Materials and Methods s13

MATERIALS AND METHODS

Immunohistochemistry To detect Akh expression in the neuroendocrine cells of adult flies, Akh RNAi transgenic flies or w-control flies were crossed against Akh-Gal4 driver flies and, after hatching, the virgin female progeny was kept for five days at 25°C. At this time, the corpora cardiac (cc) and attached tissues were manually dissected in 1×PBS. After fixation with 4% paraformaldehyde for 2 h on ice, tissues were washed 20 min in 1×PBS and 2×20 min in TNT (0.1 mol/L Tris HCL pH 7.5, 0.3 mol/L NaCl, 0.5% TritonX-100), and then blocked in TNT + 4% NGS (normal goat serum) for 1 h at room temperature. Tissues were incubated at 4°C overnight with -Akh antibody (1:300, Kim and Rulifson, 2004) and mouse -GFP antibody (1:600, A- 11121, Invitrogen/Thermo Fisher Scientific, USA), and washed in 1×PBST (PBS + 0.6% TritonX-100) for 3×10 min at room temperature before incubation with secondary antibodies (Alexa-568-goat anti-rabbit, 1:1000, Invitrogen/Thermo Fisher Scientific; Alexa-488-goat - mouse, 1:1000, Jackson ImmunoResearch Laboratories, USA) in TNT + 4% NGS for 2h at room temperature. Samples were washed in 1×PBST for 3×10 minutes at room temperature and kept overnight in 1×PBS at 4°C. For imaging, tissues were mounted in 60% glycerol/1×PBS and analysed on a Zeiss LSM-780 microscope (Zeiss, Germany) at 25× magnification in fixed gain mode. Maximum intensity projections of image stacks were done using the ZEN 2010 software (Zeiss).

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Supplementary figure legend

Fig. S1. Stim, Plc21C, Gγ1, Gαq, AkhR and Akh gene regulation in response to transgenic gene knockdown or gene overexpression at the transcriptional (A) and protein (B) level.

A: Transcriptional regulation of Plc21C, G1, Gq, AkhR, Akh and Stim genes in response to fat body targeted (FB-Gal4) or ubiquitous (da-GS) RNAi-mediated in vivo knockdown or overexpression (gof) detected by qRT-PCR analysis. B: Depletion of Akh peptide (red) from the corpora cardiaca (cc, highlighted by GFP expression in green) in response to cc-targeted Akh gene knockdown. Shown are maximum intensity projections of confocal fluorescence images of -GFP (left panel) and -Akh (middle panel) immunohistochemistry. Arrows point to the cell bodies of cc cells. Scale bars represent 20 µm.