Identification of Genes Rapidly Induced by Harmine in 3T3-F442A Preadipocytes
A, Microarray analysis of 3T3-F442A preadipocytes treated with either harmine (10 μm) or PPARγ ligand, GW7845 (30 nm), for 24 h at confluence. PPARγ target genes are shown in the upper panel. Genes that passed the criteria of being up-regulated (>2.5-fold) by harmine and not regulated by GW7845 (<1.3-fold induction or reduction) are shown in the lower panel. B, Comparison of the effects by harmine and GW7845 treatment on Id2, PPARγ, and PPARγ target gene expression. 3T3-F442A preadipocytes were treated with harmine (10 μm) or GW7845 (30 nm) for 48 h at confluence, and mRNA levels were measured by real-time PCR. C, Time course of induction of Id2 and PPARγ by harmine (10 μm) in 3T3-F442A cells as determined by real-time PCR. 3T3-F442A preadipocytes were treated with harmine for the indicated time at confluence. D, Induction of Id2, C/EBPδ, and PPARγ2 expression by adipogenic cocktail (DMI; 1 μm dexamethasone, 0.5 mm IBMX, 5 μg/ml insulin) in 3T3-L1 cells. E, Effect of actinomycin D treatment on induction of Id2 expression by harmine. 3T3-F442A preadipocytes were pretreated with actinomycin D (5 μg/ml) 1 h before harmine treatment. F, Transient overexpression of Id2 induces expression of PPARγ in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were transiently transfected with an Id2 expression vector (pCMX-Id2), PPARγ expression vector (pCMX-PPARγ), or empty vector (pCMX). mRNA was collected 48 h later and gene expression was determined by real-time PCR. Data are representative of three independent experiments. Ctrl, Control; GW, GW7845; Har., harmine; Norm., normalized; PEPCK, phosphoenol pyruvate carboxykinase.

Promotion of Adipocyte Differentiation by Stable Overexpression of Id2 in 3T3-L1 Preadipocytes
3T3-L1 preadipocytes were infected with pBabe-based retrovirus harboring the Id2 gene and large stable pools selected with puromycin (2 μg/ml). Expression of Id2, PPARγ on d 0 and d 6 (A) and PPARγ target genes on d 6 (B) are increased in Id2-overexpressing 3T3-L1 cells. C, Oil red O staining of Id2 overexpressing 3T3-L1 cells and C3H10T1/2 cells. Differentiation of 3T3-L1 and C3H10T1/2 cells into adipocytes was induced by DMI, or GW7845 (GW; 30 nm). DI, DMI and insulin.

Suppression of Id2 Expression by the Wnt Pathway in Preadipocytes
A and B, 3T3-F442A cells were treated with Wnt-3a or control conditioned media for 48 h. mRNA levels were measured by real-time PCR. C, Harmine reverses the inhibitory effect of Wnt-3a on Id2 expression. 3T3-F442A preadipocytes were treated with Wnt-3a conditioned media and/or harmine. D, Effect of harmine derivatives (10 μm) (12) on expression of PPARγ and Id2 in 3T3-F442A preadipocytes. E, dnTCF blunts the induction of Id2 expression by harmine. 3T3-F442A cells stably expressing vector or dnTCF protein were generated by retroviral transduction (12). Id2 expression 48 h after harmine treatment was determined by real-time PCR. Ctrl, Control.

Knockdown of PPARγ Blocks the Adipogenic Effect of Harmine but Does Not Alter Expression of Id2
Stable 3T3-F442A cells expressing two independent shRNAs against PPARγ were generated. A and B, Two independent shRNAs effectively reduce PPARγ mRNA expression (A) and protein levels (B). C, Expression of Id2 is not affected by knockdown of PPARγ, demonstrating that Id2 is upstream of PPARγ. D, Expression of downstream target genes of PPARγ, including aP2, adiponectin, and CD36, is reduced in PPARγ shRNA-expressing cells, both in the presence and absence of harmine. Gene expression was determined by real-time PCR. Ctrl, Control; shPPAR, short hairpin PPAR.

Id2 Knockdown Inhibits 3T3-L1 Adipogenesis
A, Transient transfection of 3T3-L1 cells with a pool of four Id2 siRNA sequences reduces mRNA levels of Id2 but not of the related factors Id1 and Id3 or 18S control. B, Id2 knockdown does not affect induction of the early transcription factors C/EBPβ or C/EBPδ by DMI. C, Oil red O staining of Id2-silenced 3T3-L1 cells (Id2 RNAi) and control (Ctrl RNAi) after 6 d of differentiation. Top, Oil red O staining; bottom, microscopic view. D, Quantitative real-time PCR analysis of expression of the adipogenic markers, adiponectin, aP2, CD36, and PPARγ, in knockdown cells vs. controls. Knockdown of Id2 blunts the induction of PPARγ, aP2, and adiponectin expression by DMI and harmine during adipocyte differentiation. Data are representative of three independent experiments. Ctrl, Control; RNAi, RNA interference.

Id2 Is Positively Correlated with Body Mass in Mice and Humans
A and B, Correlation between Ids (Id1–4) expression and body weight in genetic and diet-induced obese mice. Total RNA was collected from epididymal adipose tissues, and Id2 expression was measured by real-time PCR. A, Id2 expression positively correlates with body weight in high-fat-fed mice. C57BL/6 mice were fed with regular chow (n = 10) or high-fat diet for 11 months (n = 10). B, Id2 expression is up-regulated in ob/ob mice. C57BL/6 and ob/ob mice (3 months of age) were used in this study. **, P < 0.05, n = 8–10. C, Expression of Id2 in primary cultured abdominal sc mature adipocytes from nine male and 10 female nondiabetic obese humans. Id2 expression was measured in lean (BMI, 25 kg/m²) and obese (BMI, 55 kg/m²) Pima indians. Id2 expression in humans is also positively correlated with body weight. The microarray data are retrieved in Entrez Gene Expression Omnibus DataSets originally performed by Lee et al. (28). WT, Wild type.

Reduced WAT Development in Id2−/− Mice
A, Transverse sections at the level of the neck were from Id2+/+ and Id2−/− 6 d after birth. B, Reduced WAT development in Id2−/− pups. Dorsal view of 4-d-old Id2−/− mouse with its littermate control. Note the decreased deposition of fat interscapular regions for the Id2−/− pups. C and D, Reduced adipocyte differentiation in Id2−/− MEFs. Primary MEFs were prepared from E13.5 wild-type and Id2−/− embryos. C, Oil red O staining of Id2+/+ and Id2−/− MEFs. MEFs were differentiated with adipogenic cocktail for 8 d. Top, Oil red O staining; bottom, microscopic view. D, Expression of adipogenic markers, adiponectin, aP2, LPL, and PPARγ, in Id2−/− and Id2+/+ MEFs. Id2 expression is absent in Id2 null cells. The levels of adipogenic markers are reduced whereas C/EBPδ is not changed in Id2−/− cells. At d 6, mRNA was collected and analyzed by real-time PCR. Data are representative of three independent experiments.