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.