Fig. 5.
a Subcellular localization of CRABP1 and CRABP2 in MCF-7 cells treated with RA. Cells were cultured in medium with serum for 24 h and then treated with 0.5 μM RA in serum-free medium for 6 h. An equivalent amount of DMSO was added to control cells. Cells were immunostained with anti-CRABP1 (red, upper panel) or anti-CRABP2 (red, lower panel) antibodies as described in Materials and Methods. DAP1 (blue) staining was used to visualize the nucleus. b Expression of RA-responsive genes in MCF-7. MCF-7 cells underwent two rounds of transfection with scrambled or CRABP1 siRNAs. Cells were treated with increasing concentrations of RA [lanes 1 to 6 (0, 5 × 10−5, 5 × 10−4, 5 × 10−3, 5 × 10−2, 5 × 10−1 μM RA)]. RNA was purified from each culture and semi-quantitative RT-PCR was carried out using gene-specific primers (Additional file 1: Table S1). c Summary of the effect of CRABP1 and RA on downstream genes and pathways. d Western blots showing the subcellular distribution of CRABP2 in SK-Br-3 cells upon CRABP1 overexpression and RA treatment. Densitometric analysis was used to quantitate CRABP2 signal intensity in the cytoplasm and nucleus relative to the cytoplasmic marker (β-tubulin) and nuclear marker (lamin A/C), respectively. Changes in band intensities are shown as fold change in relation to lane 1 (for cytoplasmic CRABP2) and lane 5 (for nuclear CRABP2)