Dosage of Dyrk1a shifts cells within a p21-cyclin D1 signaling map to control the decision to enter the cell cycle.

Cell culture, transfection, antibodies and plasmids BJ-5ta cells were obtained from ATCC and cultured in 4 parts DMEM (Invitrogen) and 1 part Medium 199 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin-glutamine (PSG) (Invitrogen) and 0.01 mg/ml hygromycin B (Invitrogen) between passage number 6 and 15. Human trisomy 21 skin fibroblasts (AG5397, AG6692 and AG7438) and the age-matched normal skin fibroblasts (GM5659, GM5565 and GM5658) were obtained from the Coriell Institute for Medical Research and cultured in DMEM supplemented with 10% FBS, non-essential amino acids (Invitrogen), 55mM β-mercaptoethanol (Sigma) and PSG. Primary human fibroblasts were used for experiments up to passage 10 after receipt of the cells. Synthetic siRNAs used for this study were from Dharmacon and were used at 10-20 nM with Lipofectamine 2000 reagents (Invitrogen) according to the manufacturer's protocol. Harmine (EMD Biosciences) was used at 5μM for 6-8h unless otherwise noted. Nutlin-3 and MG132 were purchased from EMD Biosciences and were used at 10 and 50μM, respectively. Etoposide (Sigma) was used at 20μM. Sources for siRNAs, antibodies, reagents, and other constructs used in this study are provided in the Supplemental Methods .

Time-lapse microscopy

Cells were reversed transfected with indicated siRNAs for 48h prior to imaging in a 96-well dish (Costar) at a density of 4,000 cells per well. For Figure 1F , Harmine was applied immediately before imaging. Time-lapse imaging was performed in full growth media supplemented with 20mM Hepes (pH 7.4) and 1mM ascorbic acid. Images were taken in CFP and RFP channels every 12 min on an ImagExpress5000A microscope (Molecular Devices) with a 10× objective. Total light exposure time was kept under 380 msec for each time point. For tet-on experiments ( Figure 2 & Figure S2 ), images were taken in YFP channel at the end of imaging to obtain mCit-Dyrk1a intensity which remains mostly constant throughout the cell cycle. CycD1 intensity shown in Figure 2F was obtained by subsequent immunostaining and imaged in Far-Red channel following time-lapse imaging. Cells were imaged in a sealed plate at 37°C chamber.

Image Analysis

Images for the 2D signaling map experiments were analyzed using custom made MATLAB image analysis programs ( Salmeen et al., 2010 ). Briefly, nuclear centroids were identified in images of Hoechst staining. A nucleus mask was generated for each cell by expansion from the centroid to reach 30% of maximum intensity. After local background subtraction, the nuclear p21, CycD1 and phospho-Rb Serine 807/811 mean intensity were measured. The threshold level used to determine phospho-Rb positive cells was set using a k-means clustering algorithm on a well-to-well basis. Detailed analysis related to the 2D signaling map and imaging processing of the time-lapse microscopy is described in the Supplemental Methods .

Statistical analysis

Error bars represent the standard deviation, standard error of the mean or 95% bootstrap confidence interval as indicated in the legends. Statistical comparisons (p values) were obtained from two-sided t tests. The Pearson's correlation coefficients (R) were calculated as indicated.

Supplementary Material 01