Sparkcontrol software

ATPase assays were performed using the EnzChek Phosphate Assay Kit (Invitrogen). 50 μL reactions contained 200 nM recombinant condensin II in reaction buffer (40 mM Tris [pH 7.5] 15 mM NaCl, 10 mM KCl, 7.5 mM MgCl_2, and 2.5 mM ATP), supplemented with 1 × MESG substrate and 1 × purine nucleoside phosphorylase, with or without 1.59 nM (50 μg/μL) bacteriophage λ DNA (48,502 bp; TaKaRa). The ratio between condensin II and λ DNA was 1 condensin II: 385 bp of DNA. Reactions were placed at 37°C on a 384-well microplate (Greiner Bio-One), and analyzed by spark multimode microplate reader with SparkControl software (Tecan). The initial ATPase rate from timepoint 2.5 min to 7.5 min was determined as ATP molecules hydrolyzed per condensin II complex per min.

Overnight cultures of the relevant strains (Competitor = EcM-Gm-CFP, Engineered strain = E. coli JW3910: p63_AF043 + pMPES_AF01) were grown, from single colonies picked from selective LB agar plates, in 5 mL of supplemented M9 media (0.4% glycerol, 0.2 % casamino acids) containing all necessary antibiotics. The overnight cultures were diluted 1:1000 into fresh M9 media containing 10 μg/mL Gentamicin and relevant inducers and grown for 6 h. The cultures were diluted to an OD (700 nm) of 0.1 using fresh M9 media containing 10 μg/mL Gentamicin and relevant inducers. The cultures were then mixed at the specified ratio. 10 μL of the culture was inoculated into 115 μL of media containing Gentamicin and relevant inducers in the wells of a 96 well microtitre plate and the plate was covered with a Breathe-Easy sealing membrane. The plate was placed in a plate reader (Tecan Spark) and grown for shown number of hours at 37 °C with continuous double orbital shaking (2 mm, 150 rpm). Measurements of absorbance (600 nm and 700 nm) and fluorescence (CFP: ex = 430 nm, em = 490 nm, GFP: ex = 485 nm, em = 530 nm, mCherry: ex = 575 nm, em = 620 nm) were taken every 20 min using Tecan Spark Control software. Data were processed using FlopR^{40 (link)} to produce population and subpopulation curves, and plotted using R⁶⁰ with ggplot2⁶¹ and dplyr^{62 (link)}.

Proline content was determined according to Carillo and Gibon [56 ]. Proline was extracted from the 0.1 g frozen tissue powder in 40% ethanol overnight at 4 °C. After cold extraction, the homogenate was centrifuged for 5 min at 14,000× g. An aliquot of extract (0.05 mL) was incubated with 0.1 mL of a ninhydrin reagent (1% (w/v) ninhydrin in 60% (v/v) acetic acid and 20% ethanol (v/v)) at 95 °C for 20 min on a TS-100 Thermo-Shaker (Biosan, Riga, Latvia). After cooling and brief centrifugation, an 0.1 mL aliquot of the reaction mixture was transferred to a 96-well microplate, and the absorbance was measured at 520 nm and 25 °C using Spark multimode microplate reader with SparkControl software (Tecan, Männedorf, Switzerland). Proline content was determined using a standard curve with proline, and the results were expressed in nmol/mg FW.

To monitor cell numbers and growth in proliferating, quiescence‐induced (serum starvation or glutamine limitation), or quiescence‐exiting cells in situ in 96‐well plates, we used automated time‐lapse microscopy imaging in a plate reader to measure cell confluence, i.e., the area of the well bottom covered by cells. Using a TECAN Spark 10 M plate reader, we acquired bright‐field microscopy images of each well in 1.5‐h intervals in standard cell culture conditions (5% CO₂ and 37°C). Images were analyzed and confluence estimated online in SparkControl software (TECAN, Männedorf, Switzerland). Confluence data during quiescence induction and subsequent regrowth are provided in Dataset EV2.

Cell confluency measurements were performed using Spark Cyto 400 multimode reader with cell imager and environmental control module (Tecan) and SparkControl Software (Tecan). Relative cell confluence was calculated by normalizing the complete virus induced cell lysis to 0%. The dimensionless roughness factor calculated by the normalized mean standard deviation of pixel intensities over all separated areas provides a measuring tool for virus induced CPE within a well. Automatic monitoring was performed at least with multiple biological replicates and confirmed by bright-field microscopy. The standard deviation within the replicates was indicated by the error bars. RFI was used for fast IC₅₀ determination. For the fluorescence measurements, the median of the RFI was determined from five measuring points within a single well. The error bars show the deviation determined from eight replicates (wells) per dilution. Red object count (ROC) was determined alternatively to confluency and roughness during live cell imaging experiments.