Fig. 10.

Reconstitution of TRPM2-depleted cells with wild-type TRPM2 but not the Ca²⁺-impermeable mutant E960D restored viability in TRPM2-depleted cells and phosphorylation and expression of Pyk2 and CREB. TRPM2-depleted SH-SY5Y cells (KO) were stably transfected with empty vector, V5-tagged wild-type TRPM2 or the V5-tagged Ca²⁺-impermeable TRPM2 mutant E960D. Scrambled control cells (Scr) were transfected with empty vector. Two different single cells clones from each group of transfected cells (Scr, KO) were untreated or treated with doxorubicin for 24 h and cell lysates prepared. A: reconstitution of cell viability with wild-type TRPM2 but not empty vector or the E960D pore mutant is shown. Viability was measured in three experiments with XTT. Measurements were standardized to results for untreated cells in each group, and the means ± SE of six replicates from one representative experiment are shown. *P ≤ 0.05. B: Western blotting was performed with antibodies to V5, the TRPM2 COOH terminus, phosphorylated and total Pyk2 and CREB. A representative Western blot from one of three similar experiments is shown. Results were ratioed to the average densitometry measurement of Scr control cells (time 0 or 24 h) and statistical differences in three experiments calculated with one-way ANOVA. *P < 0.05. In TRPM2-depleted cells treated with doxorubicin for 24 h, wild-type TRPM2-L but not the E960D mutant significantly increased phosphorylation (P = 0.005) and expression (P < 0.0001) of Pyk2, as well as phosphorylation (P ≤ 0.02) and expression (P = 0.03) of CREB, when compared with vector (V) alone or E960D. In untreated TRPM2-depleted cells, reconstitution with wild-type TRPM2 but not E960D similarly significantly increased phosphorylation and expression of Pyk2 and CREB above the level found in TRPM2-depleted cells transfected with empty vector. CREB, cAMP-responsive element-binding protein; Doxo, doxorubicin; KO, knockout; p, phosphorylated; Pyk2, proline-rich tyrosine kinase 2; TRPM2, transient receptor potential melastatin channel subfamily member 2; TRPM2-L, full-length TRPM2; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.