Fig 1.
Schematic representation of a transwell co-culture protocol used to study the effects of (A): normal adipocyte-conditioned cell culture media (AD-CM) and (B): cancer-associated adipocyte-conditioned cell culture media (CAA-CM) on high-concentration S1P treatment of human triple-negative breast cancer (TNBC) cells.
Fig 2.
Cell viability (% of vehicle control) of normal mammary gland epithelial cell line MCF-12A (A) and triple-negative breast cancer (TNBC) cell lines MDA-MB-231 (B) and HCC1937 (C) under S1P treatment (concentration: 100–5000 nM) in complete media (control), AD-CM and CAA-CM for 48 h and 72 h, respectively. Cell treatment with methanol was used as the vehicle control. The cell viability was measured in triplicate (n = 3) using the MTT assay. The experimental data were analyzed by one-way ANOVA for each individual type of cell culture media series and two-way ANOVA followed by Tukey’s post-hoc test on three types of cell culture media series. A significant difference was defined by p < 0.05 compared to the vehicle control under different types of cell culture media series (*complete media, #AD-CM and $CAA-CM). Under the same S1P concentration, a represents a significant difference (p < 0.05) between the indicated two groups.
Fig 3.
Morphological change of nucleus (DAPI staining: shown in small white arrows) and percentage of nucleic alteration in MCF-12A (A), MDA-MB-231 (B) and HCC1937 (C) cells under different treatment conditions for 48 h. The MCF-12A and MDA-MB-231 cells were treated with 2000 and 5000 nM S1P, respectively, in complete media, AD-CM or CAA-CM; whereas the HCC1937 cells were treated with 1000 and 2000 nM S1P, respectively, in complete media, AD-CM or CAA-CM. This experiment was carried out in triplicate and cells treated with methanol were used as the vehicle control. Percentage of nucleic alteration (mean ± SD) was calculated by dividing the number of cells with chromatin condensation, nuclear fragmentation and nuclear condensation over the total number of cells. The experimental data were analyzed by one-way ANOVA for each individual type of cell culture media series and two-way ANOVA followed by Tukey’s post-hoc test on three types of cell culture media series. A significant difference was defined by p < 0.05 compared to the vehicle control under different types of cell culture media series (*complete media, #AD-CM and $CAA-CM). Under the same S1P concentration, “a” represents a significant difference (p < 0.05) between the indicated two groups.
Fig 4.
Functional profiling (MF: molecular function; BP: biological process; and CC: cellular component) of the 57 secretome DEGs in differentiated SGBS adipocytes treated with 100 nM S1P.
This figure was generated using g:Profiler (https://biit.cs.ut.ee/gprofiler/gost). The color scheme used for the figure can also be found at the g:Profiler website.
Table 1.
Upregulated and downregulated genes encoding secreted proteins in differentiated SGBS adipocytes treated with 100 nM S1P (n = 3).