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First published online November 24, 2004
doi: 10.1242/10.1242/jcs.01546

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Fad24, a mammalian homolog of Noc3p, is a positive regulator in adipocyte differentiation

¹ Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
² Research Division, Nissui Pharmaceutical Limited, 1075-2 Hokunanmoro, Yuki, Ibaraki 307-0036, Japan

View larger version (56K): [in a new window]   Fig. 1. Time course of mRNA expression of fad24 and various adipogenic genes during adipocyte differentiation by northern blot and Q-PCR analyses. (A) Northern blot analysis of fad24 expression. Total RNA from different time points after induction was prepared from 3T3-L1 cells. Isolated total RNA (15 µg) was loaded and subjected to northern blot analysis of fad24. ß-Actin is also shown as a loading control. (B) Q-PCR analysis of fad24 expression. The expression level of fad24 was determined at various time points of adipocyte differentiation of 3T3-L1 cells by Q-PCR and normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean±s.d. (n=3). (C) Northern blot analysis of expression of various adipogenic genes. Total RNA from various time points after induction was prepared from 3T3-L1 cells. Isolated total RNA (15 µg) was loaded and subjected to northern blot analyses of various adipogenic genes. Staining with ethidium bromide (EtBr) for ribosomal RNA is also shown as a control.

View larger version (16K): [in a new window]   Fig. 2. Cloning and schematic representation of mouse fad24. The full-length cDNA for mouse fad24 was isolated by 5'-RACE and RT-PCR. Su, R-5' and RT indicate the fragments obtained from the original subtraction, 5'-RACE and RT-PCR, respectively. The combined sequence is shown as fad24 and start and stop codons are indicated. The predicted number of amino acids for mouse FAD24 is 807.

View larger version (83K): [in a new window]   Fig. 3. The deduced amino acid sequences of mouse and human FAD24. (A) The deduced amino acid sequences encoded by the 2421 bp open reading frame of mouse fad24 and 2400 bp open reading frame of human fad24 are aligned. bZIP-like structure and NOC domains are underlined. (B) The various bZIP proteins are aligned. The conserved leucine residues and basic amino acid residues are boxed. (C) NOC domains from various species are aligned. The highly conserved consensus amino acid residues are boxed.

View larger version (51K): [in a new window]   Fig. 4. Tissue distribution of fad24 and the expression profile of fad24 during myogenesis. (A) Distribution of fad24 mRNA in various human tissues. A filter of multiple tissue northern (MTNTM) blots containing 2 µg of poly(A)+ RNA per lane was used to detect the tissue distribution of human fad24 mRNA. ß-Actin was used as a control. A 1.8 kb actin isoform (lower band) is predominant in the heart and skeletal muscle lanes. However, the expression level corresponding to the 2.0 kb band is almost the same, but slightly lower in the heart, skeletal muscle and liver. PBLs, peripheral blood leukocytes. (B) Expression of fad24 in the stromal vascular cells and adipocytes. Epidermal fat pads were isolated from mice. Stromal vascular cells and adipocytes were fractionated and total RNA was isolated. Total RNA (15 µg) was subjected to northern blot analysis, and the expression of fad24 was determined. aP2, whose expression is upregulated in adipocytes, is shown as a control. Staining with EtBr for ribosomal RNA is also shown as a control. (C) Expression of fad24 during differentiation of myoblasts. Total RNA (20 µg) was subjected to northern blot analysis, and the expression of fad24 was determined. The expression of myogenin, whose expression is upregulated in myocytes, is shown as a control. Staining with EtBr for ribosomal RNA is also shown as a loading control.

View larger version (45K): [in a new window]   Fig. 5. The functional analysis of fad24 by RNAi. (A) The endogenous expression of the fad24 gene. Total RNA obtained from 3T3-L1 cells transfected with shRNA expression vector for fad24 (white bars) or with scrambled shRNA expression vector as a control (gray bars) at each time point. The expression level of fad24 was determined by Q-PCR, and normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean±s.d. (n=3). (B) Differentiation of 3T3-L1 cells transfected with shRNA expression vector for fad24. The cells transfected with shRNA expression vector for fad24 (sifad24) or with scrambled shRNA expression vector as a control (Control) were stimulated with inducers. After 8 days of induction, the cells were fixed and stained on six-well plates with Oil red O to detect oil droplets. Bar, 200 µm. (C) The amount of triacylglycerol was measured on 24-well plates. Each column represents the mean±s.d. (n=3). (D) Effect of shRNA expression for fad24 on the expression of various adipogenic genes. Total RNA obtained from sifad24 cells (white bar) or control cells (gray bar) at each time point was subjected to Q-PCR. Expression levels were normalized with ß-actin expression determined by Q-PCR. Each column represents the mean±s.d. (n=3). (E) The expression of C/EBPß and C/EBP at earlier time points of differentiation. Total RNA obtained from sifad24 cells (white bars) or control cells (gray bars) at each time point was subjected to Q-PCR. Expression levels were normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean±s.d. (n=3).

View larger version (55K): [in a new window]   Fig. 6. Functional analysis of fad24 using FAD24-overexpressing NIH-3T3 cells. (A) The exogenous expression of the fad24 gene was determined by northern blot analysis. Total RNA (25 µg) obtained from a stable transformant was subjected to northern blot analysis for fad24. The retroviral exogenous gene expression and endogenous gene expression are indicated. (B) Differentiation of FAD24-overexpressing NIH-3T3 cells in the presence of BRL49653, a ligand for PPAR. NIH-3T3 cells stably expressing fad24 or control cells (infected with empty vector) were treated with inducers containing BRL49653. After 8 days of induction, the cells were fixed and stained with Oil Red O to detect oil droplets. Bar, 100 µm. (C) Northern blot analyses of adipocyte marker genes during the differentiation of FAD24-overexpressing NIH-3T3 cells. Total RNA from cells after the induction was isolated and 25 µg per lane was subjected to northern blot analysis for each adipocyte marker gene. Staining with EtBr for ribosomal RNA is shown as a control. (D) Expression of C/EBPß and C/EBP at earlier time points during the differentiation of FAD24-overexpressing NIH-3T3 cells. Total RNA obtained from FAD24-overexpressing NIH-3T3 cells (white bars) or control cells (gray bars) at each time point was subjected to Q-PCR. Expression level was normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean±s.d. (n=3).

View larger version (62K): [in a new window]   Fig. 7. Intracellular localization of EGFP-FAD24 fusion protein. (A) Intracellular localization of FAD24. NIH-3T3 cells were transiently transfected with an EGFP-FAD24-expressing plasmid or empty vector. One day after transfection, the cells were fixed and stained with DAPI. EGFP signals were detected with a fluorescence microscope. Bar, 25 µm. (B) Colocalization of FAD24 with a nuclear speckle marker. HeLa cells transiently transfected with EGFP-FAD24 or EGFP-truncated FAD24 (FAD24 bZIP), which lacks a bZIP-like domain, were fixed and stained with the nuclear speckle marker SC35. The fluorescence of FAD24 (green) and SC35 (red) were detected with a fluorescence microscope. (C) Colocalization of FAD24 with the nucleolus. HeLa cells transiently transfected with EGFP-FAD24 or EGFP-truncated FAD24 (FAD24 bZIP were fixed and green EGFP signal was detected with a fluorescence microscope. Bar in B, 10 µm for B and C.

View larger version (39K): [in a new window]   Fig. 8. Localization of FAD24 in the presence of RNA polymerase II inhibitor. HeLa cells transiently transfected with EGFP-FAD24 were treated with -amanitin (50 µg/ml, 5 hours) and then fixed and stained with the nuclear speckle marker SC35. Fluorescence of FAD24 (green) and SC35 (red) were detected with a fluorescence microscope. Bar, 10 µm.

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