Figure 1.

CTCFL is differently expressed in MDA-MB-435, MDA-MB-231, and MCF-7 cell lines. (A) qPCR analysis of CTCFL transcripts in selected human breast cell lines. Levels of CTCFL mRNA were calculated using absolute normalization of C_t and normalized to GAPDH levels (ΔC_t). Experiments were performed in triplicate, and each value represents mean ± SD of three independent experiments, *P < .05. (B) One representative Western blot analysis of three of total cell lysates showing the total content of CTCFL in the different breast cancer cell lines. Actin was used as quantitative loading control. In the lower panel, the blot was incubated with CTCFL-specific peptide blocking CTCFL Ab, as negative control. (C) CLSM analysis of fixed and permeabilized cells stained for intracellular CTCFL (gray). Offset and gain values of the photomultiplier channel were regulated with respect to the setup selected for MDA-MB-435 cell analysis to make fluorescence intensity comparable across experiments on different samples. Nuclei were stained with DAPI (blue). The specificity of the polyclonal Ab was assessed by staining cells with a combination of anti-CTCFL Ab and the immunogenic peptide. A representative example of four independently repeated experiments is shown. Scale bars, 20 µm.