Figure 7

Multiscale interactions of CG-1A and CG-8. (a) CG-1A gene expression assayed by qRT-PCR in 18 h cultures treated with 10 μg/ml CG-8 (blue bar) was 4-fold that of untreated controls (black bar). (b) CG-8 gene expression in 18 h cultures treated with 10 μg/ml CG-1A (blue bar) was 6-fold that of untreated controls (black bar). Difference between relative mean mRNA values was statistically analyzed by Student's t-test. Results are shown as mean ± S.E.M. (* p < 0.05). (c) Scheme of the principle of optical aggregometry (turbidimetry). (d) Graph comparing the aggregation of 5-day leg bud autopod cells induced by addition of CG-1A, CG-1A pre-incubated with lactose or maltose (osmolarity control), and CG-8 as well as its N-terminal domain. Lysing cells with Triton X-100 (green bar) provides a baseline for maximal light transmission under the conditions of the assay. The maximum possible transmission achieved by Triton X-100 treatment is considered 100%. Results are shown as mean ± S.E.M. (e) Plot showing the cell aggregation in real time induced by CG-1A. (f) Plot showing real-time cell aggregation induced by CG-1A in cells preincubated with either CG-8 (green) or its N-terminal domain (orange). Control aggregation (dotted blue line) occurred in cells not pre-treated with CG-8. The aggregation values in assays with the N-terminal domain of CG-8 represent the mean of two independent experiments. (g) Plot showing real-time change in cell aggregation when CG-1A was added first (to initiate the aggregation) and then CG-8 was added at different time-points (red: CG-8 added at 0.5 s, green: CG-8 added at 1 s; blue: CG-8 added at 2 s). The ordinate axis units shown in e, r and g are arbitrary units and are comparable. Values for e, f and g are mean ± S.E.M.