Fig. 5

Early developmental responses are altered in tpc2⁻ cells. a Schematic diagram showing the time course of the expression of selected genes during early Dictyostelium development, including prestarvation genes (yakA and dscA), cAMP pulse-independent genes (carA and pdsA) and pulse-dependent genes (acaA and csaA). Ax2 and tpc2⁻ cells were harvested from exponential growth, washed twice with HKC-LoCa buffer, resuspended at a density of 1.25 × 10⁷ cells/ml and shaken at 100 rpm at 22 °C for up to 6 h. Every 2 h, 1.4 × 10⁷ cells were sampled for mRNA extraction and gene expression quantified by real time RT-PCR. Results are the average of two independent experiments ± SEM. b Ax2 and tpc2⁻ cells were seeded at a density of 0.7 × 10⁶ cells/ml in HL5 and grown in shaking suspension at 22 °C. Cells were counted at the ninth hour and then every 24 h after seeding, and the cell number is plotted against the time. Results are the average of three independent experiments ± SEM. c Conditioned media were collected by seeding Ax2 and tpc2⁻ cells at a density of 0.7 × 10⁶ cells/ml in HL5 and growing in shaking suspension at 22 °C for 48 h, and cells were removed by centrifugation. Exponentially growing Ax2 and tpc2⁻ cells were harvested and resuspended in conditioned media from either tpc2⁻ or Ax2 cells at a cell density of 2 × 10⁶ cells/ml. Cells were incubated at 22 °C and counted at the times shown. Results are the average of three independent experiments ± SEM. d Ax2 and tpc2⁻ cells stably expressing the autophagy marker RFP-GFP-Atg8 [33] were imaged either during exponential growth or following 10 min of starvation in HKC-LoCa buffer. Representative punctae are highlighted by arrows. Scale bars, 10 μm. The number of punctae per cell and the number of red/green and red-only punctae were quantified. One experiment representative of three is shown. Further images are shown in Additional file 5: Figure S5