Fxcycle pi rnase

To analyze apoptosis, cells were treated as indicated and allowed to recover for 4 days. Floating cells were collected, and then attached cells were collected with trypsin and combined. After centrifugation and washing, the cells were incubated with Alexa Flour 488 annexin V and 1 µg ml^–1 PI in 1× annexin-binding buffer for 15 min in the dark (Thermo). After resuspending in additional binding buffer, the cells were analyzed on an Accuri C6 (Beckman) using FL1 and FL3.
For cell cycle analysis, 23 h after treatment, cells were pulsed with 20 µM EdU and incubated for an additional hour (Thermo). Cells were collected with trypsin, washed with 1% BSA in PBS and then fixed with Click-IT fixative D. After washing with 1% BSA in PBS, the cells were permeabilized with 1× component E for 15 min, before performing Click chemistry with Alexa Flour 488 azide for 30 min in the dark. Cells were washed with 1× component E, and then resuspended in 500 µl FxCycle PI/RNase (Thermo) for 15 min before analyzing on Accuri C6. Standard gating for cells versus debris and singlet was conducted.

For hepatocyte nuclei isolation, liver lobes from mice were homogenized in cold 1% formaldehyde in PBS with a loose pestle and Dounce homogenizer. Samples were then fixed for 10 min at room temperature followed by incubation for 5 min with glycine at a final concentration of 0.125 M. Samples were centrifuged at 300 × g for 10 min, at 4°C. Pellets were washed in PBS and re-suspended with 10 ml cell lysis buffer (10 mM Tris–HCl, 10 mM NaCl, 0.5% IGEPAL) and filtered through 100 μm cell strainers. A second round of homogenization was performed by 15–20 strokes with a tight pestle. Nuclei were pelleted at 2000 × g for 10 min at 4°C and re-suspended in 0.5 ml PBS and 4.5 ml of pre-chilled 70% ethanol and stored at −20°C before downstream GFP content and ploidy analysis by flow cytometry.
Right before flow cytometry, 1 million nuclei were re-suspended in PBS and stained with FxCycle PI/RNase (Thermo Fisher, F10797, Waltham, MA) staining solution for 15–30 min at room temperature. Cells were analyzed on an FACS ARIA II (BD). Data were processed with FACS Diva 8.0 software (BD) and FlowJo v10 (FlowJo). Doublets were excluded by FSC-W × FSC-H and SSC-W × SSC-H analysis. Single-stained channels were used for compensation and fluorophore minus one control was used for gating.

Cell pellets were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with methanol at −20°C. Cells were stained for one hr with Phospho-Rb Ser 807/811 (D20B12 XP, Cell Signaling Technology, Danvers, MA) at 1:500 dilution prior to Alexa Fluor 488 secondary antibody (ab150073, Abcam, Cambrdige, MA) staining at 1:500 dilution for one hr. Antibodies were diluted in PBST with 1% BSA. PI DNA staining was performed using FxCycle PI/RNAse (Thermo Fisher Scientific F10797). FACS for phospho-Rb and PI was performed on a BD FACSCelesta instrument and analyzed with BD FACSDiva v8 software (BD Biosciences, San Jose, CA). GFP: Ex 488, Em 530/30; mCherry Ex 561, Em 610/20. FACS enrichment of stable cell lines was performed using BD FACSAria Fusion with the following optics: CFP: Ex 445, Em 470/15; YFP: Ex 488, Em 530/30; and mCherry Ex 561, Em 610/20.