnology).

Techniques: Incubation, Translocation Assay, Staining, Phospho-proteomics, Western Blot, Activation Assay, Control