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Fig. 1 | BMC Developmental Biology

Fig. 1

From: Drosophila ML-DmD17-c3 cells respond robustly to Dpp and exhibit complex transcriptional feedback on BMP signaling components

Fig. 1

Identification of ML-DmD17-c3 (D17) cells, and characterization of their responsiveness to Dpp stimulation. (a) Graphical representation of gene expression values derived from modENCODE data [34] for each of six cell lines used in this study. The functional category and respective genes are listed to the left. Those with low (500–1000, yellow), medium (1000–2000, orange) and high (> 2000, red) expression are shaded proportionally to their expression values within each category. Expression values below 500 units are considered unreliable (white). It is only appropriate to compare expression values across cell lines within a gene, and not between genes (https://dgrc.bio.indiana.edu/cells/TilingDescription). (b) Quantification of relative Dad expression, normalized to Act5C expression, for each of the six cell lines used in this study, in the absence (empty bars) or presence (filled bars) of 5 nM recombinant Dpp. Baseline expression within each cell line was scaled to 1. Values given represent the mean and standard deviation of two independent assays, each with 2–3 technical replicates. P values were calculated for the pairwise comparison of means using Student’s t-test; *** P < 0.001. (c) Quantification of relative dad13-luciferase activity, normalized to CMV-Renilla activity, for S2 and D17 cells, in the absence (empty bars) or presence (filled bars) of 5 nM recombinant Dpp. Baseline expression was scaled to 1 for each cell line, and the fold-induction of dad13-luciferase activity is given within the filled bars; note the logarithmic axis. Values given represent the mean and standard deviation of two independent assays, each with 2–3 technical replicates. P values were calculated for the pairwise comparison of means using Student’s t-test; ** P < 0.01. (d) Quantification of relative bam and brk expression, normalized to Act5C expression, in D17 cells, in the absence (empty bars) or presence (filled bars) of 5 nM recombinant Dpp. Baseline expression was scaled to 1 for each gene. Values given represent the mean and standard deviation of two independent assays, each with 2–3 technical replicates. P values were calculated for the pairwise comparison of means using Student’s t-test; *** P < 0.001. (e) Quantification of relative Dad expression, normalized to Act5C expression, in D17 cells treated with the indicated concentrations of recombinant Dpp. Each assay is represented by a filled circle and independent assays are grouped by color, as indicated; median responses are indicated by black horizontal bars. The region contained within the dashed box is expanded to the right of the primary graph. Note that we observed larger variance between trials than within trials; we cannot explicitly account for these differences at this time. Data were analyzed using a general linear model using SPSS (IBM) and “Trial” was treated as a random factor. A posthoc Bonferroni test was used to calculate pairwise P values; * P < 0.05, ** P < 0.01, *** P < 0.001. (f, g) Representative images of immunocytochemical detection of pMad (green), cytoskeleton (magenta) and nuclei (blue) of untreated D17 cells (f) and of those treated with 5 nM recombinant Dpp (g) at low magnification and high magnification (insets)

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