Fig. 2: High cell density represses global mRNA production and reduces the expression level of membrane-associated synNotch signaling proteins which have short half-lives.
From: Control of spatio-temporal patterning via cell growth in a multicellular synthetic gene circuit
A Average of total RNA amount per L929 cell after 24 h of culture at the indicated densities as measured by spectrophotometer (Nanodrop). Lighter dots indicate individual experiment medians, darker dots the average of those. n = 3 experiments. B Fluorescence intensity levels read via FACS of different proteins at increasing cell density. L929 fibroblasts were analyzed by FACS 24 h after seeding at the indicated densities (1 x is 1250 cells/mm2). parental: unmodified L929; receivers: anti-GFP synNotch driving mCherry cells; sender: L929 cells expressing GFP-lig (PDGFR-GFP) on their surface; cytoplasmic GFP cells: L929 cells engineered to overexpress cytoplasmic GFP. SynNotch levels are measured via immunofluorescence with an antibody that recognizes a small peptide tag at the N-terminus of the synNotch protein (anti-myc-tag). 4000 or more cells per distribution are displayed, black dots indicate the medians of each distribution. Fluorescence levels were normalized as fold changes from the negative control, and plotted with a log10 scale. Lighter dots: individual experiment medians; darker and larger dots: average of those. n = 3 experiments. C Representative micrograph pictures of bright field and green fluorescence of L929 cells expressing GFP-lig, 24 h after being seeded at the indicated densities. 1x is 1250 cells/mm2. Scale bar 500 µm. See Supplementary Fig. S7 for results of similar experiments in mES cells. D, E Degradation kinetics of cytoplasmic GFP versus membrane-bound GFP-lig (see section “Measure of GFP and PDGFR-GFP half-life” in the “Methods”). L929 (D) and mES cells (E) are engineered to express cytoplasmic or membrane-bound GFP in a dox-controllable manner. The graphs report expression values for GFP over time measured via FACS after its expression is repressed via the small molecule Doxycycline at day 0. Normalized experimental means ± s.e.m. are reported. n = 3 experiments. Dark green: GFP levels; Light green: GFP-lig. Black horizontal dotted line: half maximum fluorescence. Colored vertical dotted lines were traced to infer proteins’ half-lives. Vertical error bars: s.e.m. Solid lines are normalized averages of 3 medians from 3 individual experiments. All experiments were repeated at least 3 times with similar results. Source data are provided as Source Data file.
