Journal: OncoTargets and therapy

Article Title: Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis

doi: 10.2147/OTT.S160813

Figure Lengend Snippet: Effect of NMS-E973 on the viability of glioblastoma cells. Notes: ( A ) U87 and ( B ) SW1088 cells were treated with NMS-E973 for 72 hours. Cell viability was measured by MTT assay. ( C ) Representative images of colony formation assay in U87 and SW1088 cells after exposure to NMS-E973. ( D ) Quantification of colony numbers in the colony formation assay. Expression of significant difference: * P <0.01; ** P <0.001.

Article Snippet: After NMS-E973 treatment, cell lysates (50 µg) were incubated with 200 nM Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC (caspase-3, -8, and -9, respectively; Cayman Chemical, Ann Arbor, MI, USA) in a buffer solution comprising 20 mM HEPES at pH 7.4, 100 mM NaCl, 0.1% CHAPS, DTT (10 mM), and ND 10% of sucrose at 37°C for 60 minutes.

Techniques: MTT Assay, Colony Assay, Expressing

Journal: OncoTargets and therapy

Article Title: Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis

doi: 10.2147/OTT.S160813

Figure Lengend Snippet: NMS-E973 induces apoptosis in glioblastoma cells. Notes: ( A ) U87 and ( B ) SW1088 cells were treated with NMS-E973 (1 µM) for 2 days. The number of condensed and fragmented nuclei was counted for apoptosis analysis. ( C ) Representative images of Annexin V/propidium iodide staining in U87 and SW1088 cells after exposure to NMS-E973. ( D ) Annexin V+-cell percentage (indicated in the two right quadrants) was plotted. Expression of significant difference: ** P <0.001.

Article Snippet: After NMS-E973 treatment, cell lysates (50 µg) were incubated with 200 nM Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC (caspase-3, -8, and -9, respectively; Cayman Chemical, Ann Arbor, MI, USA) in a buffer solution comprising 20 mM HEPES at pH 7.4, 100 mM NaCl, 0.1% CHAPS, DTT (10 mM), and ND 10% of sucrose at 37°C for 60 minutes.

Techniques: Staining, Expressing

Journal: OncoTargets and therapy

Article Title: Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis

doi: 10.2147/OTT.S160813

Figure Lengend Snippet: Effect of NMS-E973 on caspase-9, -3, and -8 activation in glioblastoma cells. Notes: Glioblastoma cells were treated with NMS-E973 (1 µM) at indicated times. Cell-free caspase-3 activities of U87 ( A ) and SW1088 ( B ) were analyzed with certain substrates, Ac-LEHD-AFC (caspase-3). Caspase-3 activities of U87 ( C ) and SW1088 ( D ) were analyzed with certain substrates, Ac-DEVD-AFC (caspase-9). Caspase-8 activities of U87 ( E ) and SW1088 ( F ) were examined with certain substrates, Ac-IETD-AFC (caspase-8). Figure values were calculated as the mean ± SD of three experiments done in total. * P <0.01, ** P <0.001.

Article Snippet: After NMS-E973 treatment, cell lysates (50 µg) were incubated with 200 nM Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC (caspase-3, -8, and -9, respectively; Cayman Chemical, Ann Arbor, MI, USA) in a buffer solution comprising 20 mM HEPES at pH 7.4, 100 mM NaCl, 0.1% CHAPS, DTT (10 mM), and ND 10% of sucrose at 37°C for 60 minutes.

Techniques: Activation Assay

Journal: OncoTargets and therapy

Article Title: Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis

doi: 10.2147/OTT.S160813

Figure Lengend Snippet: PUMA is induced by NMS-E973 in glioblastoma cells. Notes: Glioblastoma cells were treated with NMS-E973 (1 µM) for 24 hours. ( A ) The mRNA levels of the indicated gene in U87 cells were evaluated by RT-PCR. ( B ) Expression of the indicated protein in U87 cells was measured by Western blot. ( C ) The mRNA levels of the indicated gene in SW1088 cells were evaluated by the RT-PCR. ( D ) Expression of the indicated protein in SW1088 cells was measured by Western blot. U87 cells ( E ) or SW1088 cells ( F ) were transfected with siRNA against p53 prior to NMS-E973 (1 µM) treatment for 24 hours. Expression of the indicated protein in U87 cells was measured by Western blot. Expression of significant difference: * P <0.01, ** P <0.001. Abbreviation: RT-PCR, reverse-transcriptase PCR.

Article Snippet: After NMS-E973 treatment, cell lysates (50 µg) were incubated with 200 nM Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC (caspase-3, -8, and -9, respectively; Cayman Chemical, Ann Arbor, MI, USA) in a buffer solution comprising 20 mM HEPES at pH 7.4, 100 mM NaCl, 0.1% CHAPS, DTT (10 mM), and ND 10% of sucrose at 37°C for 60 minutes.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection

Journal: OncoTargets and therapy

Article Title: Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis

doi: 10.2147/OTT.S160813

Figure Lengend Snippet: PUMA is required for NMS-E973-induced apoptosis in glioblastoma cells. Notes: Glioblastoma cells were transfected with either a scrambled siRNA or PUMA siRNA for 24 hours and then treated with NMS-E973 (1 µM) for 48 hours. Expression of indicated protein in U87 ( A ) cells and SW1088 ( B ) cells was measured by Western blot. ( C ) Representative images of Annexin V/propidium iodide staining in U87 and SW1088 cells with siRNA transfection and following with NMS-E973 (1 µM) treatment for 48 hours. ( D ) Annexin V+-cell percentage (indicated in the two right quadrants) was plotted. ( E ) The condensed and fragmented nuclei were counted in number for apoptosis analysis. Expression of significant difference: ** P <0.001.

Article Snippet: After NMS-E973 treatment, cell lysates (50 µg) were incubated with 200 nM Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC (caspase-3, -8, and -9, respectively; Cayman Chemical, Ann Arbor, MI, USA) in a buffer solution comprising 20 mM HEPES at pH 7.4, 100 mM NaCl, 0.1% CHAPS, DTT (10 mM), and ND 10% of sucrose at 37°C for 60 minutes.

Techniques: Transfection, Expressing, Western Blot, Staining

Journal: OncoTargets and therapy

Article Title: Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis

doi: 10.2147/OTT.S160813

Figure Lengend Snippet: PUMA contributes to the antitumor activities of NMS-E973 in a xenograft model. Notes: ( A ) Tumor volume at indicated time points after treatment was calculated and plotted (n=6 in each group). ( B ) The relative weight of tumor at the end of the experiment. ( C ) The indicated proteins were analyzed by Western blotting in representative tumors. ( D ) Representative images of paraffin-embedded tumor sections were analyzed by active caspase-3 staining. ( E ) Representative images of paraffin-embedded tumor sections analyzed by TUNEL staining. ( F ) Quantification graph regarding ( D ). ( G ) Quantification graph regarding ( E ). Expression of significant difference: * P <0.01, ** P <0.001.

Article Snippet: After NMS-E973 treatment, cell lysates (50 µg) were incubated with 200 nM Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC (caspase-3, -8, and -9, respectively; Cayman Chemical, Ann Arbor, MI, USA) in a buffer solution comprising 20 mM HEPES at pH 7.4, 100 mM NaCl, 0.1% CHAPS, DTT (10 mM), and ND 10% of sucrose at 37°C for 60 minutes.

Techniques: Western Blot, Staining, TUNEL Assay, Expressing