Fig 2: Aberrant gene expression profiling and functional annotation enrichment analyses. (A) Heatmap of aberrant gene expression profiling. (B) GO (including BP, CC, and MF) and KEGG analyses of aberrant gene expression profiling. (C) GO BP enrichment of DRGs using R. (D) KEGG pathways enrichment of DRGs using R. (E) Construction of the PPI network of DRGs via Cytoscape software. (F) Multiple GSEA of varied expression of NAGLU using GO BP analysis (c5). (G) Multiple GSEA of varied expression of NAGLU using KEGG analysis (c2).

Fig 3: NAGLU knockdown causes aberrantly lysosomal accumulation in HUVEC targeting VEGFR2/ERKs and promotes EAS. (A) The protein expression levels of HB-EGF in HUVEC after transfection with si-NAGLU and si-NC as measured by Western blotting. TUBB was used as a control. (B) VEGFR2 and ERKs phosphorylation levels in HUVEC si-NAGLU and HUVEC si-NC were measured by Western blot. To monitor the equal loading of protein in the gel lanes, the upper blot was stripped and tested using anti-VEGFR2 and anti-ERKs antibodies, respectively. (C) VEGFR2 inhibition reduces ERK1/2 phosphorylation levels in HUVEC si-NAGLU. HUVEC si-NAGLU and HUVEC si-NC were both treated or untreated with 30 μM VEGFR2 inhibitor SU5614 for 12 h and then measured by Western blot. (D) VEGFR2 inhibition reduces aberrantly lysosomal accumulation in HUVEC si-NAGLU. Representative images of lysosomes labeled by the LysoTracker probe in HUVEC si-NAGLU and HUVEC si-NC, both treated with 30 μM SU5614 for 12 h. Scale bars: 10 μm. (E) MAPK/ERKs inhibition reduces aberrant lysosomal accumulation in HUVEC si-NAGLU. Representative images of the lysosomes labeled by the LysoTracker probe in HUVEC si-NAGLU and HUVEC si-NC, both treated or untreated with 50 μM PD98059 for 24 h. Scale bars: 10 μm.

Fig 4: Schematic diagram depicting the signaling pathways by which NAGLU silencing and subsequent HSPG accumulation promote hypertrophy in H9C2 cardiomyoblasts through EGFR

Fig 5: NAGLU silencing promotes ERK1/2 phosphorylation in H9C2 cardiomyoblasts.a ERK1/2 phosphorylation 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 upper blot was stripped and reprobed using anti-ERK1/2 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. b EGFR inhibition reduces ERK1/2 phosphorylation in NAGLU-silenced H9C2. ERK1/2 phosphorylation levels in H9C2 sh-CTR and H9C2 sh-NAGLU both untreated and treated for 90 min with 10 μM of the EGFR inhibitor AG1478 as measured by western blotting analysis. The upper blot was stripped and reprobed using anti-ERK1/2 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, d Expression levels of mRNAs coding for ANP (c) and BNP (d) in H9C2 sh-CTR and H9C2 sh-NAGLU, both untreated and treated for 90 min with 50 μM of the MEK/ERK pathway inhibitor PD98059, as measured by quantitative RT-PCR analysis. The amount of ANP and BNP 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