(A) The upper panel shows insertion of the gene trap vector into the mouse HEXIM1 gene, resulting in the mutant HEXIM1 allele. The insertion also resulted in an in-frame fusion with the β-geo (encoding β-galactosidase and neomycin phosphotransferase) reporter gene. The lower panel shows a Southern blot wherein genomic DNA was digested with KpnI (K) restriction enzyme and analyzed by hybridization with a 3’ end probe. (B) PCR genotyping of tail DNA from E17.5 embryos. F1, F2, and R2 refer to PCR primers (see Data Supplement for primer sequences).

(A) H&E stained sections of E17.5 mutant HEXIM1 embryos. (B) Higher power view (C) Increased apoptosis was evident in the wall of the left and right ventricular myocardium and intraventricular septum of HEXIM1_1-312 mice at E15.5. LV=left ventricle; RV=right ventricle; IVS=intraventricular septum.

(A) Northern blot analyses of VEGF expression in H9C2 cells transfected with control vector or expression vectors for WT or mutant HEXM1_1-312. The 18S ribosomal band was used to normalize for unequal loading. The autoradiograph is representative of 3 experiments. The lower panels show Western blot analyses of WT and mutant HEXIM1 expression using the FLAG antibody. NSB = non-specific band. (B) H9C2 cells were transfected with an expression vector for HEXIM1 and luciferase reporter gene containing VEGF gene promoter. The cells were also transfected with a Renilla luciferase internal control plasmid to normalize for transfection efficiency. Fold activation over cells transfected with control expression vector is displayed as an average of three independent experiments.

(A) β-galactosidase staining of sections from E11.5 embryos (B) Cross sections of E17.5 embryos stained for β-galactosidase. (C) Staining of tissue sections from E13.5 WT and mutant embryos were performed using HEXIM1 antibody to the C-terminal or N-terminal region of HEXIM1.

(A) PECAM-1 immunostaining of E15.5 WT and HEXIM1 mutant whole-mounted embryos and heart sections. (B) The hearts of E15.5 WT and HEXIM1 mutant mice were immunostained with VEGF antibody. (C) Immunostaining of the hearts of E17.5 WT and HEXIM1 mutant mice with FGF9 antibody. (D) VEGF and FGF9 expression were also determined using western blot analyses.

(A) H9C2 cells were transfected with control or expression vector for HEXIM_1-312 and subjected to ChIP assays. The -2079/-1252, -944/-633, and coding sequence regions of VEGF gene promoter were immunoprecipitated with HEXIM1 (B) (left) H9C2 cells were transfected with expression vector for WT HEXIM1 or HEXIM_1-312 and subjected to ChIP assays. The coding sequence region of VEGF gene promoter was immunoprecipitated with serine 2 phosphorylated RNAPII (right). (B) For IP experiments (left), WT and mutant HEXIM1 were immunoprecipitated from equal concentrations of whole cell extracts using FLAG polyclonal antibody followed by Western blotting with anti-C/EBPα. Normal rabbit IgG was used as control. “Input” lane represents 10% of the total volume of lysate used in each reaction. For GST-pull down assays (right) equal amounts each of in vitro translated and [³⁵S]methionine-labeled WT C/EBPα were incubated with equal amounts each of either glutathione-S-transferase (GST) control, WT or mutant HEXIM1. “Input” lane represents 10% of the total volume of in vitro translated product used in each reaction. (C) (left) The -2079/-1252 region of VEGF gene promoter was immunoprecipitated with C/EBPα or SP1 antibodies. The “input control” represents PCR for 2% of total chromatin used in each reaction. (right) H9C2 cells were transfected with expression vectors for WT HEXIM1, HEXIM1_1-312, and C/EBPα, and luciferase reporter gene containing the -2079/-1252 region of the VEGF gene promoter. (D) VEGF expression in (left) H9C2 cells transfected with control and C/EBPα siRNA using Northern blot analyses and in (right) WT (n=7) and C/EBPα (n=7) knockout mice using Western blot analyses. Densitometric quantifications of VEGF protein expression levels were expressed relative to expression levels of sarcomeric actin. NSB = non-specific band.