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Fig. 6. In vivo validation of CG2165 using transgenic RNAi. (A-E) Wild-type (A,B) and CG2165 RNAi (C-E) primary muscle phenotypes at 18°C cultured for 4 days (A,C), 8 days (E) and 11 days (B,D), revealed by phalloidin staining for Actin. (F) Western blots probed with rabbit anti-Drosophila PMCA (top) and mouse anti-tubulin (bottom, as loading controls). Note that the expression of PMCA was significantly reduced in muscle-specific CG2165 RNAi larvae (lane 2) compared with wild type (lane 1). (G) Comparison of the body size in wild-type (top) and muscle-specific CG2165 RNAi (bottom) first instar larvae of the same age (30 hours AEL at 25°C). Note the short size and hypercontracted appearance of muscle-specific CG2165 RNAi larvae. (H,I) Confocal fluorescent micrographs showing the ventral internal muscles of first instar larvae of UAS-Dcr-2; Dmef2-Gal4 (H) and muscle-specific CG2165 RNAi (I) stained for Mhc and Actn. Arrows in I point to the rounded-up muscles. Note that although both control and muscle-specific CG2165 RNAi VL4 muscles (brackets) contained a comparable number of sarcomeres longitudinally, the length of CG2165 RNAi VL4 muscles is only half that of the wild type (lines labeled L), and thus the sarcomere size was only 50% of that in wild type. Also note that the transverse distance (T) between two VL4 muscles in the same segment in muscle-specific CG2165 RNAi larvae is much greater than that in the wild type (lines labeled T). (J) Fluorescent micrograph showing a larva with almost complete rounded-up muscles as revealed by staining for Mhc. (K) Fura PE 3 ratiometric calcium imaging micrographs of primary muscles derived from embryos of wild type (top) and UAS-Dcr-2/+; Dmef2-Gal4/UAS-CG2165 hp (bottom) and cultured at 25°C for 3 days. The color indicates the ratio between the emission intensities excited at 340 nm and 380 nm, and reflects a measurement of calcium concentration. (L) Bar chart showing [Ca²⁺]_i as average±s.e.m. for wild-type control cells (0.344±0.0162 µM; n=35 muscle cells in two representative experiments, white bar), and for muscle-specific CG2165 RNAi (0.0105±0.0012 µM; n=71 in three representative experiments, gray bar). Scale bars: 50 µm in A for A-E; 20 µm in H for H,I; 75 µm in J.

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