Fig 2: Analysis of poly(ADP-ribose)-mediated caspase-independent cell death in breast adenocarcinoma cells after TRPM2 inhibition and chemotherapeutic treatments. Immunoblot detection of (A) apoptosis-inducing factor (AIF) and (B) poly(ADP-ribose) glycohydrolase (PARG) in MDA-MB-231 breast adenocarcinoma cells after RNAi silencing. Loading controls for immunoblots were provided by the immunodetection of ß-actin. Con, untransfected cells; Scr, cells transfected with negative control scrambled siRNA oligos. (C) Quantification of cell death by flow cytometry was performed in MDA-MB-231 cells after RNAi knockdown of AIF or PARG, pretreatment with 20 µM ACA for 30 min and treatment with 100 µM MNNG. *p<0.05, one-way ANOVA and unpaired Student's t-test; error bars represent the SEM.

Fig 3: Cell death analyses of MDA-MB-231 breast adenocarcinoma cells after TRPM2 inhibition and chemotherapeutic treatments. (A) Quantification of cell death by flow cytometry in MDA-MB-231 breast adenocarcinoma cells. Cells were pretreated for 30 min with 20 µM N-(p-amylcinnamoyl)anthranilic acid (ACA), a TRPM2 inhibitor, and subsequently treated with 100 µM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 30 min. (B) Quantification of cell death by flow cytometry in MDA-MB-231 cells pretreated with 20 µM ACA for 30 min and subsequently treated with 2 µM doxorubicin (Dox) for 20 h. (C) Flow cytometric quantification of cell death in MDA-MB-231 cells pretreated with 100 µM aristolochic acid (AA; a phospholipase A2 inhibitor), 20 µM 2-aminoethoxydiphenyl borate (2-APB; a TRPM2 inhibitor) or 100 µM 2-APB for 30 min and subsequently treated with 100 µM MNNG for 30 min. All error bars represent the standard error of the mean (SEM). *p<0.05 via one-way analysis-of-variance (ANOVA) and unpaired Student's t-test.

Fig 4: TRPM2 levels in breast cell lines and knockdown of TRPM2 levels by RNAi silencing. (A) Immunoblot (a) and densitometric quantification (b) of TRPM2 protein levels in two noncancerous breast cell lines (HMEC, MCF-10A) and two cancerous breast cell lines (MCF-7, MDA-MB-231). Immunoblot detection of TRPM2 protein levels in human embryonic kidney cells (HEK 293) provided the positive control. (B) RNAi silencing of TRPM2 in noncancerous HMEC cells (a), and quantification of protein levels by densitometry (b). (C and D) RNAi silencing of TRPM2 in MCF-7 and MDA-MB-231 breast adenocarcinoma cells (a) and quantification of protein levels by densitometry (b). All densitometric values represent TRPM2:actin ratios. Con, untreated negative control cells; Scr, treatment with scrambled negative control siRNA oligos.

Fig 5: IR stimulates Ca2+ entry through TRPM2 channels especially in Bcl-2-overexpressing Jurkat cells. (a) Whole-cell current tracings recorded in Jurkat-Bcl-2 cells irradiated with 0 Gy (1st and 2nd tracings) or 5 Gy (3rd and 4th tracings, 2 h after IR). Records were obtained in unpaired experiments as described in Figure 1(a) before (1st and 3rd tracings) and during bath application of the TRPM2 inhibitor ACA (20 µM, 2nd and 4th tracings). (b) Relationship of the mean (± SE, n = 7–10) current density and the voltage recorded as in (a) in Jurkat-Bcl-2 cells irradiated with 0 Gy (open circles) or 5 Gy (closed triangles). (c) Mean (± SE, n = 6–12) conductance density of control (0 Gy, open bars) and irradiated (5 Gy, 2–5 h after IR) Jurkat-vector (left) and Jurkat-Bcl-2 cells (right) recorded as in (a) in the absence or presence of ACA. Conductance densities were calculated for the outward currents as shown by the blue and red line in (b) for control and irradiated cells, respectively. (d, e) Mean (± SE, n = 197–336) fura-2 340/380 nm ratio (d) and delta fura-2 ratio (e) as measures of cytosolic free Ca2+ concentration and Ca2+ entry in Ca2+-depleted cells, respectively. Ca2+-specific fura-2 fluorescence was recorded by imaging in control (0 Gy, open circles and bars) and irradiated (5 Gy, closed triangles and bars, 1.5–5 h after IR) Jurkat-vector and Jurkat-Bcl-2 cells using extracellular Ca2+ removal/readdition protocol. Ca2+ readdition was performed in the absence (vehicle) or presence of ACA (20 µM). *** indicates p = 0.001, ANOVA.