Fig. 1

Fig. 1 Combined treatment with SL-176 and GSK-J4 induces synergistic cytotoxicity in neuroblastoma cells.

Waterfall plots showing the compounds with the highest differential drug sensitivity score (ΔDSS) in combination with SL-176 as compared to vehicle in IMR-32 cells (A) and SK-N-AS cells (B). Dashed lines indicate ΔDSS = 5, the level considered as cut-off for plausible synergy detected through this assay. Combinations with ΔDSS ≥ 3 are shown. Colors indicate drug classes and # indicates suspected false positives. C Dose-response curves for GSK-J4 in IMR-32 and SK-N-AS cells in combination with either vehicle or fixed-dose SL-176 (11 µM). Addition of SL-176 shifted the GSK-J4 dose-response curve one order of magnitude to the left. D Morphology of IMR-32 cells treated for 72 hours either with vehicle, SL-176 (3 µM), GSK-J4 (0.3 µM) or the combination. Dose-response curves (E), dose-response matrices (F) and synergy landscapes (G) for four different neuroblastoma cell lines with different genetic characteristics, treated with GSK-J4+/– SL-176. Dose response data shown is the mean of at least three independent experiments from WST-1. Synergy was assessed with the ZIP method using the SynergyFinder tool. H δ scores of the areas with the most synergistic dose combinations of SL-176 and GSK-J4 for eight neuroblastoma cell lines and human dermal fibroblasts (nHDF). I combining GSK-J4 treatment (0.3 µM for SK-N-SH and 0.1 µM for SK-N-BE(2)) with siRNA-knockdown of PPM1D yielded significantly lower cell confluency after 72 hours for two out of three constructs for each cell line (One-way ANOVA with Dunnett’s post-test, SK-N-SH: neg. siRNA+GSK-J4 vs siPPM1D1 P = 0.00496, neg. siRNA+GSK-J4 vs siPPM1D2 P = 0.0055 and SK-N-BE(2): neg. siRNA+GSK-J4 vs siPPM1D1 P = 0.0016, neg. siRNA+GSK-J4 vs siPPM1D2 P = 0.0387). Mean ± SD for four (SK-N-SH) or three (SK-N-BE(2)) independent experiments, performed in triplicates. Knockdown was confirmed to be significant at 60-72% remaining PPM1D expression for SK-N-SH and 11-44% remaining PPM1D expression for SK-N-BE(2).