Figure 4. RNLS activates STAT3, and RNLS overexpression favors cancer cell survival.

(A) Activation of STAT3 by RNLS in Panc1 cells; Panc1 cells in culture treated with either BSA or RNLS, and STAT3 phosphorylation assessed by western blot; p-Ser⁷²⁷-STAT3: phosphorylation at serine 727, p-Y⁷⁰⁵-STAT3: phosphorylation at tyrosine 705; representative study. (B) Quantification of STAT3 phosphorylation with RNLS; signals normalized to total STAT3; n = 3, *indicates p < 0.05. (C) PDAC lines BxPC3, Panc1 and MiaPaCa2 are serum starved for 48 hrs, then incubated with 30 μg/ml of either bovine serum albumin (BSA) or rRNLS for 3 days; total and live cell number determined using trypan blue and an automated cell counter; n = 4, **indicates p < 0.0001. (D) Effect of rRNLS on survival of Panc1 cells in the absence or presence of 10 μM U0126 (Erk inhibitor) or 50 μM AG490, (JAK2 and STAT3) for 72 h. The doses of AG490 and U0126 chosen had been reported to inhibit the phosphorylation of JAK³⁷ and ERK1/2³⁸ respectively in Panc1 cells. The time point of 72 hrs was chosen to allow the cell time to proliferate. Cell proliferation was measured by the WST-1 method, and depicted as % change of DMSO-treated Panc1 cells. n = 6, *=p < 0.01. (E) siRNA mediated inhibition of PMCA4b expression blocks RNLS mediated MAPK signaling; Left and middle panels: MiaPaCa2 cells transfected with either non-targeting or PMCA4b siRNA, maintained in serum free medium for 3 days and treated with either 25 μg of BSA or 25 μg of RNLS peptide RP-220 for the indicated time; RP-220 mediated ERK and STAT3 activation assessed by western blot and representative immunoblots are shown; p-ERK = phosphorylated ERK, p-Y⁷⁰⁵-STAT3 = phosphorylated STAT3, p-S727-STAT3 = phosphorylated STAT3, BSA = bovine serum albumin, RP-220 = RNLS peptide agonist; Right panel: quantification of phosphorylated ERK (p-ERK), signals normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control; n = 4, *=P < 0.03, **=p < 0.0001.