Fig. 1. NAGLU silencing in H9C2 cardiomyoblasts causes lysosomal storage defects.

a NAGLU mRNA expression levels in control H9C2 stably transfected with a non-targeting shRNA (H9C2 sh-CTR) and selected NAGLU-depleted H9C2 stable clones (H9C2 sh-NAGLU) as measured by quantitative RT-PCR analysis. The amount of NAGLU mRNA was normalized with respect to the amount of 18S ribosomal RNA housekeeping gene. The data reported are the mean ± SD of three independent experiments. *P < 0.05. b NAGLU protein levels in H9C2 sh-CTR and H9C2 sh-NAGLU as measured by western blotting analysis. To monitor equal loading of protein in the gel lanes, the blot was probed using anti-β-actin antibody. The data reported are the mean ± SD of three independent experiments of equal design. Densitometric analysis of the bands was performed, and the data obtained are reported on the histogram below. *P < 0.05. c NAGLU enzymatic activity in the extracts from H9C2 sh-CTR and H9C2 sh-NAGLU clones. Cell lysates were assayed for protein content and enzyme activity (mean ± SD, n = 3) as described in “Materials and methods” section. *P < 0.05. d Representative images of lysosomes labeled with LysoTracker probe in H9C2 sh-CTR and H9C2 sh-NAGLU. Higher magnification pictures are shown (small panels). Scale bar: 6 μm. In NAGLU-depleted H9C2 more abundant and larger intracellular vacuoles than control H9C2 are visible. Quantification of LysoTracker staining is the mean ± SD of three independent experiments. *P < 0.05. e Representative images of HS labeled by lectin-FITC in H9C2 sh-CTR and H9C2 sh-NAGLU. 3D reconstructions of confocal sections along Z-axis are shown (black and white panels). Scale bar: 10 μm. In NAGLU-depleted H9C2, an increased accumulation of HS in the periphery of the cells and on the cell membrane is detected compared to control H9C2. Quantification of HS staining is the mean ± SD of three independent experiments. *P < 0.05