Panel A: Fluorescence images of A A V-injected mouse retina expressing wild type R P G R show staining for G T 335 in yellow, R P G R in magenta, and D A P I in blue. White arrowheads mark individual connecting cilia used for analysis. Below the image, five line graphs labeled W T 1 to W T 5 show the intensity profiles of G T 335 in yellow and R P G R in magenta. The horizontal axis represents the distance from the beginning of the connecting cilium in micrometers, and the vertical axis represents fluorescence intensity in arbitrary units. Panel B: Fluorescence images of A A V-injected mouse retina expressing the L 1302 A R P G R mutant show G T 335 in yellow, R P G R in magenta, and D A P I in blue, with numbered arrowheads indicating analyzed cilia. Below the image, five line graphs labeled L 1302 A 1 to L 1302 A 5 display the intensity profiles of G T33 5 in yellow and R P G R in magenta. The horizontal axis represents the distance from the beginning of the connecting cilium in micrometers, and the vertical axis represents fluorescence intensity in arbitrary units. Panel C: Fluorescence images of A A V-injected mouse retina expressing the W 1294 C R P G R mutant show G T 335 in yellow, R P G R in magenta, and D A P I in blue, with numbered arrowheads indicating analyzed cilia. Below the image, five line graphs labeled W 1294 C 1 to W 1294 C 5 show the intensity profiles of G T 335 in yellow and R P G R in magenta. The horizontal axis represents the distance from the beginning of the connecting cilium in micrometers, and the vertical axis represents fluorescence intensity in arbitrary units.
Traced cilia and representative intensity profiles from one biological replicate quantified in Fig. 3 J. (A–C) WT, (B) L1302A, and (C) W1294C RPGR AAV-injected mouse retina. Top for each condition: micrographs of immunostained mouse retina with GT335 in yellow, RPGR in magenta, and DAPI in blue. White arrows point to each of 10 traced connecting cilia, with yellow lines indicating traces. Cilia were manually traced and intensities extracted in ImageJ. Bottom for each condition: five intensity profiles of GT335 (yellow) and RPGR (magenta) along line traces, numbered according to panel above. Mice injected with WT RPGR AAV have strong GT335 signal at the proximal portion of their connecting cilia where RPGR is localized, while both mutant forms of RPGR show low GT335 signal where RPGR is localized.