Journal:

Article Title: Cooperative Interaction between GATA-4 and GATA-6 Regulates Myocardial Gene Expression

doi:

Figure Lengend Snippet: Characterization of AS4 and AS6. (A) Schematic representation of the constructs used to generate the recombinant adenoviruses. NLS-lacZ, a 358-bp fragment from the extreme N-terminal portion of GATA-4, or a 359-bp fragment from the 5′ UTR of GATA-6 was cloned downstream of the CMV promoter and upstream of an SV40 poly(A) sequence in order to generate the recombinant adenoviruses. (B) Transgene expression is dose dependent. Ventricular cardiomyocytes were infected at MOIs of 16, 4, and 1 (corresponding to the progressively narrowing triangle) with Ctl, AS4, and AS6. RNA was isolated 3 days postinfection. Northern blot analysis showed that both antisense transgenes were efficiently expressed in a dose-dependent manner. (C) AS4 and AS6 are specific and efficient at decreasing GATA-4 and GATA-6 protein levels. Ventricular cardiomyocytes were infected as described above, and nuclear extracts were isolated 3 days postinfection and analyzed by Western blotting. L cells overexpressing GATA-4 or GATA-6 were used as controls for the specificity of the antibodies. (D) Quantification of the effects of AS4 and AS6 on GATA-4 and GATA-6 protein levels. The data represent the means of two independent Western blots performed as described for panel C and quantified by densitometry. At an MOI of 4, GATA-4 levels were decreased specifically by AS4 (80% reduction), while GATA-6 levels were reduced specifically by AS6 (60% reduction).

Article Snippet: Serial dilutions of recombinant Ad5 (Ctl, AS4, or AS6) were prepared in OptiMEMI (Canadian Life Technologies Inc.).

Techniques: Construct, Recombinant, Clone Assay, Sequencing, Expressing, Infection, Isolation, Northern Blot, Western Blot

Journal:

Article Title: Cooperative Interaction between GATA-4 and GATA-6 Regulates Myocardial Gene Expression

doi:

Figure Lengend Snippet: In vivo transcriptional targets for GATA-4 and GATA-6 in postnatal cardiomyocytes. (A) irANF secretion is decreased in a time-dependent manner by AS4 and AS6. Ventricular cardiomyocytes were infected at an MOI of 4 with Ctl, AS4, and AS6. Secreted irANF was assayed in the cardiomyocyte culture medium after a 24-h accumulation period at 2, 3, and 5 days postinfection. Since the effect of the antisense DNA on irANF secretion is clearly visible at 3 days postinfection, subsequent analyses were performed at this time point. (B) irANF secretion is decreased in a dose-dependent manner by AS4, AS6, and AS4 plus AS6. Ventricular cardiomyocytes were infected at MOIs of 16, 4, and 1 (corresponding to the progressively narrowing triangle) with Ctl, AS4, AS6, and AS4 plus AS6. (C) ANF mRNA levels are decreased in a dose-dependent manner by AS4, AS6, and AS4 plus AS6. Ventricular cardiomyocytes were infected as for panel B, and total RNA was analyzed by Northern blotting. The ANF mRNA levels are expressed relative to that of Ctl-infected cardiomyocytes. The GAPDH mRNA levels were unaffected. (D) Many cardiac genes are decreased by AS4 and AS6. Ventricular cardiomyocytes were infected at an MOI of 4 with Ctl, AS4, and AS6, and Northern blot analysis was performed on poly(A)+ RNA (left). Relative mRNA levels were quantified with a PhosphorImager (right). Note how α-MHC and β-MHC mRNAs were preferentially down-regulated by AS4, whereas cardiac (c.) α-actin, myosin light-chain 1 (MLC1), and GAPDH mRNA levels were not affected by AS4 or AS6.

Article Snippet: Serial dilutions of recombinant Ad5 (Ctl, AS4, or AS6) were prepared in OptiMEMI (Canadian Life Technologies Inc.).

Techniques: In Vivo, Infection, Northern Blot