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. 2023 Mar 22;13:4683. doi: 10.1038/s41598-023-28693-y

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Ca_V1.3-mediated Ca²⁺ signalling emerges in LNCaP during CSFBS culturing. (A) Pseudocolour micrographs of Cal-520 AM-loaded LNCaP (cultured in media containing normal FBS) during live-cell recordings. Pseudocolour scale indicating increased fluorescence intensity from blue–green–yellow–red, indicating increased intracellular [Ca²⁺]. Fluorescence micrographs in normal Hanks' (0 s) and during perfusion with high K⁺ Hanks containing Bay K 8644 (10 µM, denoted by solid horizontal bar) (100 s) are shown. Five cells (1–5, white arrows) were selected and ΔF/F0 measured then plotted on a fluorescence intensity-time graph (coloured lines, lower panel) (N = 5 recordings). (B) Pseudocolour micrographs showing CSFBS-cultured LNCaP (10 days) pre- (0 s) and during exposure (100 s) to high K⁺/Bay K 8644. Fluorescence intensity of 5 selected cells demonstrating Ca²⁺-transients in 4 of the 5 cells (lower panel) (N = 3 recordings). (C) Mean percentage of responding cells per recording is shown at several time points during CSFBS along with matched time controls. Data analysed with 1-way ANOVA with Šídák's multiple comparison test, p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) (N = 3). (D) Nifedipine (1 µM) significantly reduced the percentage of high K⁺/Bay K 8644-responding cells after 10 day CSFBS. Data was analysed with unpaired t-test where p < 0.001 (***) (N = 5 recordings). (E) Significant knockdown of CACNA1D by siRNA transfection was achieved in CSBFS-treated LNCaP (14 days, Mann–Whitney, N = 4, p < 0.05 (*). (F) The percentage of high K⁺/Bay K8644-responsive LNCaP in siRNA-LNCaP was markedly reduced compared with scrambled controls, (p = 0.0578, unpaired t-test, N = 3).