Figure 6.

hnRNP L overexpression increases VEGFA expression in normoxia by inhibiting activity of CARE-binding miRNAs. (A) Overexpressed hnRNP L binds VEGFA mRNA in normoxia. U937 cells were transfected with pcDNA3-c-Myc-hnRNP L or empty vector (Vect.) under normoxia for 24 h. Cytosolic lysates were immunoblotted with anti-c-Myc tag and anti-GAPDH antibodies (top two panels). Cytosolic lysates were subjected to immunoprecipitation with anti-hnRNP L monoclonal antibody. Expression of VEGFA and GAPDH mRNAs in lysates (input, left panels) and after immunoprecipitation (right panels) was determined by RT–PCR (bottom two panels). (B) Overexpression of hnRNP L in normoxia prevents miRNA-mediated repression of HSR-bearing reporter. FLuc reporter bearing the VEGFA HSR was cotransfected into HEK293T cells with CARE-binding miRNAs (or control miRNA, Cont.), pcDNA3-c-Myc-hnRNP L or vector, and RLuc as transfection efficiency control. Lysates were subjected to immunoblot analysis with anti-c-Myc tag (top) and -GAPDH antibodies (middle). The relative level of FLuc was normalized by RLuc expression and expressed as percentage of control (bottom). (C) Overexpressed hnRNP L alleviates miRNA-mediated repression of endogenous VEGFA in normoxia. U937 cells were cotransfected with pcDNA3-c-Myc-hnRNP L or vector and CARE-binding miRNAs for 24 h under normoxia. Lysates were subjected to immunoblot analysis with anti-c-Myc tag, VEGFA, and GAPDH antibodies (the samples were run on the same gel but the lanes were rearranged for clarity). VEGFA expression was quantitated by densitometry and expressed as percent of normoxic control. Results are expressed as mean±s.d., for n=3 independent experiments. An asterisk indicates a significant difference, P<0.05, two-tailed t-test.