Cellcarrier 96 ultra

Primary acinar cells and 266-6 cells were used with Perkin Elmer CellCarrier-96 Ultra (Waltham, MA, USA). Cell carriers were imaged using a 40× or 10× Air objective on Perkin Elmer Opera Phenix Plus HCS System (Waltham, MA, USA) equipped with an environmental controller and gas mixer to maintain cells at 37 °C and 5% CO₂. The bright field was imaged every 5 min overnight. Images were processed and videos were generated using Perkin Elmer Harmony^® (Waltham, MA, USA). The outcome analysis was processed in Perkin Elmer Harmony^® (Waltham, MA, USA) using a custom script with a graphic user interface representing data at the field and plate levels.

The adventitial layer was removed from aortas of Myh11-Confetti animals and after overnight incubation in Opti-MEM, tissue sections for explant analysis (1 mm²) were cut, pinched with sharp forceps to create an internal injury site and embedded in Matrigel in an 8-well chamber slide (Ibidi). Explanted sections were fixed before or after 8 days of culture and mounted in RapiClear 1.47 (Sunjin Lab).
Enzyme digested medial cells from VSMC-lineage labelled animals and wild-type animals were mixed (1:3 ratio for Myh11-Confetti; 1:9 for fluorescence-activated cell sorting (FACS)-isolated SCA1+EYFP+ or SCA1−EYFP+ cells). A total of 5000 cells were seeded per well of a 96-well imaging plate (CellCarrier-96 Ultra, Perkin Elmer) and imaged using an Opera Phenix high-content screening system (Perkin Elmer). Image analysis was done using Harmony software (Perkin Elmer) and Fiji.^{25 (link)} Patches were defined as an area with three or more contiguous cells of same reporter colour and patch area determined by generating a mask using thresholding after enhancing local contrast (CLAHE).²⁶

HT1080 cells were plated on CellCarrier-96 Ultra (Perkin Elmer) at 8 × 10³ cells/well and treated with spautin-1, HCQ, bafilomycin A1, SAR405, or rotenone in the presence or absence of 2DG (10 mM) for 4 h. For siRNA experiments, HT1080 cells were transfected with control siRNAs or combination of USP10 and USP13 siRNAs. After 48 h incubation, the cells were treated with vehicle or spautin-1 (10 μM) under control or 2DG-stressed conditions for 4 h. The cells were subjected to fluorescent immunostaining using anti-ATF4 rabbit monoclonal antibody (Cat#: 11815, Cell Signaling Technology), anti-XBP1s mouse monoclonal antibody (Cat#: 27901, Cell Signaling Technology), Alexa Flour Plus 647 goat anti-rabbit IgG secondary antibody for ATF4 (Cat#: A32733, Thermo Fisher Scientific), Alexa Flour Plus 488 goat anti-mouse IgG secondary antibody for XBP1s (Cat#: A32723, Thermo Fisher Scientific), and nuclear staining with Hoechst 33342, as described previously^{25 (link)}. Fluorescent images (nine fields per well) were acquired using a 20 × water objective lens by Operetta CLS (Perkin Elmer). Quantification of ATF4 and XBP1s mean intensities in nuclei was performed using Harmony high-content analysis software (Version 4.9, Perkin Elmer).

Cells (2 × 10⁴, 100 μL/well) were seeded into sterile microplates (Cell Carrier-96 ultra, PerkinElmer, Waltham, MA, USA) and grown for 24 h. After applying the indicated cell treatments (see figure legends), cells were fixed in 3% formaldehyde/PBS solution for 15 min at room temperature, washed 3× with PBS, and incubated in blocking solution (5% BSA in PBS) for 15 min at room temperature. The anti-phospho-H2AX antibody (Table S2) was diluted in blocking solution and added to wells (50 μL/well, 2 h at room temperature). The secondary antibody (Alexa Fluor 488, Table S2) was diluted in blocking solution and incubated for 1 hour. After antibody incubations, cells were washed twice with PBS and incubated for 5 min with PBS containing 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI, Table S2) at room temperature for nuclear staining. Cells were then washed three times with PBS and were kept in 100 μL of PBS until imaging. Images were acquired using an Opera Phenix High Content Analyzer (PerkinElmer, Waltham, MA, USA) with a 10× air objective (NA 0.3). Image analysis was performed with the built in Harmony software (version 4.8).

Spheroids were transferred to glass bottom microplates (Cell Carrier-96 ultra, PerkinElmer, Waltham, MA, USA) and treated with cisplatin (25, 50, and 100 μg/mL) on day 2 and day 4. Afterward, the cells were stained with Hoechst (Table S2) and Annexin V-FITC (Table S2.) for 1 hour in growth medium. Images were acquired using an Opera Phenix High Content Analyzer (Perkin Elmer, Waltham, MA, USA). Fluorescence intensity was detected at 350 (Hoechst) and 488 nm (Annexin V-FITC).