Figure 1 Celastrol inhibits cell viability and proliferation of neuroblastoma cells A: molecular structure of celastrol; B: QDDQ-NM, SK-N-SH and SH-SY5Y neuroblastoma cell lines and three normal cell lines were incubated with different concentrations (0-20 µM) of celastrol, CCK-8 assay was applied to determine cells viability. aP < 0.05; bP < 0.01 versus 0 μM celastrol treatment group; C: C1-C4: QDDQ-NM cell lines were incubated with different concentrations (0-20 µM) of celastrol; C5-C8: SK-N-SH cells were incubated with different concentrations (0-20 µM) of celastrol; C9-C12: SH-SY5Y cells were incubated with different concentrations (0-20 µM) of celastrol; C13-C16: BJ cells were incubated with different concentrations (0-20 µM) of celastrol; C17-C20: CCD-1079SK cells were incubated with different concentrations (0-20 µM) of celastrol; C21-C24: HUVEC cells were incubated with different concentrations (0-20 µM) of celastrol, cell morphology changes were detected by optical microscope. Scale bar = 100 μm; D: colony formation results after celastrol treatment (0-20 µM) in QDDQ-NM (D1-D4) and SH-SY5Y cells (D5-D8). aP < 0.05; bP < 0.01; cP < 0.001 versus control group; E: number of colonies after different concentrations of celastrol treatment 0, 5, 10, 20 µM. CCK-8: cell counting kit-8.

Figure 2 Celastrol inhibits cell cycle progression and induces cell apoptosis in QDDQ-NM and SH-SY5Y cells A: flow cytometry assay we applied to detect the cells cycle distribution in QDDQ-NM cells after treatment with different concentrations of celastrol (A1) 0 µM, (A2) 5 µM, (A3) 10 µM,(A4) 20 µM for 1 d; B: quantitative analysis of cell cycle distribution was shown after celastrol treatment; C: the protein expressions of P21 were detected by immunoblotting in QDDQ-NM and SH-SY5Y cells after different concentrations of celastrol treatment; D: QDDQ-NM cells with Hoechst (bright blue) and PI (red) fluorescence signals were identified as apoptotic cells, (D1, D4, D7, D10) Hoechst stanning for 0, 5, 10, 20 µM celastrol treatment; (D2, D5, D8, D11) PI stanning for 0, 5, 10, 20 µM celastrol treatment; (D3, D6, D9, D12) Merged stanning for 0, 5, 10, 20 µM celastrol treatment; scale bar = 50 μm. E: the percentage of cell death rate after celastrol treatment. F, G: flow cytometry assay was applied to determine the apoptosis of QDDQ-NM (a) and SH-SY5Y (b) cells after celastrol treatment. aP < 0.05, bP < 0.01, cP < 0.001 compared to control.

Figure 3 Celastrol facilitates apoptosis in QDDQ-NM and SH-SY5Y cells by activating caspases pathway A: mRNA expressions of Caspase-9, Bcl-2, PUMA, Bax, Noxa, and Caspase-3 were examined via RT-PCR assay; B: quantitative analysis of RT-PCR results; C-F: Western blotting and quantification results of the expressions of anti-apoptosis and pro-apoptosis proteins in QDDQ-NM and SH-SY5Y cells after celastrol treatment. G: cell viability of QDDQ-NM and SH-SY5Y were examined by CCK-8 assay after combined treatment with Z-VAD-FKM (caspase inhibitor) and celastrol. PUMA: p53 up-regulated modulator of apoptosis; RT-PCR: reverse transcription polymerase chain reaction; CCK-8: cell counting kit-8. aP < 0.05, aP < 0.01 versus control and cP < 0.01 versus celastrol treatment group.

Figure 4 ROS production was involved in celastrol-mediated apoptosis A, B: ROS production was examined under fluorescence microscope by DCFH-DA staining and the relative DCF fluorescence intensities was quantified by Imagelab; A1-A3: QDDQ-NM cells for control group, celastrol group, celastrol with NAC group; A4-A6: SH-SY5Y cells for control group, celastrol group, celastrol with NAC group; C: flow cytometry assay was also applied to assess the effect of NAC on celastrol-mediated ROS production; D: the percentage of cell viability after celastrol treatment. aP < 0.01 compared to control, bP < 0.01 compared to celastrol-pretreated group. E, F: chromatin-condensed nuclei (red arrow) indicated cell apoptosis after staining with Hoechst 33442 dye. E1-E4: QDDQ-NM cells for control group, NAC group, celastrol group, celastrol with NAC group; E5-E8: SH-SY5Y cells for control group, NAC group, celastrol group, celastrol with NAC group. The images were detected by the fluorescence microscope. Scale bar = 50 μm. The proportion of Hoechst 33442+ cells were quantified by Imagelab. G, H: Annexin V and PI were used to stain the apoptosis cells by flow cytometry. The percentage of apoptosis cells of QDDQ-NM and SH-SY5Y were quantified by Imagelab. aP < 0.01, compared to untreated cells; bP < 0.01, cP < 0.05, compared to celastrol treatment cells. ROS: reactive oxygen species; DCFH-DA: 2, 7-Dichlorodihydrofluorescein diacetate; DCF: 2′, 7′-Dichlorodihydrofluorescein; NAC: N-Acetyl-L-cysteine.