A total of 50 ng genomic DNA was then used as a template to amplify the promoter region of the ADFP gene in 20 luciferase activities of the lysates were determined, and the firefly luciferase activity levels were normalized to that of luciferase

A total of 50 ng genomic DNA was then used as a template to amplify the promoter region of the ADFP gene in 20 luciferase activities of the lysates were determined, and the firefly luciferase activity levels were normalized to that of luciferase. RNA isolation and PCR analysis The cells were dissolved in TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and total RNA was extracted, according to the manufacturer’s protocol. were detected in presence of siRNA targeting gene. Furthermore, the present study demonstrated the role of ADRP IKBKB in low density liporotein (LDL) and very-LDL uptake-induced lipid accumulation under hypoxia. The knockdown of ADRP did not reduce HIF1-induced lipid accumulation under hypoxia. Together, these results showed that ADRP may be not involved in HIF1-induced lipid accumulation. gene was amplified from human genomic DNA using polymerase chain reaction (PCR) and cloned into a luciferase reporter vector (pGL3-Basic; Promega Corporation, Madison, WI, USA). Briefly, human genomic DNA was extracted using a Quick Genomic DNA Extraction kit (Guangzhou Dongsheng Biotech Co., Ltd., c-Kit-IN-2 Guangzhou, China) according to manufacturer’s instructions. A total of 50 ng genomic DNA was then used as a template to amplify the promoter region of the ADFP gene in 20 luciferase activities of the lysates were determined, and the firefly luciferase activity levels were normalized to that of luciferase. RNA isolation and PCR analysis The cells were c-Kit-IN-2 dissolved c-Kit-IN-2 in TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and total RNA was extracted, according to the manufacturer’s protocol. Total RNA (1 was oxygen regulated, the MCF7 cells were incubated under normoxic (21% O2) or hypoxic (1% O2) conditions, or in medium containing c-Kit-IN-2 100 as a hypoxia-inducible gene. In agreement, a significant decrease in the mRNA and protein levels of ADRP (Fig. 1C and D) were detected in the MCF7 cells transfected with siRNA targeting gene reduced the hypoxic induction of in a HIF1-dependent manner. Open in a separate window Figure 1 is induced by hypoxia in an HIF-dependent manner. (A) mRNA levels of ADRP were analyzed using qPCR in MCF7 cells cultured in normoxia or hypoxia, or in medium containing 100 gene for a consensus HRE sequence, as previously described (3). Several putative HREs were identified (Fig. 2A), however, only the HRE at ~-33 in position is conserved in human, mouse and rat (Fig. 2B). To determine whether this was a functional HRE, the promoter region of the gene was amplifed and inserted it into the luciferase reporter plasmid, pGL3-promoter. The plasmid of the pGL3-promoter was used as a negative control. A construct of the pGL3-promoter with the insertion of the identified HRE of the gene was used as a positive control. As shown in Fig. 2C, the region between ?754 and +635 markedly increased luciferase activity in the MCF7 cells under hypoxia. Analysis of the deletion constructs suggested that the conserved HRE c-Kit-IN-2 was functional (Fig. 2C). Mutation of the conserved HRE significantly impaired the induction of luciferase activity by hypoxia, however, mutation of the putative HRE in exon 1 did not impair the luciferase activity by hypoxia (Fig. 2D and E), which also suggested that the conserved HRE may be a functional HRE. Open in a separate window Figure 2 Identification and validation of HREs in the gene promoter. (A) Sketch map of the promoter region. The putative HREs are indicated by arrows. The nucleotide sequences are numbered in relation to the transcription initiation site, which is designated ‘+1’. P1, P2, P3 and P4 indicate the primers used for polmterase chain reaction amplification of the immunoprecipitated chromatin fragments in Fig. 2E. (B) Comparison of the conserved HRE and flanking nucleotides identified in the human, rat and mouse ADRP gene promoter regions. (C and D) Luciferase reporter assays were performed in MCF7 cells transfected with the constructs containing the indicated sequences from the human ADRP gene promoter region. Each transfection experiment was performed in triplicate. The relative mean luciferase activity in the cells under hypoxia is shown as the fold over the mean activity in the cells under normoxia. Error bars.