Summary of the primer sets used in qRT-PCR.

<p>Summary of the primer sets used in qRT-PCR.</p

Aggregation stream formation under the submerged condition.

<p>Ax2 or <i>stlA</i> null cells were plated at 5×10⁵ cells/cm² under the submersion. Photographs were taken at the indicated time. <i>StlA</i> null cells showed shorter aggregation streams and smaller aggregation territories than these of Ax2 cells (see arrows). In the presence of 200 nM MPBD, <i>stlA</i> null cells showed normal aggregation streams. These observations were confirmed in experiments repeated more than 3 independent times. Bars indicate 500 µm (top panels) and 200 µm (bottom panels).</p

Effect of exogenous cAMP pulses on chemotaxis in <i>stlA</i> null cells.

<p>(A) Cells were starved at 2×10⁷ cells/ml in PB with or without 20 nM cAMP pulses every 6 min for 6 h. Droplets of pulsed cells were spotted next to 500 nM cAMP droplets on hydrophobic agar plates. Photographs were taken after 1 h. Bars: 200 µm. (B) Chemotaxis rate of the <i>stlA</i> null mutant with or without cAMP pulsing. Cells were assayed for chemotaxis towards indicated concentrations of cAMP using the two droplet chemotaxis assay and their chemotaxis rates were calculated (see Materials and Methods). Values are the means and SEM (bars) of 10 independent experiments (n = 10). *<i>p</i><0.001 (two paired t-test).</p

Developmental phenotype of <i>stlA</i> null mutant in the early stage.

<p>(A) Cells were developed on the PB agar plate at a density of 1×10⁶ cells/cm². <i>StlA</i> null cells (bottom panels) showed aggregation delay compared to wild type strain, Ax2 cells (top panels). (B) Cells were developed on nitrocellulose filters at 1×10⁶ cells/cm². The cells were observed when they finished forming multicellular aggregates. (C) Fruiting body of Ax2 and <i>stlA</i> null mutant developed on the PB agar plate at a density of 1×10⁶ cells/cm². Bars indicate 500 µm (A and B) and 1 mm (C), respectively.</p

cAMP pulsed <i>stlA</i> null cells show normal aggregation in the absence of MPBD.

<p>Cells were stimulated by 20 nM cAMP pulses every 6 min for 6 h and then were developed on nitrocellulose filters at 1×10⁶ cells/cm². Time points on panels indicate time elapsed since cells were washed following the completion of cAMP pulsing. cAMP pulsed <i>stlA</i> null cells show normal development without developmental delay. We observed the same phenomena in experiments repeated 3 independent times. Bar: 400 µm.</p

Representative result of chemotactic response to folate.

<p>Cells were harvested, washed twice, and resuspended in PB (2×10⁷ cells/ml). The cell droplet was spotted next to a droplet of 500 µM folate on a hydrophobic agar plate. Photographs were taken after 4 h from placing the cell droplet by phase- contrast microscopy. We took photographs of the cell droplet dividing into two pieces and then connected those photographs because we were not able to take it with one piece. Pictures presented are the connected photographs. Bars: 1 mm.</p

cAMP relay response in <i>stlA</i> null mutant.

<p>Cells were developed on non-nutrient agar plates until aggregation territories started to form. Cells were resuspended in PB and stimulated with 5 µM 2′-deoxycAMP and 5 mM DTT or with 5 mM DTT only. (A) cAMP accumulation in Ax2 or <i>stlA</i> null cells without 2′-deoxycAMP stimulation. The cAMP accumulation in <i>stlA</i> null cells was less than half of that in Ax2 cells. (B) cAMP accumulation in each cell that ACA was activated by 2′-deoxycAMP stimulation. <i>StlA</i> null cells showed a slower production rate than Ax2 cells. However, the accumulation in <i>stlA</i> null cells was almost the same as that in Ax2 cells eventually. Values are the means and SD (bars) of 3 independent experiments (n = 3).</p

Expression patterns of cAMP signalling genes in <i>stlA</i> null mutant.

<p>Expression levels of each gene were analyzed by qRT-PCR. The levels were normalized to that of the endogenous control <i>ig7</i> (mitochondrial large rRNA). In addition, the expression value of Ax2 at some time point (<i>carA</i>, <i>acaA</i>, <i>pdsA</i>, <i>gpaB</i> and <i>csA</i>: 6 h, <i>regA</i>: 3 h, <i>piaA</i> and <i>dagA</i>: 12 h) in each gene was used as an internal standard and values were normalized it. Values indicated in graphs are the means and SD (bar) of 3 independent experiments, which were performed by using independent total RNA.</p

Chemotaxis defects in <i>stlA</i> null cells and the restoration by MPBD addition.

<p>(A) Cells were starved for 6.5 h in PB in the presence or absence of 200 nM MPBD and then the cell droplets were spotted next to 250 nM cAMP droplets on a hydrophobic agar plate. Cell droplets did not contain MPBD as starved cells were washed twice to eliminate MPBD before using this assay. The distribution of cells within droplets was observed and photographs were taken after 1 h. Bars: 100 µm. (B) Chemotaxis rate of the <i>stlA</i> null mutant with or without various doses of MPBD during starvation. The rate was calculated as the percentage of the number of ‘positive’ droplets to that of total droplets by the two droplet chemotaxis assay (see Materials and Methods). Cells were assayed for chemotaxis towards indicated concentrations of cAMP. Values are the means and SEM (bars) of 10 independent experiments (n = 10). The statistical analysis was performed by one-way ANOVA with Bonferroni's post-test. Significant differences (<i>p</i><0.05) were shown in pairs as follows: Ax2–<i>stlA</i> null (towards 10, 50, 100, 250, and 500 nM cAMP), Ax2–<i>stlA</i> null with 1 nM MPBD (towards 100, 250, and 500 nM cAMP), Ax2–<i>stlA</i> null with 10 nM MPBD (towards 100 and 250 nM cAMP), <i>stlA</i> null–<i>stlA</i> null with 10 nM MPBD (towards 100, 250 and 500 nM cAMP), <i>stlA</i> null–<i>stlA</i> null with 50 nM MPBD (towards 100, 250 and 500 nM cAMP), <i>stlA</i> null–<i>stlA</i> null with 100 nM MPBD (towards 100, 250 and 500 nM cAMP), <i>stlA</i> null with 1 nM MPBD–<i>stlA</i> null with 10 nM MPBD (towards 100 nM cAMP), <i>stlA</i> null with 1 nM MPBD–<i>stlA</i> null with 50 nM MPBD (towards 100, 250 and 500 nM cAMP), and <i>stlA</i> null with 1 nM MPBD–<i>stlA</i> null with 100 nM MPBD (towards 100 and 500 nM cAMP).</p

MOESM3 of Phylogeny-wide conservation and change in developmental expression, cell-type specificity and functional domains of the transcriptional regulators of social amoebas

Additional file 3: Table S2. Transcription factor conservation. Conservation and change in the presence, developmental expression and functional domain architecture in transcription factors across five Dictyostelid genomes