FIG 4.

PU.1 is expressed in myeloid cells and activates the MRC1 promoter. (A) Immunoblot analysis of various cell types for expression of PU.1. A sample of HEK293T cells transfected with pcDNA-PU.1 (lane 2) was included as control. Tubulin expression was used as internal reference. (B) Comparison of exogenous and endogenous PU.1 expression. HEK293T cells (3 × 10⁶) were transfected with increasing amounts (0, 0.2, 0.65, or 2.2 μg) of a vector encoding untagged PU.1 (lanes 1 to 4). DNA concentrations were chosen to match the DNA concentrations (i.e., μg DNA per 10⁶ cells) of panel C. All samples were adjusted to 5 μg total transfected DNA using empty vector DNA (pUC19). Lane 1 is a mock-transfected control. Cell extract (20 and 40 μL) from uninfected MDMs was included as reference. Whole-cell extracts were prepared 24 h posttransfection and processed for immunoblotting using antibodies to PU.1 (α-PU.1) or tubulin (α-Tub). Relative expression of PU.1 was calculated by dividing the signal obtained for PU.1 by the signal for tubulin in each lane. Results are indicated below the PU.1 blot. (C) HEK293T cells were plated in 24-well plates (1 × 10⁵ cells/well) and transfected in triplicate with 0.1 μg each of pGL-hMRC1-Luc (hMRC1; red line) or promoterless pGL3-basic (mock; black line) together with increasing amounts (0.06 μg, 0.2 μg, and 0.4 μg) of pcDNA-PU.1 DNA. Total amounts of transfected DNA were adjusted to 0.5 μg in each sample using empty vector DNA as needed. Production of luciferase was measured 24 h later. Means and error bars representing SEM calculated from triplicate transfections are shown. (D) Cartoon of constructs employed in a dual-reporter assay. Firefly luciferase is under the control of the PU.1 responsive hMRC1 promoter; Renilla luciferase is under the control of the HSV-thymidine kinase (TK) promoter. (E and F) Dual-reporter assay to assess the specificity of PU.1 activation of the MRC1 promoter. HEK293T cells were plated in 24-well plates (1 × 10⁵ cells/well) and transfected in triplicate with 0.1 μg of pGL-hMRC1-Luc with or without 0.1 μg of pRN-TK-Luc, together with increasing amounts (0, 0.02, 0.06, and 0.2 μg) of pcDNA-PU.1. After 24 h, samples were analyzed for firefly and Renilla expression using the Promega dual-luciferase reporter assay system (Promega; catalog no. E1910). (E) Results of firefly luciferase activity. There were no statistically significant differences in firefly luciferase activities between samples with and without transfection of the Renilla luciferase regardless of PU.1 amounts (all comparisons were nonsignificant [ns]). (F) Results of Renilla luciferase activity. Renilla luciferase activities at increased PU.1 amounts were not statistically significantly different from no PU.1. Mean and error bars reflecting the SEM calculated from three analyses are shown. For details of the statistical analyses, see Fig. S1 in the supplemental material.