Columbus image data storage and analysis software

The ciPTEC were plated in an optic 96‐well imaging plates and treated with DMKG in presence or absence of bicalutamide, cysteamine or their combination. Subsequently, the Cell Event Caspase‐3/7 Green Detection Reagent (8 µM) was added and incubated for 30 min before imaging. Caspase‐active cells were identified as described in manufacturer’s instruction. Each well was imaged using the CV7000 confocal microscope and analysed with Columbus™ Image Data Storage and analysis software (PerkinElmer, Groningen, The Netherlands).

To measure the extent of nascent peptides labeled with OPP in granules containing DDX6 and TIAR, 5 images of cells under each condition were captured by confocal microscopy (Leica SP5 AOBS, Leica, Germany). The images were analyzed using Columbus™ Image Data Storage and Analysis software (PerkinElmer, Waltham, MA, USA).
The methodology applied was based on the study of Ganassi and collaborators [32 (link)]. Briefly, the nuclei and cytoplasm were delimited based on DAPI and OPP fluorescent staining, respectively. The DDX6 and TIAR granules were identified based on fluorescence intensity using the common threshold tool. Then, the surrounding cytoplasm (which consisted of the cytoplasm region but not the granule region) was delineated. The mean OPP intensity was measured in the granules and in the cytoplasm surrounding region. OPP enrichment was calculated as the ratio of the mean intensity in each granule to the mean intensity in the surrounding region. The obtained data were plotted in frequency distribution histograms. Granule ratios of 1.5 or greater were considered to be enriched in OPP-labeled nascent peptides, as described by Ganassi and collaborators [32 (link)]. Bar graphs showing the percentages of OPP-enriched granules per cell were also plotted.

The endocytic uptake was monitored in ciPTEC following incubation for 1.5 h at 37°C with 50 μg/ml of either BSA‐AlexaFluor‐647 (A34785, Thermo Fisher Scientific) or DQ Red BSA (D12051, Invitrogen). The cells were then fixed and stained with Hoechst 33342 (1 µM) for 10 min and imaged using a CV7000 confocal microscope (Yokogawa Electric corporation, Tokyo, Japan). Data were quantified with Columbus™ Image Data Storage and analysis software (PerkinElmer, Groningen, the Netherlands). Data are expressed as the number of BSA/DQ‐BSA spots per cell.