Delta vision imaging system

Based on Park et al.’s paper [6 (link)], to detect cell death, HCT116 or SW480 cells were treated with Morusin (5 μM) for 24 h, and incubated with TUNEL assay mixture for 60 min by using the DeadEnd™ Fluorometric TUNEL system kit (Promega, Madison, WI, USA). Then, TUNEL-stained cells were visualized by a Delta Vision imaging system (Applied Precision, Issaquah, WA, USA).

Colon cancer cells treated by Ptero (40 µM) and/or MLT (1 mM) for 24 h were fixed with 4% formaldehyde and were permeabilized in 0.1% Triton X-100, according to the paper [13 (link)]. The fixed cells were washed with 1X PBS, blocked with 2% BSA in 1X PBS for 30 min at room temperature (RT), and incubated with the specific antibodies of NEDD9, SOX10, and Ki67 (1:1000; Abcam, Cambridge, UK) overnight at 4 °C. After washing, the cells were incubated with Alex Fluor 489 goat mouse-IgG antibody (Invitrogen) and Alexa Fluor 546 goat rabbit-IgG antibody (1:1000) for 1 h at RT. After washing twice, the nuclei of the cells were stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma) and then were visualized under a FLUOVIEW FV10i confocal microscope (Olympus). Images of NEDD9- and SOX10-stained cells were taken by a Delta Vision imaging system (Applied Precision, Issaquah, WA, USA).

Cells expressing SOD1(A4V)‐GFP were exposed to heat stress for 2 h and fixed with 4% PFA (diluted in PBS) for 10 min. This was followed by washing with PBS and then permeabilization with 0.2% Triton X‐100 (diluted in PBS) for 10 min. The cells were then washed with 2× SSC. Hybridization was then performed by incubating the cells for 2 h at 37°C in 4× SSC containing 10% formamide, 5% dextran sulfate, 1% BSA, 0.5 mM EDTA, and 100 nM biotinylated oligo‐dT probe (23‐mer). This was followed with washing in 2× SSC (3 × 10 min) and blocking in 3% BSA (diluted in 4× SSC) for 1 h at room temperature. The cells were then incubated with primary antibodies (goat anti‐biotin, Sigma‐Aldrich, B3640; rabbit anti‐G3BP1, Thermo Fisher Scientific, PA5‐29455) diluted in 1% BSA (in 4× SSC) for 1 h. The cells were then washed in 4× SSC and incubated with secondary antibodies conjugated with Alexa Fluor fluorophores (Invitrogen) diluted in 1% BSA (in 4× SSC) for 1 h. The cells were then washed in 4× SSC, then washed in 2× SSC, and finally mounted in DAPI‐Fluoromount G (SouthernBiotech). The cells were then imaged using the DeltaVision imaging system (Applied Precision) as described above.

Cover-slips with live cells were assembled into a perfusion open-closed chamber, a miniature climate box system suitable for cultivation and live cell imaging of eukaryotic cells (POC chamber, H. Saur, Germany). The chamber temperature was kept at 37°C during imaging by a heater. Images (512 x 512 pixels) were collected using a 100x 1.4 NA oil immersion objective on a Nikon inverted microscope TE2000 Eclipse equipped with a motorized Z stage (Applied Precision, USA) and linked to a Micromax CCD camera (Roper Scientific, USA), part of the DeltaVision imaging system (Applied Precision, USA). Time points were collected each 10 seconds during a period of 30 minutes. Images (S1 Raw images) were subsequently deconvolved and analyzed using softWorX software (Applied Precision, USA).

HeLa cells were imaged using a DeltaVision imaging system equipped with SoftWorx software (Applied Precision) and based on Olympus IX71 microscope. The oil immersion objective used was 60×/1.42NA/UPlanSA. A stable temperature (37°C) was maintained during imaging. Cells were imaged in 3.5‐cm glass bottom Petri dishes (MatTek) in CO₂‐independent medium Leibovitz's L‐15 (Gibco) or DMEM supplemented with 20 mM HEPES. For imaging during heat stress, a Warner heating chamber (Warner instruments) was used. Deconvolution was performed using SoftWorx (conservative algorithm, medium noise filtering). Maximum intensity projections from collected Z‐stacks were generated in Fiji (Schindelin et al, 2012). Fiji was also used for brightness adjustment, cropping, creating scale bars and insets (3× zoom). MS Excel was used to plot the prevalence of SG fusion and fission. Trajectories of SGs were generated with the TrackMate plugin in Fiji.

One day after transfection of GFP-LC3 plasmid into H28 cells, the cells were treated with Tan I for 24 h, stained with 50 nm LAMP-1 or LysoTracker red (Invitrogen, L-7528) for 30 min, and then washed three times with PBS. The cells were fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 1% Triton-X 100 in PBS for 5 min. The fixed cells were incubated with an anti-LC3II antibody (Cell signaling, 3868S) in 1% BSA-PBS overnight at 4 °C. The fixed cells were washed and then stained with the corresponding Alexa Fluor fluorescent antibody (Life Technologies, A-11008) for 30 min at RT. The cell nuclei were counterstained with 1 μg/ml DAPI (Sigma, ZA0629), and the cells were mounted onto slides. The images were obtained using the Delta Vision imaging system (Applied Precision).