Fig. 5. TERS induces WNT signaling and cytoplasmic export of TERT.

(A) RT-qPCR analysis of the transcriptional activation of CTNNB1 and AXIN2 of Veh CM– or TERS CM–treated PC3 cells throughout 5 days of culture with CM resupplementation every other day. Relative quantification of gene expression of samples is normalized to Veh CM day 1 condition (n = 2 per group). (B) TOP-GFP expression of PC3 cells during TERS priming, determined by flow cytometry (at least 10,000 events were analyzed per condition). PC3.TOP cells treated with Veh CM or TERS CM for 48 hours in the absence or presence of (C) the IRE1α inhibitor 4μ8C or (D) the PERK inhibitor GSK2656157 and measured for mean fluorescence intensity (MFI). MFI expression was then normalized to Veh CM uninhibited value (n = 2; at least 10,000 events were analyzed per condition). (E) Normalized MFI expression of PC3.TOP cells treated with tunicamycin (5 µg/ml) for 48 hours during IRE1α or PERK inhibition (n = 2; at least 10,000 events were analyzed per condition). (F) Normalized MFI expression of PC3.TOP stimulated for 48 hours with recombinant WNT3a (rWNT3a) (20 ng/ml) during IRE1α or PERK inhibition (n = 2; at least 10,000 events were analyzed per condition). (G) hTERT RT-qPCR analysis of 48-hour Veh CM– or TERS CM–treated PC3 cells (n = 3 per condition). (H) Relative firefly TERT promoter-luciferase or ATF6 promoter-luciferase of dually transfected LNCaP cells. Cells were treated with LNCaP Veh CM or TERS CM for 48 hours and normalized for expression by Renilla-luciferase (n = 3 per condition). **P < 0.01, Student’s t test (paired two-tailed). (I) Immunofluorescence staining for TERT in PC3 cells treated for 48 hours. Scale bars, 25 µm. Error bars represent SEM. Data in (C), (D), and (H) are representative of two experiments; data in (A), (B), (F), (G), and (I) are from at least three independent experiments.