Harmony analysis software

L-02 cells transfected with mRFP-GFP-LC3 were fixed with 4% paraformaldehyde and stained with 10 μM Hoechst 33342. Cell images were obtained using the Operetta High Content Imaging System (Perkin-Elmer) and analyzed using Harmony Analysis Software (Perkin-Elmer). Cells were analyzed using green (GFP) or red (mRFP) fluorescence. Autophagosomes were stained yellow puncta and autolysosomes stained red puncta in merged images. Autophagic flux was determined by the increased percentage of red puncta in merged images.

CHOK1 cells stably expressing human GIPR (CHOK1 + GIPR) and CHOK1 cells were cultured in Ham’s F12 (Gibco) supplemented with 10% FBS, 1% penicillin/streptomycin/glutamine (PSG) with or without 5 μg/ml puromycin. Cells were plated at a density of 20,000 cells/well in 96-well plates and cultured for 4 h at 37 °C, 5% CO₂ to allow cell attachment prior to treatment with vehicle (culture media) or 100 nM DA-GIP. After 24 h of treatment, cells were washed three times prior to acclimation to assay buffer (F12 + 0.1% BSA) for 1 h at 37 °C, 5% CO₂. The cells were then placed on ice for another 15 min prior to treatment with a dose titration of Rhodamine GIP (8 nM to 2 µM, Phoenix Pharmaceutical) in ice-cold F12 + 0.1% BSA. Cells were incubated with Rhodamine-GIP on ice for 60 min and then washed three times with cold PBS and fixed with 4% paraformaldehyde. Cells were then washed and stained with Hoechst 33342 (Thermo Fisher) for nuclei detection. Cells were imaged with Operetta CLS high content imaging system (Perkin Elmer) and Rhodamine-GIP fluorescence was quantitated using the Harmony analysis software (Perkin Elmer). Data represented as relative fluorescence unit (RFU) with background fluorescence (calculated from CHOK1 cells) subtracted.

A custom image analysis pipeline was developed with Harmony analysis software (version 4.9, Perkin Elmer). The sampling of organoids and cell nuclei throughout the dome was performed via image analysis on individual planes as follows. Organoids were segmented based on the combined image of the three imaging channels (Hoechst 33342, Caspase 3/7 Green and PI). Gaussian filtering and region resizing were conducted to eliminate debris-derived fluorescence in the organoids’ periphery. The number of organoids and mean organoid sizes (μm²) were quantified. Organoids with an area < 250 μm² were excluded in order to eliminate single cells, small cell clumps and debris. Within the identified organoids, cell nuclei were segmented and quantified via nuclear Hoechst 33342 staining. Mean Caspase 3/7 Green and PI intensity were determined for each nucleus.

Images of immunofluorescent staining were analyzed using the Operetta high content imaging system (PerkinElmer). N/C ratio, F/G actin ratio, and cell morphology were automatically detected by the Harmony analysis software (PerkinElmer). The imaging analysis was performed in 59 fields; the size of one field was 500 µm × 650 µm, and a number of cells were evaluated. We screened troponin T-positive cells in iPSC-CMs. YAP localization, dystrophin expression, Ki67 expression, cell morphology, and actin filament status were analyzed in troponin T-positive cells.

MEFs were plated in 96‐ or 384‐well imaging plates (CellCarrier Ultra, PerkinElmer) and incubated at least 1 day at 37°C, 5% CO₂. The day of experiment, cells were incubated with NucBlue™ Live ReadyProbes™ Reagent (Thermo Fisher Scientific) and Propidium Iodide (PI, Sigma) and treated either with 4 μM Actinomycin D + 10 µM ABT‐737 ± 20 μM qVD or 0.5 μM Staurosporine ± 20 μM or 16 µM etoposide ± 20 μM qVD for the indicated time. Total cells (stained by NucBlue) and dead cells (stained by PI⁺) were imaged every hour for the indicated time with the Operetta CLS High‐Content microscope (PerkinElmer) at 40× Air/0.6 NA. PI and NucBlue were excited with the 530–560 and 355–385 nm LEDs, respectively. PI⁺/total cells over time were quantified using the Harmony Analysis Software (PerkinElmer).

BEAS-2B cells were transfected with mRFP‐GFP-LC3 for 24 h. After transfection, mRFP‐GFP‐LC3‐BEAS-2B cells were fixed with 4% paraformaldehyde and stained with 10 μmol/L Hoechst 33342. Cell images were obtained using an Operetta High Content Imaging System (Perkin‐Elmer) and analysed using Harmony analysis software (Perkin‐Elmer). Cells were detected using green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP). Puncta in autophagosome and autolysosomes were stained yellow and red, respectively, in merged images. The assessment of autophagic flux involved quantifying the increased percentage of red puncta in merged images.