Figure 4

Screening of TAMs and BMDMs from 4T1 and 67NR tumor-bearing mice for gene expression. Heat maps of expression of selected genes related to macrophage function and mechanisms of action of VD₃ in (A) tumor-associated macrophages (TAMs; freshly isolated, plated for additional 3 days, and then treated with LPS, followed by analysis; N = material pooled from 2 to 4 separations, with each separation consisting of TAMs isolated from 3 mice) and (B) bone marrow-derived macrophages (BMDMs; isolated bone marrow cells were seeded with the presence of 50 ng/mL of M-CSF for 7 days, then treated with LPS, followed by analysis; N = 3 mice). On heat maps: pink indicates values higher than those shown on the scale; crossed-out boxes indicate no detectable expression. (C) Real-time PCR analysis of selected genes in 4T1 BMDMs; N = 3. It was impossible to isolate the appropriate number of TAMs from 4T1 tumors from mice fed a deficient diet (100 IU). (A–C) The RQ data are normalized to the 1000 IU group (normal diet—control group). (D) Western-blot analysis of epithelial cell adhesion molecule (EpCAM) expression in 4T1 BMDMs; N = 5. (E) An example membrane showing western blot analysis of EpCAM. (F) Number of 4T1 colonies in the bone marrow. Bone marrow cells were seeded in the presence of 10 µM of 6-thioguanine and cultured for 14 days. The 4T1 colonies were stained with crystal violet and counted; N = 4. (G) An example image of stained 4T1 colonies. Data are shown as the mean with standard error of the mean (SEM). Statistical analysis: Dunn’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.