Figure 6

NBL and oral squamous cell carcinoma cell lines were pretreated for 12 hr with vehicle control, or either dopamine or L-mimosine at either 0.01 mM or 0.03 mM. Cells were then treated with increasing concentrations of cisplatin and incubated for either 24 hr (Figure 6?figure supplement 1a-f) or 48 hr (data shown here). (A?F) An alamarBlue assay was used to determine cell viability. Results are displayed as % compared to untreated control. (A and D) LAN5, (B and E) SK-N-AS, (C and F) HSC-3. *=p<0.05, **=p<0.01, ***=p<0.005, and ****=p<0.001, as per two-way ANOVA with a Tukey post-test. N=3. (G) SK-N-AS cells were treated the same as in A-F, with the concentrations of protective agent specified. 48 hr following cisplatin treatment, cells were prepared for PE-conjugated Annexin V/SYTOXBlue-based flow cytometry. Graph of fold change in Annexin+ cells (gated to biological control) is displayed, relative to vehicle control. *=p<0.05, ****=p<0.001, as per two-tailed student?s t-tests comparing treatment groups to control. N=4. Representative flow plots and gating strategies can be found in Figure 6?figure supplement 2a-i H?J) Cancer cells were treated as in A-G, with the concentrations of protective agents specified. Twenty four hours following cisplatin treatment, cells were fixed, permeabilized and labeled with anti-phospho-histone H2A.X (Ser139) and DAPI to label ?H2AX positive foci and nuclear material, respectively. (H and I) Quantification of ?H2AX staining, reported as ?H2AX integrated density/DAPI integrated density, with each data point corresponding to an individual nucleus. *=p<0.05, **=p<0.01, ****=p<0.001, as per Kruskal-Wallis testing with a Dunn?s multiple comparison test. N=3. (H) SK-N-AS cells, (I) LAN5 cells. (J) Representative confocal microscopy of SK-N-AS cells with indicated treatments, displaying DAPI, gamma H2AX, and an overlay.