Journal:

Article Title: Mutation of hepatocyte nuclear factor-1? inhibits Pkhd1 gene expression and produces renal cysts in mice

doi: 10.1172/JCI200420083

Figure Lengend Snippet: DNase hypersensitive site mapping and deletion analysis of the mouse Pkhd1 promoter. (A) Structure of the 5′ end of the Pkhd1 gene. Boxes indicate exons. Bent arrow indicates the transcription initiation site at +1. Bar indicates the 3′ probe used for indirect end labeling. Vertical arrows indicate hypersensitive sites. (B) Southern blot of genomic DNA from mIMCD-3 cells (right) and 10T1/2 cells (left) after digestion with graded concentrations of DNase I. Open arrow indicates the parental 8.1-kb EcoRI fragment. Closed arrows indicate sub-bands corresponding to hypersensitive sites located at the positions indicated on the right. (C) Northern blot showing endogenous expression of Pkhd1 (upper panel) and HNF-1β (middle panel) in mIMCD-3 cells (lane 2) and absence of expression in 10T1/2 cells (lane 1). Lower panel shows expression of GAPDH as a loading control. (D) Deletion analysis of the Pkhd1 promoter. Left panel shows plasmids containing fragments of the Pkhd1 promoter linked to a promoterless luciferase (Luc) reporter gene. Bent arrow indicates the transcription initiation site at +1, gray boxes indicate exons, and black boxes indicate the consensus HNF-1 site. Right panel shows luciferase activity in transfected mIMCD-3 cells (white bars) and 10T1/2 cells (gray bars). Data are presented as mean ± SE of six to nine independent transfections. *P < 0.05 compared with empty pGL3-Basic.

Article Snippet: These results indicate that the consensus HNF-1 site is required for the activity of the Pkhd1 promoter in transfected mIMCD-3 cells. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 Mutational analysis of the Pkhd1 promoter and binding of HNF-1. (A) Luciferase activity in mIMCD-3 cells transfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector).

Techniques: End Labeling, Southern Blot, Northern Blot, Expressing, Luciferase, Activity Assay, Transfection

Journal:

Article Title: Mutation of hepatocyte nuclear factor-1? inhibits Pkhd1 gene expression and produces renal cysts in mice

doi: 10.1172/JCI200420083

Figure Lengend Snippet: Mutational analysis of the Pkhd1 promoter and binding of HNF-1. (A) Luciferase activity in mIMCD-3 cells transfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector). Data are presented as mean ± SE of nine independent transfections. *P < 0.01 compared with WT promoter. (B) EMSA performed with a 44-bp DNA fragment containing the consensus HNF-1 site and reticulocyte lysates programmed with HNF-1α (lanes 2–7), HNF-1β (lanes 9–14), or unprogrammed lysates (lanes 1 and 8). Binding reactions were performed in the presence of anti–HNF-1α Ab (lane 3), anti–HNF-1β Ab (lane 10), or 100-fold excess unlabeled competitor (Comp.) DNA fragment (lanes 4–7, 11–14). (C) EMSA performed using the 44-bp DNA fragment and nuclear (Nuc) extracts from mIMCD-3 cells (lanes 2–8) or no protein (lane 1). Binding reactions were performed in the presence of anti–HNF-1β Ab (lane 3), irrelevant Ab (lanes 4), or 100-fold excess unlabeled DNA fragment (lanes 5–8). In B and C, arrows indicate retarded band, and arrowheads indicate supershifted band. †Complex that does not contain HNF-1β. (D) Luciferase activity in HeLa cells cotransfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector) and expression plasmids encoding HNF-1α, HNF-1β, or empty pcDNA3. Data are presented as mean ±SE of six independent transfections. *P < 0.01 compared with cells cotransfected with empty expression plasmid.

Article Snippet: These results indicate that the consensus HNF-1 site is required for the activity of the Pkhd1 promoter in transfected mIMCD-3 cells. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 Mutational analysis of the Pkhd1 promoter and binding of HNF-1. (A) Luciferase activity in mIMCD-3 cells transfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing

Journal: PLoS ONE

Article Title: Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels

doi: 10.1371/journal.pone.0139417

Figure Lengend Snippet: Cells were co-transfected with 10 ng of the Renilla luciferase expression vector pRL-SV40 and 0.2 μg each of the pGL3-rs2919872G and pGL3-rs2919872A plasmids; the promoterless pGL3-Basic vector served as the negative control. Intracellular luciferase activity was measured 48 h after transfection. The relative luciferase units (RLU) were determined by comparison with the promoterless pGL3-Basic plasmid, which was assigned an arbitrary value of 1. Each transfection was performed in duplicate and the data are expressed as the mean ± SD of three separate experiments. (* P < 0.05).

Article Snippet: Cells were seeded (2×10 5 cells/well) in 12-well plates and transfected with the empty pGL3-Basic vector (a promoterless control) or with pGL3- rs2919872G or pGL3- rs2919872 A constructs using Lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, Negative Control, Activity Assay