FIGURE 5.

HuR is required for activation of RAR and suppression of tumor development by CRABP2. A, M-2^−/− cell lines stably expressing EGFP (Ctrl) or EGFP-CRABP2 (CRABP2) (Fig. 3A) were transduced with lentiviral particles encoding a non-targeting shRNA (shCtrl) or shRNA targeting HuR (shHuR). Cells were selected with puromycin to generate cell lines stably expressing the respective shRNAs. Top, overexpression of CRABP2 and reduced expression of HuR demonstrated by immunoblotting. Bottom, quantitation of HuR immunoblots. B, cells were cultured in delipidated medium for 48 h, treated with vehicle or RA (200 nm) for 3 days, and counted. Data are mean ± S.E. (n = 3). *, p ≤ 0.01. C, 3 × 10⁶ M-2^−/− cells expressing reduced levels of HuR and ectopically expressing EGFP (Ctrl) or EGFP-CRABP2 (CRABP2) (A) were injected subcutaneously into the opposite flanks of female NCr^nu/nu mice. Tumor growth was monitored. Data are mean ± S.E. (n = 10). D, levels of denoted mRNAs in tumors were assessed by qPCR. Data are mean ± S.E. (n = 3). E, cells with varying expression levels of CRABP2 and HuR (A) were cultured in delipidated medium for 48 h and then co-transfected with an RAR response element (RARE)-driven luciferase reporter gene and a vector encoding β-galactosidase. Cells were treated with vehicle or RA (20 nm for 16 h), and luciferase activity was measured and normalized to β-galactosidase (βgal) activity. Data are mean ± S.D. (n = 3). *, p ≤ 0.01 versus RA-treated control cells. Error bars represent S.E.