Proteasome inhibition or knock-down of E6-AP increases the levels of pro-IL-1β in immortalized E6-positive cells.

<p>A) ELISA of intracellular IL-1β from untreated immortalized HPV-positive cells and cells incubated with 10 µM of the proteasome inhibitor MG132 for 6 h. B) Western blot analysis of pro-IL1β and p53 in immortalized keratinocytes after inhibition of deubiquitinases using PR-619 (10 µM) for 6 h prior to protein extraction. C) Detection of poly-ubiquitinated pro-IL-1β in immortalized keratinocytes by Western blotting. Cells were treated with MG132 for 6 h prior to protein extraction and pull-down of ubiquitinated proteins was performed by the tandem ubiquitin-binding entities technique (TUBE-PD, right panel). Left panel: input, representing 2.5% of the total protein extract. D) Confocal microscopy analysis of IL-1β (green) in immE6 cells after knock-down of E6-AP by siRNA or scrambled siRNA delivery used as a control. Nuclei (blue) were stained using Hoechst dye solution; scale bars represent 10 µm. E) Western blot analysis of pro-IL-1β after the knock-down of E6-AP. F) ELISA of intracellular IL-1β from immortalized HPV-positive cells after the knock-down of E6-AP by siRNA (+) or control knock-down.(−) G) ELISA of IL-1β secretion from immortalized HPV-positive cells after E6-AP knock-down and/or after NALP3 inflammasome activation using 50 µM of nigericine for 6 h. H) Knock-down of p53 and/or E6-AP in immortalized cells. Cells were transfected with 30 pmoles of the respective siRNA against E6-AP or p53 and incubated for 72 h prior to protein extraction and Western blot analysis. The bar graphs shown in A (ANOVA ***p<0.05), D (ANOVA ***p<0.001, **p<0.01) F and G (ANOVA ***p<0.001) represent the mean levels (± SEM) of three independent experiments.</p

Caspase-1 activity did not account for decreased IL-1β in E6-positive cells.

<p>A) RT-PCR analysis of caspase-1 mRNA in comparison to the GAPDH steady state levels in PK and HPV-positive immortalized keratinocytes. B) Fluorometric measurement of caspase-1 activity was performed incubating the cells for 4 h at 37°C with 20 µM of the specific caspase-1 substrate R110-YVAD. RFU: Relative fluorescence units. C) Quantification of intracellular IL-1β levels by ELISA after 5 h of caspase-1 inhibition using 250 nM of caspase-1 inhibitor. The graphs in B and C represent the mean values (± SEM) of three independent experiments. ANOVA **p<0.01, ***p<0.001.</p

Expression and secretion of IL-1β in human primary keratinocytes and HPV-positive cell lines.

<p>A) Quantification of secreted IL-1β (expressed as pg/ml) by ELISA in human primary keratinocytes (PK), keratinocytes immortalized by individual oncogenes (E6, E7, E6/E7) and HPV16/18-positive cervical carcinoma cell lines (SiHa, CaSki, HeLa, respectively) 24 h after infection with a recombinant GFP-expressing adenovirus 5 (Ad) in comparison to uninfected cells. B) Quantification of basal intracellular IL-1β levels by ELISA. C) Western blot analysis of pro-IL-1β in human primary keratinocytes (PK) and HPV-positive cells. Actin was used as a loading control. D) qPCR analysis of IL-1β cDNA obtained from PK and HPV-positive cells (Ordinate: expressed as fold changes using the average IL-1β steady state level of 3 different primary human keratinocyte preparations PK cells as a reference which was arbitrarily set as 1. The graphs in A, B and D represent the mean values (± SEM) of three independent experiments. ANOVA ***p<0.05.</p

Analysis of IL-1β expression in normal and HPV16-positive cervical tissues.

<p>A) Immunohistochemical analysis of IL-1β expression in normal epithelium and HPV-positive lesions differing in their progression grades CIN I to CIN III and cervical tumors; scale bars represent 25 µm. B) qPCR analysis of IL-1β cDNA derived from samples negative for intraepithelial lesion and malignancy (NT), different HPV-positive lesions (CIN I, II, III) and cervical tumors (CxCa); ordinate: expression as fold changes using SiHa cells as reference which was arbitrarily set as 1. The pictures in A are a representative example of 25 biopsies analyzed from normal tissue (n = 5) and HPV-positive lesions of different donors (n = 25). The box-and-whisker blot in B represents the mean values from three to five samples for each group depicted in the graph (± SEM). ANOVA *p<0.01.</p

Additional file 5: Figure S2. of Systems-level effects of ectopic galectin-7 reconstitution in cervical cancer and its microenvironment

Transcriptional analysis of the genes identified by the integrative analysis in Gal-7+ CxCa cells. (PDF 343 kb

Additional file 6: Figure S3. of Systems-level effects of ectopic galectin-7 reconstitution in cervical cancer and its microenvironment

Gene mapping of the mouse microenvironment and immune cells in accordance with The Immunological Genome Project. (PDF 263 kb

Glaucidium palmatum Sieb. et Zucc.

原著和名: シラネアフヒ科名: キンポウゲ科 = Ranunculaceae採集地: 青森県 下北郡 宇曽利山 (陸奥 下北郡 宇曽利山)採集日: 1966/6/18採集者: 萩庭丈壽整理番号: JH001900国立科学博物館整理番号: TNS-VS-95190