Endogenous Replication Stress in Mother Cells Leads to Quiescence of Daughter Cells.

Cell culture and reagents

MCF10A human mammary epithelial cells were maintained in DMEM/F12 (ThermoFisher) supplemented with 5% horse serum (Invitrogen), 20ng/ml epidermal growth factor (EGF, Sigma-Aldrich), 0.5 mg/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO), 100ng/ml cholera toxin (Sigma-Aldrich) and 10 µg/ml insulin (Invitrogen). Hs68 primary human foreskin fibroblasts were cultured in DMEM with 10% FBS and penicillin/streptomycin, and RPE-hTERT retinal epithelial cells were cultured in DMEM/F12 with 10% FBS and penicillin/streptomycin. For live-cell time-lapse imaging, phenol-red free DMEM/F12 was used. MCF10A, Hs68 and parental RPE-hTERT cell lines were purchased from ATCC. Aphidicolin (#A0781) and Hydroxyurea (#H8627) were purchased from Sigma-Aldrich.

Constructs

MCF10A cells expressing the CDK2 sensor (DHB-mVenus) and tagged histone H2B (H2B–mTurquoise) are as described in ( Spencer et al., 2013 ). The MCF10A DHB-mVenus-H2B–mTurquoise cells were further transduced with mCherry-BP1 lentivirus (construct courtesy of Dr. Galit Lahav). RPE-hTERT mCherry-BP1 cells (cell line courtesy of Dr. James Orth) were transduced with DHB-mVenus and H2B–mTurquoise lentivirus. Cells expressing all three fluorescent proteins were sorted using FACS.

Immunofluorescence

Cells were fixed with 4% paraformaldehyde, washed twice with PBS and incubated with a blocking/permeabilization buffer (10% FBS, 1% BSA, 0.1% TX-100, 0.01% NaN3) for an hour at room temperature. Primary antibody staining was carried out overnight at 4°C and visualized using secondary antibodies conjugated to Alexa Flour 488 or Alexa Flour 546. Where indicated, cells were incubated in media containing 10µM EdU for 15 mins, and then fixed and processed according to manufacturer’s instructions (ThermoFisher # C10340 ). Images were acquired on an IXMicro microscope (Molecular Devices) with a 10X 0.45NA objective. Antibodies used in this study are: 53BP1 (BD Biosciences #612522), p21 Waf1/Cip1 (CST #2947), phospho-Rb (Ser807/811) (CST #8516), Alexa Flour 488 Goat anti-mouse (ThermoFisher A-11001) and Alexa Flour 546 Goat anti-rabbit (ThermoFisher A-11035).

Time-lapse microscopy

Cells were plated at least 24 hr prior to imaging in phenol red-free full-growth media in a 96-well plate (Greiner bio-one #655090) such that the density would remain sub-confluent until the end of the imaging period. For experiments that required drug addition, cells were first “pre-imaged” for ~12 hr in full growth media without any drugs. At the time of drug addition, 50% of the media in the wells was replaced with media containing a 2X drug concentration and cells were imaged for additional 48 hr in presence of the drug. Images were acquired every 12 min on an IXMicro microscope (Molecular Devices) with a 10X 0.45NA objective. Total light exposure time was kept under 300 ms for each time point. Cells were imaged in a humidified, 37°C chamber at 5% CO 2 . Cell tracking was done using custom MATLAB scripts, as in ( Cappell et al., 2016 ). See Supplementary Methods for details.

Supplementary Material 1 2 3 Supplementary movie S1: Related to Figure 6 . CDK2 activity in MCF10A cells treated with DMSO MCF10A cells expressing the CDK2 sensor (DHB-Venus) were imaged every 12 min for 60 hr with a 10X 0.45NA objective. Cells were imaged for 12 hr in full-growth media and then for an additional 48 hr in full-growth media containing DMSO. Scale bar is 50µm (first frame). 4 Supplementary movie S2: Related to Figure 6 . CDK2 activity in MCF10A cells treated with 0.4µM Aphidicolin MCF10A cells expressing the CDK2 sensor (DHB-Venus) were imaged every 12 min for 60 hr with a 10X 0.45NA objective. Cells were imaged for 12 hr in full-growth media and then for an additional 48 hr in full-growth media containing 0.4µM Aphidicolin. Scale bar is 50µm (first frame).