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: The Journal of Experimental Biology

Article Title: Circadian coupling of mitochondria in a deep-diving mammal

doi: 10.1242/jeb.246990

Figure Lengend Snippet: Clock genes expression profile in synchronized hooded seals fibroblasts and tissues. (A) mRNA expression of core clock genes arntl (blue line) and per2 (black line) in hooded seal fibroblasts synchronized by temperature cycle (°C), as represented in the upper panel. Arntl and per2 mRNAs show a 24-h period and are in antiphase. P -values and phases were calculated through JTK cycle analysis ( n =4). (B) mRNA expression of core clock genes arntl (blue line) and per2 (black line) in hooded seal fibroblasts synchronized by temperature cycling and then left at constant temperature (upper panel). Arntl and per2 mRNAs maintain a 24-h period and their antiphasic relationship also in constant conditions. P -values and phases were calculated through JTK cycle analysis ( n =4). (C) Photon multiplier tube recordings of hooded seal skin fibroblasts transfected with arntl :luciferase (blue line) and per2 :luciferase (black line). Cells were synchronized with dexamethasone. (D) Arntl mRNA expression in hooded seal kidney and liver tissue, sampled in mid-light phase (ZT6) and mid-dark phase (ZT18). * P <0.05 (one-way ANOVA; n =3 except liver ZT18, where n =2). Data are expressed as means±s.e.m.

Article Snippet: The latter consisted of the pLV6 backbone and a mouse per2 promoter with adjacent luciferase sequence contained in the pGL3 basic E2 vector (Addgene plasmid 48747).

Techniques: Expressing, Transfection, Luciferase