Figure 6

Characterization of BMDMs differentiated ex vivo with 4T1 conditioned medium (CM). BMDMs were derived from healthy mice fed diets containing varying contents of VD₃ and treated with calcitriol. BMDMs were differentiated in the presence of 50 ng/mL M-CSF and 40% v/v CM-conditioned medium. CM was derived from 100 nM calcitriol-stimulated (4T1cal CM) or not-stimulated 4T1 (4T1 CM) cells for 72 h prior to medium conditioning. Before lysate collection, BMDMs were stimulated for 24 h with 100 ng/mL LPS. Flow cytometry analysis of phenotypic markers was performed. (A–C) Results are presented as MFI from 3 independent replicates. Real-time PCR analysis of Il-23, Il-6, Tgfb, Tnfa, and Irf4 was performed. (D–H) RQ parameter was calculated using BMDMs from the 1000 IU group as a reference sample in each CM treatment. Gapdh was used for analysis as an endogenous control, N = 3. Statistic: Sidak’s multiple comparisons test; *P < 0.05, **P < 0.01.