Figure 2

hGX induces TAG synthesis and LD formation in MDA-MB-231 cells in an enzymatic activity-dependent manner. (A) Quiescent MDA-MB-231 cells were treated with recombinant hGX (10 nM) in serum-free medium and neutral lipid accumulation was determined at indicated time points, using Nile red staining and flow cytometry. (B) Quiescent MDA-MB-231 cells were treated with hGX (10 nM) for 96 h in serum-free medium in the presence or absence of varespladib (Var; 50 μM) and stained with Nile red. (C) MDA-MB-231 cells were grown in complete culture medium in the presence of different concentrations of hGX and stained with Nile red at the indicated time points. (D) MDA-MB-231 cells were grown in complete culture medium in the presence of hGX (1 nM) for 48 h and TAG content was determined in cell lysates as described in Methods. The TAG content of hGX-treated cells was significantly higher than in that of control cells (**, P = 0.0086; Student’s t-test). (E) MDA-MB-231 cells were grown in complete culture medium in the presence of hGX (1 nM) for 48 h. The cells were fixed, stained with Nile red to visualize LDs (green) and DAPI to visualize nuclei (blue). The image shown is representative of two experiments. Values on the graphs are means ± SD of at least two independent experiments performed in duplicate. Results that are statistically significant over control samples are indicated (**, P < 0.01; ***, P < 0.001; one-way ANOVA with Bonferroni adjustment).