Figure 5.

NSC697923 has stronger cytotoxic effects than Dox and VP16 on chemoresistant NB cell line LA-N-6. (a) LA-N-6 cells were treated with indicated concentrations of NSC697923, Dox, or VP16 for 24 h. Cell viability was then measured by adding 10 μl of CCK-8 and reading the absorbance at 450 nm. Data were represented as mean±S.D. P-values <0.05 (*), <0.01 (**), or <0.001 (***) were indicated. (b) LA-N-6 cells were first incubated with the indicated concentrations of NSC697923, Dox, or VP16 for 24 h, stained by propidium iodide (PI) without fixing and then analyzed by flow cytometry. PI-positive cells were shown by percentage. (c and d) LA-N-6 cells were seeded in six-well plates with NSC697923 and agar, and grown for 2 weeks, then stained with MTT for 4 h, pictures were taken and colonies were counted. Colony numbers were represented as mean±S.D. P-values <0.01 (**) or <0.001 (***) were indicated. (e and f) LA-N-6 cells were treated with indicated concentrations of NSC697923, Dox, and VP16 for varying time courses, lysed, subjected to SDS–PAGE and immunoblotted with indicated antibodies. Beta-actin was detected as a loading control. (g) LA-N-6 cells were left untreated or treated with NSC697923 alone, JNK inhibitor SP600125 alone, or the combination of the two for 24 h, then cell viability was then measured by adding 10 μl of CCK-8 and reading the absorbance at 450 nm. Data were represented as mean±S.D. P–value <0.05 (*) was indicated. (h and i) LA-N-6 cells were treated with Dox or VP16 at the indicated concentrations with or without NSC697923 for 24 h. Then cell viability was then measured by adding 10 μl of CCK-8 and reading the absorbance at 450 nm. Data were represented as mean±S.D. P-values <0.05 (*), <0.01 (**), or <0.001 (***) were indicated. Data from a to e and h to i are representative of three independent experiments. Data from f and g are representative of two independent experiments