Fxcycle pi rnase

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.

Cell-surface RTK FACS was conducted as previously described^{34 (link)} (primary and secondary antibodies, Supplementary Table S4) on a FACS CANTO II machine using FlowJo software (FlowJo, Ashland, OR, USA). Annexin V analyses were conducted as per the manufacturer’s instructions. For cell cycle analyses, cells were processed and stained using FxCycle PI/RNase (Life Technologies) as per the manufacturer’s instructions.

Human umbilical vein endothelial cells were cultured and treated with 0 or 0.5 mM HP for 24 h. At the end of the treatment time, cell density was adjusted to 106 cells in 0.5 ml PBS and transferred to 4.5 ml of ice-cold ethanol overnight for fixing. Subsequently, the cell suspension was spun down (300 g at 4°C for 10 min), and ethanol was decanted. Next, the cell pellet was washed in 5 ml of PBS and incubated with 0⋅5 ml of staining solution (FxCycle PI-RNAse, LifeTechnologies) for 30 min in the dark before analyzing in the flow cytometer. Ten thousand events were collected for each sample, and their histograms were plotted for data visualization and analysis (n = 2). The percentage of cells in each phase with 0 and 0.5 mM treatment was compared using Student’s two-tailed t-test (p < 0.05 was considered significant).

Crizotinib and selumetinib were purchased from LC laboratories (Woburn, Massachusetts, USA). Bovine serum albumin (BSA), Foetal bovine serum (FBS), Rosswell park memorial institute medium (RPMI), penicillin/streptomycin were purchased from Life Technologies (Auckland, New Zealand). Precision plus protein kaleidoscope, acrylamide (1:30) were obtained from Bio-Rad Laboratories (Hercules CA, USA). CL-XPosure film, supersignal west pico were obtained from Thermofisher (Auckland, New Zealand). Propidium iodide was purchased from Sigma- Aldrich (St louis, MO, USA). FxCycle PI/RNase was from Life technologies (California, USA). Annexin V-APC and Ac-DEVD-AFC caspase-3 fluorogenic substrate was purchased from BD Biosciences (New Jersey, USA).
Antibodies against ALK(D5F3), phosphorylated-ALK (Tyr1604), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Bim, Bcl2, caspase, cleaved caspase, PARP, cleaved PARP, cyclinD1, p27 were purchased from Cell Signaling Technology (Danvers, MA, USA). Erk1/2 and β-tubulin antibodies were purchased from Sigma-Aldrich (St louis, MO, USA). HRP-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse were obtained from Calbiochem (San Diego, CA, US).