Attune

Cell proliferation of AC009283.1 knockdown cells was analyzed using two methods: (1) counting the total number of positive cells stained with trypan blue in a TC20 Automated Cell Counter (BIO-RAD), and (2) detecting proliferation rate using Cell Trace CFSE Cell Proliferation Kit (Invitrogen, Cat C34554), according to the manufacturer’s protocol, using flow cytometry (Attune, Applied Biosystems). 7.7 × 10⁴ SKBR3 cells were seeded in 24-well plates and were transfected with shRNA or NC shRNA. Cell proliferation assays were carried out at 24, 48, 72 and 96 h. At least three independent experiments were performed for each assay.

Following combination treatment, cell culture supernatants (500 µl) were used for transwell migration assay for 72 h in Boyden’s chamber (Nunc) according to manufacturer’s instructions and using THP-1 cells. Migrated cells were stained with DRAQ5 (1 µM; Thermo Fisher) and images were acquired by high-throughput confocal imaging (Operatta CLS; PerkinElmer) with a × 5 objective. Cells were then trypsinized, and total cell counting was performed using flow cytometry (Attune; Applied biosystems).

Approximately 10⁵ cells were distributed into tubes for flow cytometry analysis of each marker. The cells were washed with 1 ml of Dulbecco’s phosphate-buffered solution FACS buffer, containing 0.1% BSA, and centrifuged at 6000 ×g for 8 min. The cells were incubated with the primary antibody (1:100), for 30 min at room temperature to analyze the expression of each immune cell population. After incubation, the cells were washed once with 1 ml of FACS buffer to remove excess antibodies and were incubated with the secondary antibody (1:300) for 30 min. The cells were washed again with FACS buffer and fixed with a 4% buffered paraformaldehyde solution. Samples were analyzed using a flow cytometer (Attune, Applied Biosystems) equipped with two lasers (red and blue). The cell populations were estimated by evaluating the percentage of cells expressing each of the markers, relative to the total number of cells acquired. To accomplish this procedure, a panel of three antibodies and two secondary antibodies were used (Table 1).

Single-cell GFP intensity was measured by flow cytometry on an Attune^® acoustic focusing cytometer (Applied Biosystems). GFP+ cells were determined as cells with >2x the maximum signal observed from non-transduced sister cultures for the BL1 channel (488 nm excitation, 530/30 nm emission filter). Mean BL1 values of all GFP+ cells in a culture were used for comparisons.

To measure E2F activity in individual cells, REF/E23 cells were collected at indicated time points by trypsinization, fixed with 1% formaldehyde in Dulbecco’s phosphate-buffered saline (DPBS), and measured for fluorescence intensity of the E2F-GFP reporter. To measure DNA content, cells were trypsinized, spun down and resuspended in Nuclear Isolation Medium (0.5% bovine serum albumin and 0.1% NP-40 in phosphate-buffered saline) with propidium iodide (PI, 5 µg/ml) and 1% RNase A (R5000, Sigma), and measured for PI fluorescence intensity in individual cells. For EdU incorporation assay, EdU (1 μM) was added to culture medium at the indicated time and kept in the medium until cell harvest, when cells were trypsinized at indicated time points and subjected to a Click-iT EdU assay according to the manufacturer’s protocol (No. C10418, Life Technologies, Thermo Fisher). Flow cytometry was used to measure GFP, EdU, and PI signal intensity in individual cells. Approximately 5,000–15,000 cells from each sample were measured using a flow cytometer (LSR II from BD Biosciences, or Attune from AppliedBiosystems). Data were analysed using FlowJo software (v. 10.0).

1 × 10⁶ cells were plated in 25 cm² angled-flasks, except VCaP cells (1.5 × 10⁶ cells). 48 and 72 h after treatments, cells were trypsinized. We performed the assay with Cell cycle test kit (BD Biosciences, California USA) according to manufacturer’s instructions. Nocodazole (Sigma, St. Louis, MO, USA) at 1 μM was used to arrest the cell cycle in G2 phase. We used half of the cell population fated to apoptosis assays. In this case, cells were processed using Annexin V- FITC Kit (Thermo Fisher Scientific, V13242) according to manufacturer’s instructions. 2-amino-N-quinoline-8-yl-benzenesulfonamide (QBS) (SIGMA) was selected as an apoptosis inducer. Attune (Applied Biosystems, Life Technologies, Carlsbad, CA) was used for performing cell cycle and apoptosis assays.