Countess 2 cell counter

To measure cumulative population doublings, cells were plated at 3 × 10⁵ cells/60 mm dish or 1 × 10⁶ cells/100 mm dish. Confluent cultures were trypsinized, viable cells were counted, and cells were replated at the same density. For viable cell counting, aliquots of trypsinized cells were stained with Trypan Blue staining solution (Invitrogen) and were counted using a Countess II cell counter (Invitrogen) equipped with a Countess^TM cell counting chamber slide (Invitrogen). If tissue cultures were not confluent, cells were grown until confluence after adding fresh media. To count senescent cells, SAβ-gal assays were conducted using a Senescence β-Galactosidase Staining Kit (Cell Signaling) according to the manufacturer’s instructions (Kim et al. 2020 ). Photographs were taken using a Nikon eclipse Ts2 microscope (Nikon, Tokyo, Japan) equipped with an HK3.1 CMOS digital camera and HKBASIC Software (KOPTIC).

PC-3 cells were cultured under standard growth conditions (37 °C and 5% CO₂ in a humidified atmosphere) in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated FCS and 2 mM l-glutamine, while for HCT-116 McCoy's 5A medium supplemented with 10% (v/v) heat-inactivated FCS and 2 mM l-glutamine was used.^{21 (link)} Cells were monitored daily using light microscopy and were passed whenever the confluency reached 90%. For passaging, the adherent cells were detached with 0.05% trypsin–EDTA for 3 min at 37 °C. After detachment, a 9-fold volume of FCS-containing complete medium was added to stop the trypsin enzyme activity, and the cell suspension was collected and centrifuged for 3 min at 800 rpm. The supernatant was discarded, and the cells pellet was washed once with PBS. Afterwards, the cells were either diluted 10-folds and transferred to a new T75 culture flask in 10 mL of the respective complete culture medium, or cell seeding for cell viability assays was performed in 96-well plates. For seeding, cells were counted using a countess II cell counter (Invitrogen, Waltham, MA, USA). For PC-3, a cell suspension was prepared with a density of 6 × 10³ cells per 100 μL, while for HCT-116, 5 × 10³ cells per 100 μL were used. Finally, 100 μL per well of these cell suspensions were pipetted in 96-well plates and allowed to adhere overnight under standard growth conditions.

For PRC1^CPM and PRC1^CKO cells, growth curves were performed by plating out 10,000 cells on each well of a 6 well plate. Cells were harvested at 24 h intervals for a period of 6 days, and live cells (not stained by trypan blue) were counted using a Countess II cell counter (Invitrogen). Counts were performed for 6 independent experiments.
For alkaline phosphatase staining, cells were first fixed in 3.7% formaldehyde for 20 min at 4°C. Cells were then stained in AP staining solution (100 mM Tris-Hcl pH 9, 100 mM NaCl, 5 mM MgCl₂, 0.4 mg/mL Napthol phosphate N-5000, 1 mg/mL Fast Violet B Salt) for 10 min, rinsed with PBS and then distilled water, and air-dried. For each cell line (UNT and OHT-treated), 3 independent experiments were performed and at least 100 colonies were counted on each occasion.

T47D cells stably expressing mGFP-KRASG12V and MTMR 2, 3, 4 or 7 knockdown were seeded at 1 ×10⁴ in triplicate onto a 96-well plate in complete growth medium containing 0.5 μg/mL puromycin. Cells were cultured for 4 days. Fresh complete growth medium containing 0.5 μg/mL puromycin was supplemented every 24 h. On day 5, cell proliferation was assayed by counting cells using Countess II cell counter (Invitrogen).

Cells were infected in suspension (T cells) or as adherent monolayers (HaCaT and Vero) with HSV-2 at indicated MOI (based on Vero cell titer) at 37°C for 2 hours, washed twice, and resuspended or overlaid with fresh medium. Multistep HSV growth kinetics were assessed by infection of CD4⁺ T cells and HaCaT cells with SD90 at 0.001 PFU/cell. Culture supernatants were harvested at indicated times, centrifuged at 500g for 5 minutes at 4°C, and frozen at –80°C. Viral titer was determined by plaque assay on Vero cells. Cell viability was determined by vital dye exclusion using a Countess II cell counter (Invitrogen).