Fig 2: Effects of the potent proteasome inhibitor, MG132 (100 nM) in the presence of 10 nM DHT on expression levels of KCa1.1 proteins in antiandrogen-treated MDA-MB-453 cells. MG132 was applied 24 h after the treatment with antiandrogens. (A) Protein lysates of vehicle-, 1 µM BCT-, and 1 µM EZT-treated MDA-MB-453 cells were probed by immunoblotting with anti-KCa1.1 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter. (B) Summarized results were obtained as the optical density of KCa1.1 and ACTB band signals. After compensation for the optical density of the KCa1.1 protein band signal with that of the ACTB signal, the KCa1.1 signal in the vehicle control was expressed as 1.0 (dotted line, n = 3 for each). (C) Measurement of PAX-induced depolarization responses in vehicle-, BCT-, and EZT-treated MDA-MB-453 cells. Summarized data are shown as the PAX-induced ? relative fluorescence intensity of DiBAC4(3) in vehicle-, BCT-, and EZT-treated MDA-MB-453 cells. Cells were obtained from three different batches (64, 43, and 35 cells in each group). Results were expressed as means ± SEM.

Fig 3: Comparison of KCa1.1 expression and activity between human CS SW-1353 cells cultured as 2D monolayers and 3D spheroids. A, Phenotypic properties of SW-1353 cells cultured with ultra-low attachment PrimeSurface 96 U plates (upper panel: day 0, lower panel: day 4). Bars show 50 µm. B, Time course of the voltage-sensitive fluorescent dye imaging of NS1619-induced hyperpolarizing responses in isolated cells from the 2D monolayers (“2D”) and 3D spheroids (“3D”) of SW-1353. The fluorescence intensity of DiBAC4(3) before the application of NS1619 was expressed as 1.0. Images were measured every 5 s. C, Summarized results of NS1619-induced hyperpolarizing responses in cells isolated from at least 3 different batches in each group. Cell numbers used in experiments are shown in parentheses. The values for fluorescence intensity were obtained by measuring the average for 1 min (12 images). D, KCa1.1 protein expression in the lipid raft-enriched protein lysates of both groups. Blots were probed with anti-KCa1.1 (approximately 100 kDa, upper panel) and anti-ACTB (43 kDa, lower panel) antibodies. E, Summarized results were obtained as the optical density of KCa1.1 and ACTB band signals. After compensation for the optical density of the KCa1.1 protein band signal with that of the ACTB signal, the KCa1.1 signal in “2D” was expressed as 1.0 (n = 4 for each). F, Real-time PCR examination of the KCa1.1 transcript in both groups (n = 4 for each). Expression levels were shown as a ratio to ACTB. Results are expressed as means ± SEM

Fig 4: Effects of antiandrogens on the viability of 2D- and 3D-cultured LNCaP cells and the effects of a KCa1.1 inhibitor on the antiandrogen resistance acquired by 3D-cultured LNCaP cells. (A,B): Effects of the treatment with 10 µM BCT and 10 µM EZT for 48 h on the viability of ‘2D’- and ‘3D’-cultured LNCaP cells using the WST-1 assay (n = 5 for each). Cell viability additing 0.1% DMSO instead of antiandrogens was expressed as 1.0. (C,D): Effects of the treatment with antiandrogens for 48 h on the viability of vehicle- and 10 µM PAX-pretreated (for 24 h), 3D-cultured LNCaP cells (n = 5 for each). **: p < 0.01 vs. ‘2D’; ##: p < 0.01 vs. vehicle control.

Fig 5: Inhibition of BKCa channel protects the neonatal heart from ischemia injury.A Schematics of the ischemia-reperfusion protocol. Rat P6 pup hearts were subjected to 20 min ischemia, 5 min post-conditioned with DMSO, Iberiotoxin (100 nM), or NS1619 (10 µM) followed by 60 min of reperfusion. Hearts post-conditioned with C and E iberiotoxin exhibited significantly less infarction (white) as compared to the B and E DMSO control. In hearts post-conditioned with NS1619 displayed significantly higher infarction compared to DMSO control and IbTx treatment (E). The current versus voltage relationships are plotted with or without iberiotoxin (F). Data represented as mean ± SEM for percentage infarction from four independent experiments and electrophysiology data represented from four independent litters P6 rat pups. P-values were determined by a one-tailed paired student’s t-test; *= 0.05, ***= 0.0001.