Formaldehyde 4

PC-3 cells were seeded into a 12-well plate containing cover glasses covered by FBS, at a density of 100,000 cells/well. Twenty-four hours later, cells were treated with compounds (DMSO, DOU-PEG₂₀₀₀, DOTAU, phenazine #14, NP_{DOU-PEG2000-phenazine #14,} and NP_{DOTAU-phenazine #14}) at 100 µM as indicated above. After 48 h of treatment, cells were washed with PBS1X and fixed with formaldehyde 4% (Thermo Fisher Scientific, Illkrich, France) for 15′ at RT. Subsequently, the cover glasses were washed with PBS 1 × 2 times and mounted on the glass slides using Prolong Gold antifade reagent with DAPI (Life Technologies, Villebon-sur-Yvette, France). The samples were kept to dry in the dark for 24 h, RT, and sealed with nail polish. Images were captured with Zeiss 510 META fluorescence confocal microscope plan 40×/1.4 (Carl Zeiss, Le Pecq, France) for both phenazine #14 (absorption; 452 nm, emission; 478 nm) and DAPI (absorption; 350 nm, emission; 450 nm–490 nm).

PC-3 cells were seeded into a 12-well plate containing cover glasses treated with FBS, at a density of 100 000 cells/well. 24h later, cells were treated with compounds (14, DMSO) at 100μM as indicated above. After 48h of treatment, cells were washed with PBS1X and fixed with formaldehyde 4% (Thermo Fisher Scientific, France) during 15’ at RT. Further extensive washing was performed with PBS1X before mounting the cover glasses on glass slides by using Prolong Gold anti-fade reagent with DAPI (Life Technologies, France). Glass slides were allowed to dry in the dark at RT for 24h, and cover glasses were then immobilized with nail polish. Fluorescent images of compound 14 (absorption; 452nm, emission; 478nm) and DAPI (absorption; 350nm, emission; 450-490nm) were captured with a Zeiss 510 META fluorescence confocal microscope plan 40X/1.4 (Le Pecq, France).