Flowsight flow cytometer

Cells were imaged with an IncuCyte HD (Essen BioScience, Ann Arbor, MI, USA) every 6-12 h during culturing, recording cell confluency for growth curves. Alternatively, since DIP2C KO cells differed in size, growth curves were generated by collection and counting of cells at set time points. For cell size comparison, cell diameter data was collected from the Cedex cell counter (Roche Innovatis, Switzerland) at eight occasions for a total of >5000 cells/cell line. For colony formation analyses, 400 cells plated in triplicate in 6-well plates were stained with 5% methylene blue in methanol after 10 days and colonies quantified. The plating efficiency was calculated as the number of obtained colonies divided by the number of seeded cells. For cell cycle analysis, equal numbers of cells fixed in ice cold 70% ethanol were stained with FxCycle PI/RNase staining solution (Molecular Probes, Eugene, OR, USA) for 15 min at room temperature, washed once in PBS, and analysed using a FlowSight flow cytometer (Amnis, Merck Millipore, Darmstadt, Germany).

In total, 1 × 10⁶ cells were fixed with 5 mL of cold ethanol at −20 °C overnight. After washed twice with cold PBS, the cells were suspended with 200 μl of PBS, and supplemented with 10 μl of RNase A stock (#10405ES03, Yeasen) solution and incubated 60 min at room temperature. After incubation, cells were stained with propidium iodide (PI, # 40710ES03, Yeasen) solution (40 μg/mL in PBS) for 15 min at room temperature and the cell cycle was analyzed using Flowsight flow cytometer (Merck Millipore, Darmstadt, Germany). For the cell apoptosis experiment, the ARP1-SRD5A1-KD and H929-SRD5A1-KD cells post 2 μg/mL treatment for 48 h at a density of 2 × 10⁵ cells were collected and stained using Annexin-V-Allophycocyanin (Annexin-V-APC) (#640941, BioLegend, San Diego, USA) and PI for 15 min in dark conditions, and the apoptosis of the cells was detected using flow cytometer.

Cells were removed from tissue culture plates with trypsin and washed twice in ice-cold PBS. A total of 5 × 10⁵ cells were resuspended in 500 µL of binding buffer (KeyGen, China). This was followed by the addition of 5 µL of Annexin V-APC (KeyGen) and 5 µL of the counterstain, 7-amino actinomycin D (7-AAD). The mixture was incubated at room temperature for 10 min in the dark, followed by quantitative analysis of apoptosis using a FlowSight flow cytometer (Merck, Germany).