Figure 1

hGX sPLA ₂ stimulates the proliferation and prevents serum withdrawal-induced cell death of breast cancer cells in an enzymatic activity-dependent manner. After serum deprivation for 48 h (A) or 24 h (B) MDA-MB-231 cells were treated with recombinant hGX (10 nM) in serum-free medium containing 0.1% FAF BSA for 24 h (A) or 96 h (B) in the presence or absence of the sPLA₂ inhibitor varespladib (Var) at a final concentration of 50 μM. MDA-MB-231 cells grown for 24 h in complete culture medium were transiently transfected with empty vector and plasmids encoding the wild-type hGX or catalytic-site mutant hGX(H48Q) (C, D). The cells were then cultured in complete medium in the presence or absence of varespladib for an additional 48 h (C). Alternatively, the cells were washed at 24 h post-transfection, and incubated in serum-free medium containing 0.05% FAF BSA for an additional 96 h (D). Breast cancer cell lines were exposed to prolonged serum-deprivation without medium renewal, as described in Methods, and treated with 10 nM recombinant hGX in the presence or absence of 50 μM varespladib (E). Cell proliferation (A, C) was determined using the EdU incorporation assay on fixed cells with additional 7-AAD staining for accurate quantification of cells in the S-phase of the cell cycle. Cell death (B, D, E) was analyzed using the TMRM/YO-PRO-1 assay and the percentage of TMRM negative and YO-PRO-1 positive (late apoptotic) cells within the population was used for final analyses. The resulting values are means ± SD of at least two experiments performed in duplicate. Results that are statistically significant over control samples are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Bonferroni adjustment).