Summit 4

The analysis was performed on ENSc at time of isolation (T0) and after 7-day culture (T7) using the primary antibodies reported in Table 1. In parallel, controls were stained using only corresponding secondary or isotypic antibodies. Data were acquired using FACSCanto II Flow cytometer (BD Biosciences, San Josè, CA, USA) and FACSDiva v6.1.3 software (BD Biosciences). The positive expression of each target marker was established using the overton subtraction tool of Summit 4.3 software (Beckman Coulter Inc, Brea, CA, USA).Table 1

Primary and secondary antibodies used for the immunophenotypical characterization of ENS cells

Primary antibodies	Manufacturing company
Mouse anti-rat NG2 FITC	Santa Cruz Biotechnology, Inc
Goat anti-rat Nanog PE	BD Biosciences
Mouse anti-rat CD34 PECy7	BD Biosciences
Rabbit anti-rat Sox2	Millipore
Rabbit anti-rat Sox10	Santa Cruz Biotechnology, Inc
Rabbit anti-rat TLR4	Santa Cruz Biotechnology, Inc
Goat anti-rat Frizzled 9	Santa Cruz Biotechnology, Inc
Mouse anti-rat Nestin	Millipore
Mouse anti-rat GFAP	Millipore
Mouse anti-rat PAN neuronal	Millipore
Rabbit anti-rat p75	Millipore
Secondary antibodies
Goat anti-mouse FITC	Santa Cruz Biotechnology, Inc
Goat anti-rabbit FITC	Santa Cruz Biotechnology, Inc
Bovine anti-goat FITC	Santa Cruz Biotechnology, Inc
Donkey anti-goat PE	Santa Cruz Biotechnology, Inc

Intracellular staining was performed using BD transcription factors staining kit (BD), according to the manufacturer instructions with modifications. In brief, cells (1 × 10⁶/tube) were incubated with BD Horizon fixable viability dye450 (1:1000) diluted in PBS (4 °C, 25 min). Cells were rinsed in PBS, resuspended in fixation buffer (4 °C, 45 min), and washed with ice-cold permeabilization/washing buffer. Fixed cells were incubated with primary antibodies (see supplementary table 1), diluted in permeabilization/washing buffer, overnight at 4 °C. The following day, cells were washed in permeabilization/washing buffer and incubated in secondary antibody (anti mouse-APC,1:500, Santa Cruz), for 45 minutes. After 3x washes in permeabilization buffer, cells were resuspended in ice-cold 500ul running flow buffer consisting of 1% bovine serum albumin (BSA) (Sigma-Aldrich) and 0.5 mM EDTA (Sigma-Aldrich) diluted in PBS. Flow cytometry analysis was performed using Beckman-Coulter CyAn analyzer (Beckman-Coulter), Summit 4.3 software (Beckman-Coulter) and Flowlogic software (Inivai technologies). Single cells were gated based on forward-side scatter profiles and dead cells excluded using violet Horizon viability dye (data not shown). Undifferentiated ESCs were used as a negative control to set gates.

S. pombe DNA content and cell size were measured by flow cytometry as previously described (Costello et al., 1986 (link)). Samples were sonicated and 30,000 events processed using a Beckman Coulter Cyan ADP instrument with 488 nm excitation detection filter and 530/40 nm bandpass. HeLa cells were fixed drop-wise with 70% ethanol and 5000 events processed as above. Data were analysed using Summit 4.3 software (Beckman Coulter).

BC cell lines were transiently transfected with reagent only (mock), miR-control, miR-145-5p, miR- 145- 3p, siRNA-control, or si-UHRF1 at 10 nM in 6 well tissue culture plates, as described previously [14 (link), 17 (link)–19 (link)]. Cells were harvested by trypsinization 72 hours after transfection and washed in cold phosphate-buffered saline. For apoptosis assays, double staining with FITC-Annexin V and propidium iodide was carried out using a FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Bedford, MA, USA) according to the manufacturer's recommendations and analysed within 1 hour by flow cytometry (CyAn ADP analyzer; Beckman Coulter, Brea, CA, USA). Cells were identified as viable cells, dead cells, early apoptotic cells, and apoptotic cells using Summit 4.3 software (Beckman Coulter), and the percentages of early apoptotic and apoptotic cells from each experiment were then compared. As a positive control, we used 2 μg/mL cycloheximide.

MDA-MB-231 and MCF-10A cells were seeded in duplicate at a density of 2 × 10⁵ cells/well and incubated for 24 h before drug treatment. After that, cells were exposed to the indicated drug concentrations for 12, 24, and 48 h to analyze cell cycle distribution. The treated cells were harvested by trypsinization followed by washing with PBS buffer and centrifugation at 4°C for 5 min at 200 × g. After overnight incubation at 4°C of fixed cells in chilled absolute ethanol, centrifugation was performed at 300 × g for 5 min and 1 ml of 1× PBS was used for re-suspension of pellet. 0.5 ml of RNase A was then applied to re-suspended cells for 20 min and subsequently stained with 1 mM PI at 37°C for 15 min. DNA content in a cell population was measured by flow cytometry (BD LSRFortessa^TM cell analyzer, United States) and cell cycle distribution was analyzed with Summit 4.3 software (Beckman Coulter, Inc.).