FIG 6.

Nuclear localization of TAZ and transcriptional activation of TAZ target genes with PCBP2 depletion. (A) Nuclear localization of TAZ with PCBP2 depletion. MCF10A cells were treated with control (C) or PCBP2 (P2) siRNA and then analyzed by indirect immunofluorescence against TAZ (green) with DAPI staining of nuclei (blue). The cells were imaged with identical exposure times. (B) TAZ expression in cytosolic and nuclear fractions. Nuclear and cytosolic extracts prepared from PCBP2-depleted cells were subjected to Western blotting against TAZ, Lamin A/C (nucleus), and tubulin (cytosol) (left) and quantified (right). (C) CTGF and Cyr61 mRNA levels increase with PCBP2 depletion. Transcripts were measured by RT-PCR. (D) CTGF promoter-luciferase reporter activity with PCBP2 depletion. MCF10A cells were treated with siRNAs, followed by cotransfections with TK-Renilla and CTGF-luciferase (CTGF-Luc) or its mutant (mCTGF-Luc). Luciferase activity was normalized with TK-Renilla and is presented relative to the control (P = 0.0002). (E) Anchorage-independent growth of MCF10A cells with PCBP2 depletion. MCF10A cells were depleted of PCBP2, and then, 5,000 cells were seeded onto ultra-low-attachment plates with mammosphere formation medium. After 6 days, the cells were imaged (left), and the sizes and the numbers of all cell aggregates larger than 50 μm were recorded (right). Cumulative data from 3 experiments are shown. Horizontal bars indicate the means. All experiments were replicated three times; the error bars indicate SEM. **, P < 0.0001.