Journal: Experimental and therapeutic medicine

Article Title: Oxidized low-density lipoprotein-induced p62/SQSTM1 accumulation in THP-1-derived macrophages promotes IL-18 secretion and cell death.

doi: 10.3892/etm.2017.5221

Figure Lengend Snippet: Figure 3. Silencing p62 reduces IL‑18 secretion and promotes cell survival of macrophages with prolonged oxLDL exposure. (A) THP‑M cells treated with oxLDL (20 µg/ml) for the indicated time following transfection with 50 nM con siRNA or p62 siRNA. Western blotting experiments were performed on total protein extracts using anti‑p62 antibody, and β‑actin expression was used as a loading control. The graph represents the values of p62 band intensity after normalization to β‑actin by densitometry. *P<0.05 vs. 24 h group. (B) The graph represents values of the SQSTM1 mRNA expression following normalization for β‑actin mRNA by reverse transcription‑quantitative polymerase chain reaction. *P<0.05 vs. 24 h group. (C) ELISA of secreted IL‑18 in the media of cells treated with con or p62 siRNA and 20 µg/ml oxLDL for 0, 12, 24, 48 and 72 h. *P<0.05 and **P<0.01 vs. 0 h; ##P<0.01 as indicated. (D) THP‑M cells were pretreated with con or p62 siRNA and then treated with 20 µg/ml oxLDL for the indicated times. Cell viability was analyzed by the MTT assay. Results are expressed as a percentage of con siRNA group at 0 h. *P<0.05 vs. con siRNA group. (E) Determination of cell death by flow cytometry of Annexin V‑fluorescein isothiocyanate/PI staining in THP‑M cells transfected with con or p62 siRNA and then incubated with oxLDL (20 µg/ml) for the indicated times. Data are representatives of three independent experiments. **P<0.01 as indicated. IL, interleukin; oxLDL, oxidized low‑density lipoprotein; THP‑M, THP‑1‑derived macrophages; siRNA, small interfering RNA; con, control; PI, propidium iodide.

Article Snippet: Interleukin-18 (IL-18) levels from culture media was measured with an ELISA kit from R&D Systems, Inc., (cat. no. 7620; Minneapolis, MN, USA), according to the manufacturer's instructions.

Techniques: Transfection, Western Blot, Expressing, Control, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, MTT Assay, Flow Cytometry, Staining, Incubation, Small Interfering RNA

Journal: Medical mycology

Article Title: A fucose specific lectin from Aspergillus flavus induced interleukin-8 expression is mediated by mitogen activated protein kinase p38.

doi: 10.1093/mmy/myw066

Figure Lengend Snippet: Figure 3. Interleukin-8 expression profile upon FFL treatment: Cells were treated with indicated concentrations of FFL for different time period. Total RNA was isolated and cDNA was synthesized as mentioned, mRNA expression level was determined by Q-PCR method. (A) Cell viability assay: Cells were treated with varying concentrations of FFL for 24 hours. Cell viability was determined as explained in methods. The untreated cells were considered as 100% viable. (B) Cell number assay: Cells were stimulated with varying concentrations of FFL for 24 hours. The cell number in untreated sample was taken as 100%. Trypan blue exclusion assay was used to count the cells. (C) Time dependent expression of IL-8 mRNA in L-132 cells. Cells without treatment were used as control. (D) Dose dependent expression of IL-8 mRNA in L-132 cells. (E) Inhibition of IL-8 mRNA expression by L-fucose: The 40 ng/ml of FFL was preincubated with different concentrations of L-fucose (0.1 mM, 0.2 mM, and 0.3 mM) for 1 hour prior treatment to the cells for indicated time. RNA was isolated and mRNA expression level was determined as explained earlier. (F, G) Expression of IL-8 mRNA in L-132, U-937, and PBMCs: Cells were treated with FFL for 6 and 12 hour. The mRNA expression was determined as explained earlier. (H) Measurements of IL-8 production by sandwich ELISA in L-132, U-937, and PBMCs: Cells were treated with 40 ng/ml of FFL for indicated time. After each time course supernatant was collected and IL-8 concentration was determined as mentioned in kit. The cells treated with 100 nM TPA for 4 hours was used as positive control and untreated cells considered as negative control. The data are presented as mean ± SD of three individual experiments that gave similar results. ∗∗P < .01, ∗∗∗P < .001 versus control. #P < .05, ##P < .01, ###P < .001 versus FFL (40 ng/ml).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) Interleukin-8 concentration in cell culture supernatant was determined by sandwich ELISA using human IL-8 ELISA kit from Elabscience, Beijing (E-EL-H0048), following manufacturers protocol.

Techniques: Expressing, Isolation, Synthesized, Viability Assay, Trypan Blue Exclusion Assay, Control, Inhibition, Sandwich ELISA, Concentration Assay, Positive Control, Negative Control