Fig 7

Fig 7 Kcnk5b requires CaMKK for growth and SHH/shha expression.

(A) SHH expression in HEK293 cells transfected with GFP or Kcnk5b-GFP after 20-h treatment at the indicated concentrations of the CaMKK inhibitor STO-609 at the indicated concentrations. (B) Expression of shha in pectoral fin buds from heat-shocked non-transgenic siblings after 6-h treatment with DMSO (a) or 24 μm STO-609 (b) and from transgenic Tg[hsp70:kcnk5b-mCherry] siblings after treatment with DMSO (c) or STO-609 (d). (C) The ratios of each in situ staining area of shha to the area of its corresponding fin bud for the indicated groups. (D) qRT-PCR of shha expression in isolated fin buds in the indicated control and experimental groups. (E) Graphed assessment of fin-bud-area-to-eye-area measurement ratios. (F, G) Difference in SHH expression in HEK293 cells transfected with mCherry or human CaMKK2-mCherry (F) or camkk1b-mCherry (G). Experiments were repeated 3 or more time (N ≥ 3). For cell qRT-PCR experiments, each RNA isolation per well was measured in duplicate or triplicate. Each measured value is represented as a data point (A, F, G). For fin bud fluorescence measurements, we measured 2 or 3 locations in each tissue of 1 fin bud per embryo. For fin bud area measurements, we measured the area of 1 fin bud and eye per embryo. We measured at least 4 embryos per repeat (E). For fin bud qRT-PCR experiments, we collected 80 fin bud samples per isolation. Three or more isolations were measured. Each isolation was measured in duplicate or triplicate samples (D). Each measured value is represented as a data point. P values represent statistical analysis by Student’s two-tailed t test. P values >0.05 are designated as “not significant” (NS). Scale bars equal 0.5 μm (B). Numerical data used in this figure are included in S7 Data.