Quantigene viewrna ish cell assay

RNA in situ hybridization was carried out using the Affymetrix Quantigene ViewRNA ISH cell assay. Briefly, cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton TX-100 in PBS for 20min at 4’C. Cells were then incubated with probes specific for E2F1 or CCNE1, which were then amplified by three rounds of branched DNA hybridization that included labeling with fluorescent probes excitable at 550nm. As this assay was used following live-cell imaging (Figure 3F), the nuclear marker H2B-mTurquoise was used to identify nuclei and estimate cellular boundaries (see Image Analysis below). While thousands of cells were stained per well, only cells with desired live-cell cyclin E/A-CDK activity dynamics were gated in silico, resulting in the mRNA being quantified in at least 96 cells per replicate.

Twelve thousand NIH3T3 cells or 2 × 10⁴ human tumor cells were plated on μ-Slides VI^0.4 (Ibidi). Forty-eight hr after seeding, cells were washed in Cytoskeleton Buffer (CB) (27 (link)) (10 mM MES pH 6.1, 150 mM NaCl, 5 mM MgCl₂, 5 mM EGTA, 5 mM glucose) and permeabilized/fixed in ice-cold Pre-Fixative mix (28 (link)) (2% paraformaldehyde, 0.01% glutaraldehyde, 0.05% saponin, in CB) for 15 min at 4°C. Cells were then incubated with ice-cold Fixative mix (2% paraformaldehyde, 0.01% glutaraldehyde, in CB) for 100 min at 4°C. Fixed cells were washed with CB twice and quenched by addition of 50 mM NH₄Cl for 5 min at 20°C. For FISH, QuantiGene ViewRNA ISH Cell Assay (Affymetrix) was used according to the manufacturer’s protocol; a human CEBPB FISH probe was used for hybridization (VA1-18129). Cells were then blocked with 5% albumin in PBS-S (0.05% Saponin, in PBS) for 1 hr at 20°C and incubated with CK2α primary antibody (1:100) for 16 hr at 4°C in 5% albumin in PBS-S. Cells were washed three times for 5 min at 20°C with PBS-S and incubated for 1 hr at 20°C with Alexa 488 and Alexa 647 conjugated secondary antibodies (1:1000) in 5% albumin/PBS-S. Cells were washed four times with PBS-S for 5 min at 20°C, co-stained with 0.1 μg/mL DAPI (in PBS) for 1 min at 20°C and then washed twice with PBS. Images were acquired using a Zeiss LSM-780 confocal microscope.