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

Journal: Oncology Research

Article Title: Knockdown of Urothelial Carcinoma-Associated 1 Suppressed Cell Growth and Migration Through Regulating miR-301a and CXCR4 in Osteosarcoma MHCC97 Cells

doi: 10.3727/096504018X15201143705855

Figure Lengend Snippet: Knockdown of UCA1 inhibited cell growth and migration in MHCC97 cells. MHCC97 cells were transfected with shNC, sh-UCA1#1, and sh-UCA1#2. (A) The efficiency of transfection was quantified by RT-qPCR analysis. (B) Cell viability was measured by trypan blue exclusion assay. (C) Cell migration and (D) invasion were detected by Transwell migration assay. (E) Apoptotic cells and (F) the levels of apoptosis-related proteins were measured by flow cytometry and Western blot, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The apoptotic cell rates were determined using an Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (Beyotime Biotechnology).

Techniques: Knockdown, Migration, Transfection, Quantitative RT-PCR, Trypan Blue Exclusion Assay, Transwell Migration Assay, Flow Cytometry, Western Blot

Journal: Oncology Research

Article Title: Knockdown of Urothelial Carcinoma-Associated 1 Suppressed Cell Growth and Migration Through Regulating miR-301a and CXCR4 in Osteosarcoma MHCC97 Cells

doi: 10.3727/096504018X15201143705855

Figure Lengend Snippet: miR-301a overexpression blocked the effect of UCA1 on cell growth and migration in MHCC97 cells. MHCC97 cells were transfected with sh-UCA1 or shNC, or cotransfected with sh-UCA1 and miR-301a mimic or mimic control. (A) Cell viability, (B) cell migration, and (C) invasion were assessed by trypan blue exclusion assay and Transwell migration assay, respectively. (D) Apoptotic cells were quantified by flow cytometry assay. (E) Western blot was conducted to measure the expression of apoptosis-related core factors. * p < 0.05, ** p < 0.01.

Article Snippet: The apoptotic cell rates were determined using an Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (Beyotime Biotechnology).

Techniques: Over Expression, Migration, Transfection, Control, Trypan Blue Exclusion Assay, Transwell Migration Assay, Flow Cytometry, Western Blot, Expressing

Journal: Oncology Research

Article Title: Knockdown of Urothelial Carcinoma-Associated 1 Suppressed Cell Growth and Migration Through Regulating miR-301a and CXCR4 in Osteosarcoma MHCC97 Cells

doi: 10.3727/096504018X15201143705855

Figure Lengend Snippet: CXCR4 was involved in the regulatory effect of miR-301a on cell growth and migration. MHCC97 cells were transfected with sh-CXCR4 or sh-NC, or cotransfected with sh-CXCR4 and miR-301a mimic, or mimic control. The (A) mRNA and (B) protein expression levels of CXCR4 were tested by RT-qPCR analysis and Western blot analysis, respectively. (C) Cell viability, (D) cell migration, (E) invasion, and (F) apoptotic cells were assessed by trypan blue exclusion assay, Transwell migration assay, and flow cytometry assay, respectively. (G) The protein levels of apoptosis-related factors were measured by Western blot. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The apoptotic cell rates were determined using an Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (Beyotime Biotechnology).

Techniques: Migration, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Trypan Blue Exclusion Assay, Transwell Migration Assay, Flow Cytometry

Journal: Molecular Medicine Reports

Article Title: Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells

doi: 10.3892/mmr.2015.4715

Figure Lengend Snippet: ROS levels are increased in HepG2 cells following co-treatment with ABT-737 and curcumin. (A) HepG2 cells were separately treated with 1% dimethyl sulfoxide, 2 µ M curcumin, 10 µ M ABT-737 and 2 µ M curcumin+10 µ M ABT-737 for 24 h, following which a 2′,7′-dichlorofluorescin diacetate probe was used to measure the levels of cellular ROS. HepG2 cells were treated with 2 µ M curcumin and 10 µ M ABT-737 for 24 h in the presence or absence of the antioxidant, NAC (10 mM). Subsequently, the levels of HepG2 cell apoptosis were analyzed using (B) Hoechst 33258 staining (white arrows indicate cells undergoing apoptosis with nuclear fragmentation visualized by Hoechst staining), (C) Annexin V-fluorescein isothiocyanate/PI staining and (D) caspase-3 activity detection. Data are expressed as the mean ± standard deviation. * P<0.05. ROS, reactive oxygen species; PI, propidium iodide; NAC, N-acetyl-L-cysteine.

Article Snippet: An Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit was purchased from Beyotime Institute of Biotechnology (Beijing, China).

Techniques: Staining, Activity Assay, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells

doi: 10.3892/mmr.2015.4715

Figure Lengend Snippet: Increased levels of ROS promote the sustained activation of JNK, which leads to the apoptosis of HepG2 cells. (A) HepG2 cells were separately treated with 1% dimethyl sulfoxide, 2 µ M curcumin, 10 µ M ABT-737 and 2 µ M curcumin+10 µ M ABT-737 for 24 h, following which the total cellular proteins were extracted. Subsequently, the protein levels of p-JNK1/JNK2 and total JNK1/JNK2 were detected using western blotting. (B) HepG2 cells were treated with 2 µ M curcumin+10 µ M ABT-737 for 24 h in the presence or absence of 10 µ M SP600125 (a JNK inhibitor). Following treatment, apoptosis of the HepG2 cells was assessed using annexin V-fluorescein isothiocyanate/PI staining. (C) HepG2 cells were treated with 2 µ M curcumin+10 µ M ABT-737 for 24 h in the presence or absence of antioxidant, NAC (10 mM). Subsequently, the levels of p-JNK1/JNK2 and total JNK1/JNK2 were detected using western blotting. (D) HepG2 cells were separately transfected with control siRNA or ASK1 siRNA for 24 h, following which the cells were treated with 2 µ M curcumin+10 µ M ABT-737 for 24 h. A Trypan blue exclusion assay was then used to measure the total cell death ratio. * P<0.05 vs. control. ROS, reactive oxygen species; JNK, c-Jun N-terminal kinase; p-, phosphorylated; siRNA, small interfering RNA; PI, propidium iodide.

Article Snippet: An Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit was purchased from Beyotime Institute of Biotechnology (Beijing, China).

Techniques: Activation Assay, Western Blot, Staining, Transfection, Control, Trypan Blue Exclusion Assay, Small Interfering RNA