Fig 2: 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 3: BKCa openers control spasticity. (A) RNAseq expression of BKCa channel components in EA.hy926 cells. Inset represents the western blot of KCNMB4 in EA.hy926 cells, which was tested in four samples, and western blot of KCNMB4 expression in the spinal cord in control (n = 3) and spastic EAE spinal cord tissues (two shown, n = 6 total) showing an additional prominent band, not present in EA.hy926 cells, above the anticipated fragment size more notable during EAE. This antibody detected multiple bands in western blot analyses. (B) RNAseq expression of BKCa channel components in the spinal cords of spastic EAE (n = 3 mice) and age-matched normal mice (n = 3 mice). The results represent the mean ± SD. Inset represents the western blot of KCNMA1 in control and spastic EAE tissue (Antibody APC-021 detected a single band at the anticipated size and was repeated with antibody APC-151 with comparable results), and insets show a significant change in ratio between Kcnma1 and neurofilament light (Nefl) gene levels. (C) Percentage change in resistance to flexion of spastic hindlimbs in ABH mice in response to treatment with 5 mg·kg-1 p.o. VSN16R in water (n = 10 animals, n = 19 limbs) that were pretreated with saline or 1 mg·kg-1 i.p. paxilline (n = 7 animals, n = 14 limbs). (D) Percentage change in resistance to flexion of hindlimbs in ABH mice following injection i.p. with 1 mg·kg-1 paxilline (n = 7 animals, n = 13 limbs), 40 mg·kg-1 i.p. BMS-204352 (n = 7 animals, n = 13 limbs), 20 mg·kg-1 i.p. NS-1619 (n = 7 animals, n = 14 limbs) or 10 mg·kg-1 i.p. NS-11021 (n = 7 animals, n = 13 limbs). *Significant difference compared with baseline.

Fig 4: Increased expression of NRF2 transcripts decreases by inhibition with KCa1.1, and effects of NRF2 and HSF1 inhibitors on the expression of MRP1 transcripts in sarcoma spheroids. Real-time PCR examination of NRF2 and HSF1 in 2D- and 3D-cultured MG-63 (A, C) and SW-1353 cells (G, I) and in vehicle- and PAX (10 µmol/L)-treated (24 h) MG-63 (B, D) and SW-1353 spheroids (H, J). Real-time PCR examination of MRP1 in vehicle- and ML385 (10 µmol/L)-treated (for 24 h) MG-63 (E), and SW-1353 (K) spheroids and in vehicle- and KRIBB11 (10 µmol/L)-treated (for 24 h) MG-63 (F) and SW-1353 spheroids (L). Expression levels are shown as a ratio compared with ACTB. Results are expressed as means ± SEM. **P < .01 vs “2D” or the vehicle control

Fig 5: Comparison of KCa1.1 expression and activity between LNCaP cells cultured as 2D monolayers and 3D spheroids. (A): Phenotypic properties of LNCaP cells cultured in ultra-low attachment PrimeSurface 96U plates (upper panel: on day 0; lower panel: on day 7). Brightfield images were obtained with the Axio Observer Z1 microscope system (Carl Zeiss, Oberkochen, Germany). Bars show 500 μm. (B): Time course of the voltage-sensitive fluorescent dye imaging of KCa1.1 activator (1 μM NS1619)-induced hyperpolarizing responses in isolated cells from ‘2D’ monolayers and ‘3D’ spheroids of LNCaP. The fluorescent intensity of DiBAC4(3) before the application of NS1619 is expressed as 1.0. Images were measured every 5 s. (C): Summarized results of NS1619-induced hyperpolarizing responses in cells isolated from at least three different batches in each group. Cell numbers used in experiments are shown in parentheses. The values for fluorescent 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 KCa1.1 in both groups (n = 4 for each). Expression levels were shown as a ratio to ACTB. *: p < 0.05 vs. ‘2D’.