Cell culture slides

LNCap, DU145, and PC3 cells were grown on cell culture slides (Thermo Fisher Scientific, San Diego, CA, USA) and treated with 10 μM 5-Aza-CdR (Sigma Aldrich, St. Louis, MO, USA) for 3 days. Then, these cells were fixed in 4% paraformaldehyde, washed with PBS, blocked with 10% donkey serum, and incubated overnight with an anti-human ANO1 primary antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C. After washing with PBS, the samples were incubated with an Alexa Fluor^® 488 donkey anti-mouse IgG secondary antibody (1:200) and Alexa Fluor^® 594 donkey anti-mouse IgG secondary antibody (1:200) for 1 h at room temperature. The slides were mounted with Vectashield H-1200 including DAPI (Vector Laboratories, Burlingame, CA, USA) and visualized by a LSM700 Confocal Laser Scanning Confocal Microscope (Carl Zeiss, Jena, Germany). All experiments were performed in triplicate.

Histological and histochemical analyses of WJSC 2D cultures and WJCS-MTs were assessed on three independent samples. In the case of the WJSC 2D cultures, 1 × 10⁴ cells were seeded per chamber by using commercially available cell culture slides (Thermo Fisher Scientific, Waltham, MA, USA; Cat. nº: 154526). Cells were kept in standard culture conditions until confluence, washed in PBS, chemically fixed (3.7% neutral buffered formaldehyde), air-dried and stored at −20 °C until use. In order to conduct histological analyses of the WJSC-MTs, they were processed as a tissue. Briefly, agarose chips containing MTs were carefully harvested, chemically fixed and processed with an adapted tissue processing (dehydration, clearing and paraffin-embedding) technique which consisted in the use of slight centrifugation steps between each change as described previously [24 (link),33 (link)]. Then, 5-µm thick sections of WJSC-MTs and chamber slides containing WJSC were stained with the same histochemical and immunohistochemical panel described above (see Section 2.2).