Staurosporine

The 2 g of ethyl acetate extract was completely resuspended in H₂O (40 ml). This H₂O (re)suspension was re‐extracted using an equal volume of heptane (excluded to remove nonpolar compounds) for further purification, followed be re‐extraction with EtOAc (using two times equal volume of solvent). The EtOAc and aqueous phases were dried under vacuum and then dissolved in 5 ml of MeOH, followed by separation by size‐exclusion chromatography on Sephadex LH20 (Sigma) with methanol as the mobile phase. Fractions found to contain our molecule of interest (based on activity screening) were pooled and concentrated under vacuum prior to final purification by preparative HPLC. The isolated molecule was used for downstream structure elucidation by NMR and for biological activity assays. Commercially available staurosporine (Fisher BioReagents, Fisher Scientific) was used as an analytical standard (Figure S1 in Appendix S1).

The selective PKCε activator, FR236924, and staurosporine were purchased from Fisher Scientific. mPKCε-CAT-HA was a gift from Bernard Weinstein (Addgene; plasmid 211242).

Compounds used included trametinib (Selleck Chemicals, Houston, TX, USA), Z-VAD-FMK (UBP Bio, Aurora, CO, USA), Nec-1 (ApexBio, Houston, TX, USA), cisplatin (ApexBio), and staurosporine (ApexBio). trametinib, Z-VAD-FMK, Nec-1, and staurosporine were dissolved in DMSO (Fisher Scientific, Waltham, MA, USA) and cisplatin was dissolved in sterile water. Control samples in all experiments performed were treated with vehicle only. Vehicle concentration in growth medium did not exceed 0.2%. Cell treatment was performed by aspirating the growth media from the cells and replacing it with growth medium containing selected concentrations of drugs.