dmnp edta  (Biotium)

Journal: Oncotarget

Article Title: Triptolide suppresses the in vitro and in vivo growth of lung cancer cells by targeting hyaluronan-CD44/RHAMM signaling

doi: 10.18632/oncotarget.15879

Figure Lengend Snippet: ( A ) Chemical structure of triptolide. ( B ) Dose-dependent anti-proliferative effects of triptolide in NSCLC cells. MTT assays were performed in five NSCLC cell lines treated with DMSO or triptolide (12.5, 25 and 50 nM) for 72 h and the data were presented as percentage mean ± SD of cell viability compared to DMSO-treated cells. ( C , D ) Representative western immunoblotting results showing dose-dependent (C) and time-dependent (D) effects of triptolide on the expression of cell proliferation- and apoptosis-related proteins in NSCLC cells. Cells were treated with different concentrations of triptolide (0, 25 and 50 nM) for 72 h or A549 cells were treated with 50 nM of triptolide for different time periods (6, 12, 24, 48 and 72 h). Three independent assays were performed from different samples as described in materials and methods section. * P < 0.05, compared with the control group. Assays were performed in triplicate and repeated three times on different days. C, Control; T, triptolide.

Article Snippet: NSCLC cells were plated on a 24-well plate at a density of 20,000 cells/well, grown in culture media containing 10% FBS for 24 h and exposed to triptolide (0–50 nM) for 72 h followed by methylthiazoletetrazolium (MTT, Biotium, Hayward, CA) treatment (40 μL per well) for 4 h. For assays with siRNAs, cells were transfected with HAS2, CD44 or RHAMM siRNAs (100, 200 or 50 nM, respectively, Valencia, CA) in lipofectamin RNAiMAX reagent (Invitrogen, Carlsbad, CA) for 6 h. In some experiments, A549 cells were co-treated with HA (2.5 mg/mL) and triptolide or HAS2, CD44 or RHAMM siRNAs and grown in media containing 2.5% FBS.

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Triptolide suppresses the in vitro and in vivo growth of lung cancer cells by targeting hyaluronan-CD44/RHAMM signaling

doi: 10.18632/oncotarget.15879

Figure Lengend Snippet: ( A ) Constitutive levels of HAS1 (2A-i), HAS2 (2A-ii), HAS3 (2A-iii), HA (2A-iv) and CD44 and RHAMM (2A-v) in immortalized BEAS-2B bronchial cells and NSCLC cell lines. ( B ) Modulation of levels of HAS2 (2B-i), HAS3 (2B-ii), HA (2B-iii), CD44 and RHAMM (2B-iv; 2B-v) in NSCLC cells treated with triptolide (25 nM) or DMSO. Cells were treated with 25 nM of triptolide for 72 h with the exception of the results shown in Figure in which cell were exposed to 0, 25 or 50 nM of triptolide. ( C ) Exogenous HA attenuated triptolide-induced cytotoxicity and modulation of cell proliferation and survival-related proteins. C-i, A549 cells grown in RPMI media supplemented with 2.5% FBS were treated with DMSO, triptolide (25 nM), HA (2.5 mg/mL), or triptolide + HA for 72 h and cell viability determined by MTT assay. C-ii, images of A549 cells exposed to DMSO, triptolide (25 nM), HA, or triptolide + HA for 72 h. C-iii, Western immunoblotting assays showing attenuation by HA of triptolide-induced modulation in the expression of cell proliferation and survival-related proteins. * P < 0.05. Assays were performed in triplicate and repeated three times on different days. C, Control; T, triptolide.

Article Snippet: NSCLC cells were plated on a 24-well plate at a density of 20,000 cells/well, grown in culture media containing 10% FBS for 24 h and exposed to triptolide (0–50 nM) for 72 h followed by methylthiazoletetrazolium (MTT, Biotium, Hayward, CA) treatment (40 μL per well) for 4 h. For assays with siRNAs, cells were transfected with HAS2, CD44 or RHAMM siRNAs (100, 200 or 50 nM, respectively, Valencia, CA) in lipofectamin RNAiMAX reagent (Invitrogen, Carlsbad, CA) for 6 h. In some experiments, A549 cells were co-treated with HA (2.5 mg/mL) and triptolide or HAS2, CD44 or RHAMM siRNAs and grown in media containing 2.5% FBS.

Techniques: MTT Assay, Western Blot, Expressing

Journal: Oncotarget

Article Title: Triptolide suppresses the in vitro and in vivo growth of lung cancer cells by targeting hyaluronan-CD44/RHAMM signaling

doi: 10.18632/oncotarget.15879

Figure Lengend Snippet: ( A ) Effects of HAS2, CD44 and RHAMM siRNA on the viability of NSCLC cells. Each cell line was transfected with the individual siRNAs and cell viability was determined by MTT assay as described in the Materials and Methods section. ( B ) HAS2, CD44 and RHAMM siRNAs modulated the expression of cell proliferation-and survival-related proteins. NSCLC cells were transfected with the siRNAs and Western immunoblotting was performed as described in the Materials and Methods section. ( C ) Effect of HAS2 siRNA on HA synthesis. A549 cells were transfected with HAS2 siRNA and accumulation of HA in the culture media was determined as described in the Materials and Methods section. ( D , E ) Exogenous HA (2.5 mg/mL) rescued cells from the cytotoxic effects of siRNAs targeting HAS2 (D), CD44, RHAMM or CD44 + RHAMM (E). A549 cells were treated with HAS2, CD44, RHAMM or CD44 + RHAMM siRNAs or HA alone or siRNA + HA and cell viability determined by MTT assay. ( F ) Exogenous HA attenuated the effects of triptolide on cell proliferation-and apoptosis-related proteins. A549 cells grown in RPMI media supplemented with 2.5% FBS were treated with HAS2, CD44, RHAMM or CD44 + RHAMM siRNAs or HA alone or siRNA + HA and expression of the proteins determined by Western immunoblotting. For all experiments, at least three independent assays were carried out. * P < 0.05, compared to treatment with DMSO-treated cells; Δ, compared to DMSO-treated cells; ♯, compared to treatment with siRNA only.

Article Snippet: NSCLC cells were plated on a 24-well plate at a density of 20,000 cells/well, grown in culture media containing 10% FBS for 24 h and exposed to triptolide (0–50 nM) for 72 h followed by methylthiazoletetrazolium (MTT, Biotium, Hayward, CA) treatment (40 μL per well) for 4 h. For assays with siRNAs, cells were transfected with HAS2, CD44 or RHAMM siRNAs (100, 200 or 50 nM, respectively, Valencia, CA) in lipofectamin RNAiMAX reagent (Invitrogen, Carlsbad, CA) for 6 h. In some experiments, A549 cells were co-treated with HA (2.5 mg/mL) and triptolide or HAS2, CD44 or RHAMM siRNAs and grown in media containing 2.5% FBS.

Techniques: Transfection, MTT Assay, Expressing, Western Blot

Journal: Oncotarget

Article Title: Triptolide suppresses the in vitro and in vivo growth of lung cancer cells by targeting hyaluronan-CD44/RHAMM signaling

doi: 10.18632/oncotarget.15879

Figure Lengend Snippet: ( A , C ) Effects of triptolide (A) or CD44 siRNA (C) on the number of primary-, secondary and tertiary-pulmospheres generated from A549 cells. Cells were treated with triptolide (12.5, 25 and 50 nM) or CD44 siRNA and the frequency of pulmospheres determined as described in the Materials and Methods section. ( B , D ) Images of primary, secondary and tertiary pulmospheres generated from triptolide (B)- or CD44 siRNA (D)- treated A549 cells. At least three independent assays were carried for these assays. * P < 0.05.

Article Snippet: NSCLC cells were plated on a 24-well plate at a density of 20,000 cells/well, grown in culture media containing 10% FBS for 24 h and exposed to triptolide (0–50 nM) for 72 h followed by methylthiazoletetrazolium (MTT, Biotium, Hayward, CA) treatment (40 μL per well) for 4 h. For assays with siRNAs, cells were transfected with HAS2, CD44 or RHAMM siRNAs (100, 200 or 50 nM, respectively, Valencia, CA) in lipofectamin RNAiMAX reagent (Invitrogen, Carlsbad, CA) for 6 h. In some experiments, A549 cells were co-treated with HA (2.5 mg/mL) and triptolide or HAS2, CD44 or RHAMM siRNAs and grown in media containing 2.5% FBS.

Techniques: Generated

Journal: Oncotarget

Article Title: Triptolide suppresses the in vitro and in vivo growth of lung cancer cells by targeting hyaluronan-CD44/RHAMM signaling

doi: 10.18632/oncotarget.15879

Figure Lengend Snippet: ( A ) Effects of triptolide on the growth of orthotopically transplanted A549 cells as determined by bioluminescence imaging. Rats in which luciferase expressing A549 cells were implanted in the lung were given liposome-encapsulated triptolide (400 μg/kg) and the growth of lung tumors monitored by bioluminescence imaging as described in the Materials and Methods section. ( B ) Representative results of bioluminescence imaging studies in the vehicle and triptolide groups. ( C ) Representative images of Ki-67 expression in the lung tissues of vehicle- (top panel) or triptolide-treated (bottom panel) rats. Right panel: Bar graph showing the percent of Ki-67-positive lung tumor cells (mean ± SD) in vehicle- or triptolide-treated rats. ( D ) Levels of HA in lung tumor tissues of vehicle- and triptolide-treated rats. HA levels were measured by HA ELISA-like assay kit as described in the Materials and Methods section. ( E ) Levels of HAS1, HAS2, HAS3, CD44, and RHAMM mRNA transcripts in lung tumor tissues of vehicle- and triptolide-treated rats. ( F ) Left panel: Representative results showing Western immunoblotting analyses of CD44 expression in lung tumor tissues of vehicle- and triptolide-treated rats. Right panel: Quantification of CD44 expression in lung tumor tissues. The results from Figure were obtained from at least three independent assays performed on different days. * P < 0.05.

Article Snippet: NSCLC cells were plated on a 24-well plate at a density of 20,000 cells/well, grown in culture media containing 10% FBS for 24 h and exposed to triptolide (0–50 nM) for 72 h followed by methylthiazoletetrazolium (MTT, Biotium, Hayward, CA) treatment (40 μL per well) for 4 h. For assays with siRNAs, cells were transfected with HAS2, CD44 or RHAMM siRNAs (100, 200 or 50 nM, respectively, Valencia, CA) in lipofectamin RNAiMAX reagent (Invitrogen, Carlsbad, CA) for 6 h. In some experiments, A549 cells were co-treated with HA (2.5 mg/mL) and triptolide or HAS2, CD44 or RHAMM siRNAs and grown in media containing 2.5% FBS.

Techniques: Imaging, Luciferase, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Oncotarget

Article Title: Triptolide suppresses the in vitro and in vivo growth of lung cancer cells by targeting hyaluronan-CD44/RHAMM signaling

doi: 10.18632/oncotarget.15879

Figure Lengend Snippet: Effect of liposomal triptolide on the rat body weight, the wet lung weight and gross tumor burden in the lung

Article Snippet: NSCLC cells were plated on a 24-well plate at a density of 20,000 cells/well, grown in culture media containing 10% FBS for 24 h and exposed to triptolide (0–50 nM) for 72 h followed by methylthiazoletetrazolium (MTT, Biotium, Hayward, CA) treatment (40 μL per well) for 4 h. For assays with siRNAs, cells were transfected with HAS2, CD44 or RHAMM siRNAs (100, 200 or 50 nM, respectively, Valencia, CA) in lipofectamin RNAiMAX reagent (Invitrogen, Carlsbad, CA) for 6 h. In some experiments, A549 cells were co-treated with HA (2.5 mg/mL) and triptolide or HAS2, CD44 or RHAMM siRNAs and grown in media containing 2.5% FBS.

Techniques: