Figure 2.

NK1 therapeutic efficacy is abrogated by PI3K inhibition

(A) Human dermal adult fibroblasts (HDFa) or MPS I, MPS IIIA, and MPS IIIB patient fibroblasts were grown on coverslips and treated or not with MEK/ERK pathway inhibitor (PD98059) for 90′ at the concentration of 5x10⁻⁵ M, PI3K/Akt pathway inhibitor (LY294002) for 60′ at the concentration of 5x10⁻⁵ M, NK1 recombinant protein for 48 h at the concentration of 10⁻⁶ M, and the combination NK1/PD98059 or NK1/LY294002 before being processed for indirect immunofluorescence. The lysosomal marker LAMP1 (green) and HS (pink) were revealed by using specific antibodies. Nuclei (blue) were decorated by DAPI staining. Single focal sections are shown. Images are representative of three independent experiments made in triplicates. Scale bar: 50 μm.

(B and C) Histograms show the quantification relative to the percentage of cells with pathological enlarged lysosomes (green bars) and HS mean value of fluorescence intensity (pink bars). Means ± SEM were obtained from three independent experiments. ∗∗∗ p value <0.001. ns = not significant.

(D) Scheme of the molecular mechanism of action of NK1 which goes through HS binding, restoration of endogenous growth factor signaling, and PI3K/Akt activation rather than MEK/ERK pathway.