75 mm polypropylene tubes

Cells were treated with Ethanol and UiO-66_N at 1 μg for 24 h and 48 h respectively; then the culture medium was collected and centrifuged (400× g, 7 min) in order to recover cells in suspension and cells were washed two times with PBS and processed for cell cycle analysis by propidium iodide staining and flow cytometry. Briefly, the cells were resuspended in 0.4 mL of hypotonic fluorochrome solution (50 μg/mL propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) in 12 × 75-mm polypropylene tubes (BD Biosciences Italy, Milano, Italy). The tubes were kept at 4 °C for at least 30 min before flow cytometric analysis. The propidium iodide fluorescence of individual nuclei was measured using a FACScan flow cytometer (BD Biosciences Italy, Milano, Italy) at 488 nm. The percentages of cells in G0/G1, S, and G2/M phases and apoptotic cells were calculated using Cell FIT cell cycle analysis version 2.0.2 software.

Cell cycle was analyzed by flow cytometry. One-hundred-thousand cells were seeded into six well plates and treated with 2.5 μM PP242, 500 nM wortmannin or 1 μM rapamycin. Twenty-four hours and 48 h later, the culture medium of each sample was collected and centrifuged (400× g, 7 min) in order to recover apoptotic cells. Attached cells were washed twice with PBS and resuspended with 0.5 ml of hypotonic propidium iodide solution (50 μg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) in 12 × 75 mm polypropylene tubes (BD Biosciences). The tubes were kept at 4°C for at least 30 min before flow cytometry analysis. Flow cytometry experiments were performed using Coulter Epics XL-MCL Flow Cytometer (Beckman Coulter) and data were analyzed using FlowJo software (TreeStar).