Fig. 5: The phorbol ester PMA is an effective mTORC1 inducer that facilitate PI toxicity.

A Shown is a volcano plot representing transcriptome comparison between wt RPMI8226 cells (n = 3 independent samples) and TSC2 KO RPMI8226 cells (n = 2 independent samples). Upregulated genes in TSC2 KO cells (n = 1516, FC > 2, p < 0.05) are shown in red rectangle. Enriched genes were analyzed using ‘Drug Perturbations from GEO up’ as a reference library. Enrichment output is shown in the middle volcano plot. Overlapping genes are shown in the right graph and correspond to TSC2 KO upregulated genes set. B MM cells were treated with PMA [125 nM] alone or in combination with rapamycin [50 nM] for 2 h in the presence and absence of AA, as indicated. Shown are a typical immunoblots out of three independent experiments. C MM cells were treated with IXZ [32 nM] alone or in combination with PMA [125 nM]. Treatment was performed in a time-dependent manner, as indicated. Shown are a typical immunoblots out of three independent experiments. D MM cells were treated for 24 h with PMA [125 nM], IXZ [15 nM], rapamycin [50 nM], as indicated. Contour plots of JC-1 stained cells following the treatment are shown and each represents typical result out of two independent experiments. Red gate corresponds to cells with ‘healthy’ mitochondrial membrane potential and green gate corresponds to cells with ‘unhealthy’ mitochondrial membrane potential. E Column graphs corresponding to the experiment described in (D), error bars represent S.E.M., *p < 0.05 of unpaired two-tailed student’s t-test. F MM cells were treated for 48 h with PMA [125 nM], IXZ [15 nM], rapamycin [50 nM], as indicated. Shown is average relative viability of three independent experiment ± S.E.M., *p < 0.05 of unpaired two-tailed student’s t-test. G Average OCR measurements are shown following treatment with DMSO, PMA [125 nM], rapamycin [50 nM] and IXZ [10 nM] for 12 h, error bars represent ±S.E.M., *p < 0.05 of unpaired two-tailed student’s t-test. ns, not significant.