Fig. S6

Fig. S6 PKA localizes to cilia, and RAB23 expression does not expand zebrafish somite HH signaling or HH-dependent fates, related to Figure 5

(A) Immunofluorescence imaging of NIH/3T3 cells transfected with GFP alone and stained for GFP and cilia (TUB^AC). Nuclei are stained with Hoechst. Arrowheads indicate cilia depicted in inset. Scale bars, 10 μm and 2 μm (inset).

(B) NIH/3T3 cells were co-transfected with GPR161-FLAG, PKA-Cα-YFP, and NeonGreen-PKA-RIα and stained for cilia (TUB^AC) and indicated constructs: (i) GPR161-FLAG (FLAG), (ii) PKA Cα-YFP (YFP), (iii) NG-PKA RIα (NeonGreen). Nuclei are stained with Hoechst. Yellow arrowheads indicate cilia depicted in inset. Scale bars, 10 μm and 2 μm (inset).

(C) Quantification of relative ciliary enrichment of PKA-Cα-YFP compared to GFP alone. n = 18 cells from 3 independent experiments. Significance was determined via two-tailed unpaired t test. p values less than 0.05 were considered statistically significant and are denoted as follows: ^∗∗∗∗ < 0.0001. Data are represented as means ± SD.

(D) Immunofluorescence imaging of somites 12-15 in Gli:mCherry transgenic embryos expressing GFP alone, RAB23 S23N, or RAB23 Q68L. Images depict 24hpf somites stained for MPs (En, green) or mCherry (magenta).

(E) Quantification of Gli:mCherry-expressing cells per somite of uninjected embryos, and those expressing GFP, RAB23 S23N, or RAB23 Q68L. mCherry-expressing cells in somites 12 through 15 were counted and the average per somite determined for each embryo.

(F) Quantification of En-expressing MPs per somite of uninjected embryos, and those expressing GFP, RAB23 S23N, or RAB23 Q68L. n > 12 embryos quantified from two injections. Significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. p values less than 0.05 were considered statistically significant and are denoted as follows: ^∗ < 0.05. Data are represented as means ± SD.