Differential Regulation of TauT by Calcitriol and Retinoic Acid via VDR/RXR in LLC-PK1 and MCF-7 Cells - Advances in Experimental Medicine and Biology - page 275

27.2.4 Immunohistochemistry

Cells were cultured directly on sterile cover slips (Sigma) that were placed into
6-well tissue culture plates for 2 days. The medium was removed from cells, which
were rinsed once with 1× PBS at room temperature. The cells were fixed for 10 min
at room temperature in 3.7% buffered formaldehyde and washed once in 1× PBS.
Fixed cells were dehydrated by immersing in 70, 95, and 100% ethanol for 5 min
each followed by air drying for 10 min each. Samples were rehydrated in decreasing
ethanol series (100, 95, and 70%) for 5 min each. Then samples were immersed in
1× PBS for 5 min at room temperature. The slides were immersed in quenching
solution (3% H 2 O 2 in methanol) for 5 min and then washed twice in dH 2 O for
10 min. Slides were blocked for 20 min with the blocking buffer. Primary antibodies
(antibody against VDR, RXR) were applied to slides and incubated for 1 h. Slides
were washed for 10 min with PBS, treated with a secondary antibody, and incubated
for 1 h. Afterwards the slides were rinsed with PBS for 10 min.

27.2.5 Statistical Analysis

All experiments using tissue cultures were performed in triplicate. Statistical com-
parisons were made using one-way ANOVA and Student's t -test to determine
significant differences in the means between experimental groups.

27.3 Results

27.3.1 Regulation of Expression of TauT by 1, 25(OH) 2 D 3

and Retinoic Acid (RA) in Renal Cells

To investigate whether 1,25(OH) 2 D 3 regulates TauT expression, LLC-PK1, MDCK,
and 293 renal cells were transfected with a full-length TauT promoter-reporter gene
(p923) and cultured for 24 h in the presence or absence of 1,25(OH) 2 D 3 (10 nM).
As shown in Fig. 27.1a , LLC-PK1 and 293 cells showed higher reporter gene
expression than did MDCK cells. Treatment with 1,25(OH) 2 D 3 had no influence on
the activity of the TauT promoter in all three lines of renal cells.
We then used LLC-PK1 cells to test the dose response of the TauT promoter to
1,25(OH) 2 D 3 , because it is known that LLC-PK1 cells express vitamin D receptors
(VDRs) (Barletta et al. 2004 ). Again, we found that there was no dose-response of
the TauT promoter to the 1,25(OH) 2 D 3 (0-20 nM) treatment (Fig. 27.1b ). However,
taurine uptake was elevated after 9- cis RA and all- trans retinoic acid (atRA) addition