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Synergism of SL-176 and GSK-J4 is confirmed in long-term experiments and in neuroblastoma spheroids. A confluency of NB cells followed for 144 hours after adding vehicle or drug treatment. Cells were treated with GSK-J4 (0.3 µM in IMR-32, SK-N-AS and SK-N-BE(2) and 0.6 µM in SK-N-SH), SL-176 (3 µM in IMR-32 and 6 µM in SK-N-SH, SK-N-AS and SK-N-BE(2)) or their combination. In the “washout” group, the medium was exchanged at 72 hours. Data represent the mean ± SD from four independent experiments. B representative bright-field microscopy images of IMR-32 tumor spheroids exposed to vehicle, SL-176 10 µM, GSK-J4 1 µM, or the combination, for 6 days. Scale bar, 100 µm. Representative bright-field images and overlay with green fluorescence of IMR-32 (C) and SK-N-AS (D) spheroids after 6 days of treatment. Scale bars, 400 µm. E SL-176 and GSK-J4 combination treatment affects both size and viability of IMR-32- and SK-N-AS-derived spheroids in a dose-dependent manner. GCU, green calibrated unit. *p < 0.05; **p < 0.01; ***p < 0.001. Experiments were performed three times (SK-N-AS) or four times (IMR-32), and 3-8 spherocytes were analyzed in each run. F immunohistochemistry of IMR-32 and SK-N-AS spheroids detecting cleaved caspase-3 and p21. Scale bars, 100 µm.
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