Fig. 3: Co-silencing IRS-1 and RPS6 potentiates apoptotic cell death and ablates cellular proliferation. | Cell Death & Disease

Fig. 3: Co-silencing IRS-1 and RPS6 potentiates apoptotic cell death and ablates cellular proliferation.

From: Inhibiting insulin and mTOR signaling by afatinib and crizotinib combination fosters broad cytotoxic effects in cutaneous malignant melanoma

Fig. 3

a Immunoblotting showing efficient knockdown of siRNAs against IRS-1 and RPS6 used in this study. b Immunoblotting showing IRS-1 siRNA (#3, #4) and RPS6 siRNA (#1, #3) in single and combination knockdowns. These siRNAs were chosen for further validation studies. In all subsequent experiments sicombo #1 (siIRS-1 #3 + siRPS6 #1) and sicombo#2 (siIRS-1 #4 + siRPS6 #3). c Colony formation assay showing reduced proliferation after knocking down RPS6 alone or in combination with IRS-1. d Quantification of c (error bars represent mean ± SD; n = 3). e FACS analysis quantification showing concomitant silencing of RPS6 and IRS-1 for 48 h further induces apoptotic cell death than silencing either gene alone (error bars represent mean ± SD; n = 3). f, g FACS analysis quantification showing CMM cells lose sensitivity towards combination drug treatment upon concomitant silencing of RPS6 and IRS-1. Silencing was performed for 24 h followed by 24 h drug treatment with afatinib 2 µM + crizotinib 2 µM. h Protein expression and localization patterns of IRS-1 and RPS6KB1 in a cohort of tumor samples (n = 60) from CMM patients. Boxed area are enlarged to show more detailed staining of IRS-1 and RPS6KB1.

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