As lite software

FRAP assay was conducted using the FRAP module of the Leica SP8 confocal microscopy system. The EGFP-N or mCherry-N was bleached using 488-nm or 568-nm laser beam separately. Bleaching was focused on a circular region of interest (ROI) using 100% laser power and time-lapse images were collected. Fluorescence intensity was measured and normalized relative to pre-bleaching time points by Leica AS Lite software. GraphPad Prism 8 is used to plot and analyze the FRAP results.

Spheroids were incubated with 7.34 µM of mTHPC or M-Lipidots in 100 µL cell culture medium for up to 28 h in 96 well plates in the dark. Spheroids were picked with a 1 mL pipette and transferred to microcentrifuge tubes. After washing twice with PBS spheroids were fixed in 4 % (w/v) FA/PBS for 1 h, washed in PBS and analyzed in 18-well µ-slides (IBIDI) by widefield fluorescence microscopy (Leica DMI 6000) or confocal laser scanning microscopy (Leica, SP5). Per time point, 3–5 images were acquired using differential interference contrast (DIC) and epifluorescence and mean fluorescence was calculated from regions of interest (ROIs) which were drawn around the cell assemblies in the DIC channel with Leica AS lite software. Confocal laser scanning microscopy (Leica SP5) was performed on 3–5 fixed spheroids per condition with a 20× objective (HC Plan APO). After spheroid integrity was confirmed by DIC imaging, optical sectioning was performed with an argon laser at 488 nm for excitation of mTHPC. Pictures from the center of the spheroids were taken and processed with the imaging software Imaris (Bitplane, Belfast, UK).