Fig 2: The effect of 1,25(OH)2D3 on the expression of VDR and PPARγ. RAW264.7 cells were stimulated with 1,25(OH)2D3 in a dose- (0.1 nM, 1 nM, 10 nM, and 100 nM) dependent manner. After 24 h, the cells were collected for (a) RT-PCR and (b) western blotting analysis. β-actin was used as an internal control. A concentration of 11.1 mM glucose was used as a control. HG: 25 mM glucose. Data are presented as the mean ± SD (n = 3-4 per group). * P < 0.05 versus control; # P < 0.05 versus HG.

Fig 3: A-F Effect of vitamin D3 on the localization of VDR in the testis of different groups. Control rat showed localization of VDR in Leydig cells (L), Sertoli cells, Spermatogonia (Spg) and Primary spermatocytes (Psc). (A) The testis of D-gal-induced aged rat showed no immunostaining of VDR. (B) The treatment of vitamin D3 resume the localization of VDR in the testis. (C,D) The alone treatment of vitamin D3 to normal rat also showed distinct localization of VDR in the testis. (E,F) The figure showed 40X magnifications.

Fig 4: Fluorescence microscopy analysis of vitamin D receptor (VDR) and fibroblast activation protein (FAP) expression in cultured NFs isolated from lungs of healthy BALB/c mice fed various amounts of vitamin D3 and/or treated with calcitriol. (A–D,I) VDR expression; (E–H,J) FAP expression. Example images from selected groups are presented for (K) VDR and (L) FAP. NFs were treated ex vivo with transforming growth factor-ß (TGF-ß) or culture media from the culture of 4T1 and 67NR cells. Cells were reviewed and photographed using an Olympus IX81 fluorescence microscope. Fluorescence was determined from images using ImageJ according to the protocol of Luke Hammond (QBI, The University of Queensland, Australia; accessed on 18 October 2021; https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html). The areas of interest (whole cell for FAP staining and cell nucleus for VDR staining) and areas of background were measured, and Corrected Total Cell Fluorescence (CTCF) was calculated using the following formula: CTCF = integrated density–(area of selected cell × mean fluorescence of background readings). Data are shown as (A–H) mean with individual data; (I–J) mean ± standard error of mean. Statistical analysis: Kruskal–Wallis test followed by Dunn’s test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig 5: The iNOS, MR, VDR, and PPARγ immunofluorescence expression after VDR siRNA intervention (200x).