Evos floid cell imaging station

Bright-field microscopy of cells was performed using EVOS FLOID cell imaging station (ThermoFisher Scientific). For immunofluorescent staining, the cells grown on coverslips were fixed with 4 % (w/v) paraformaldehyde, permeabilised with 0.5 % (v/v) Triton X-100 in PBS and followed by incubations with primary antibodies, the appropriate fluorophore-conjugated secondary antibodies, mounted on glass slides and imaged using a 3i Marianas spinning disk confocal microscope, fitted with a Plan-Apochromat × 63/1.4 NA Oil Objective, M27 and a Hamamatsu ORCA-Flash4.0 v2 sCMOS Camera (all from Intelligent Imaging Innovations, GmbH, Gottingen, Germany).

HaCaT cells were grown to confluence on 24-well microplates (Iwaki) and scratched with or without 0.1 μg/ml poly I:C. After 24 h of incubation, bright field images were obtained using EVOS FLoid Cell Imaging Station (Thermo Fisher Scientific, Boston, Massachusetts, USA).

Recovered embryos were observed using a fluorescence stereomicroscope fitted with a filter for detecting EGFP (EVOS® FLoid® Cell Imaging Station; Thermo Fisher Scientific Inc.) and Rhodamine-derived fluorescence.

BHK-21 cells (2.5 × 10⁴ cells) were infected with VSV at an MOI of 0.1 in the presence of various concentrations of each small molecule in a 96-well plate and incubated for 24 h as described in Section 2.4. The culture supernatants were subjected to a plaque assay to determine their virus titers [26 (link)]. Similarly, BHK-21 cells were infected with VSV-AcGFP and cultured for 24 h. After addition of Hoechst 33342 to culture medium to a final concentration of 5 µg/mL, cells were incubated for 30 min at 37 °C in a CO₂ incubator, and then observed under a fluorescent microscope with a 20× objective (EVOS FLoid Cell Imaging Station, Thermo Fisher Scientific).

PDAC tissues were fixed in 10% neutral buffered formalin for 24 h and transferred to 70% ethanol. Tissues were embedded in paraffin, and 3–5 μm sections were processed for hematoxylin and eosin (H&E) staining using standard protocols as previously described [31 (link)]. H&E stained sections were used for the assessment of clinicopathological features of the tumor.
Cells cultured in 96-well plates were fixed in 4% formaldehyde, followed by morphology assessment or immunocytochemistry as described previously [25 (link)]. Briefly, the cells were stained with hematoxylin and eosin (H&E); images were captured under the light microscope and assessed for morphology. For immunocytochemistry, cells were incubated overnight with various primary antibodies, followed by staining with Alexa Fluor-conjugated secondary antibodies. DAPI was used for nuclear staining. Images were captured using EVOS FLoid Cell Imaging Station (Thermo Fisher Scientific). Antibody details are provided in Supplementary Materials Table S1.

HepG2 cells (2 × 10⁵) were treated with 2 µM ETTC or DMSO for 24 h. Nuclear morphological changes were investigated by using 4′,6-diamidino-2-phenylin-dole (DAPI) staining [32 (link)]. Cells were washed with PBS and incubated with 300 nM DAPI for 10 min. Images were acquired with the Evos Floid Cell Imaging Station (Thermo Fisher Scientific).

Mia Paca-2, hTERT-HPNE, SKOV-3, LNCaP, and HEK 293 cells were grown to approximately 80% confluency on 8-well chamber slides (Nest Scientific, Rahway, NJ, USA). The cells were incubated with 10 µM biotin-GSG-MCA1, biotin-GSG-MCA2, biotin-GSG-J18, or dimethyl sulfoxide (DMSO; vehicle) for 1 h at 37 °C and 5% CO₂. The cells were washed with PBS and then fixed using 10% formalin for 10 min. Next, the cells were washed thrice with PBS and blocked using 10% FBS, 0.3 M glycine, 0.05% Tween-20 in PBS for 1 h. The cells were then probed by streptavidin–fluorescein isothiocyanate (FITC) for 1 h, and the cells were washed with 0.05% Tween-20 in PBS. The slides were mounted using Pro Longwear Anti-Fade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) and left to cure overnight in the dark. Bound peptides were visualized using an epifluorescent EVOS FLoid Cell Imaging Station (ThermoFisher Scientific, Waltham, MA, USA). The fluorescent intensity per cell was quantified by calculating the corrected total cell fluorescence (CTCF) using ImageJ software (v1.52a, National Institute of Health, Bethesda, MD, USA) [34 ]. The mean background (non-cell) fluorescent intensity was used to adjust the mean fluorescent intensity per cell (region of interest; ROI).