1. Volume 23, Issue 10, Pages 1228-1240 (October 2016)
Histone Demethylase LSD1 Promotes Adipocyte Differentiation through Repressing Wnt Signaling 
Yan Chen, Jeesun Kim, Ruipeng Zhang, Xiaoqin Yang, Yong Zhang, Jianwu Fang, Zhui Chen, Lin Teng, Xiaowei Chen, Hui Ge, Peter Atadja, En Li, Taiping Chen, Wei Qi 
Cell Chemical Biology 
Volume 23, Issue 10, Pages (October 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 1228-1240DOI: (10. 1016/j. chembiol
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3. Figure 1 LSD1 Inhibitors Block Brown Fat Differentiation
(A) qRT-PCR analysis of BAT maker expression in differentiated brown fat cells treated with DMSO, 10 μM AZ505, 5 μM EPZ003696, 10 μM Cpd. 1, or 5 μM Cpd. A separately.
(B) Structures of Cpd. A and Cpd. B.
(C) Morphological differentiation at day 8. Cells were treated with DMSO, Cpd. A, or Cpd. B, and were stained with oil red O.
(D) Proliferation curve of C3H10T1/2 cells treated with DMSO, Cpd. A, or Cpd. B.
(E) qRT-PCR analysis of BAT makers and adipogenesis transcriptional factors in differentiated brown fat cells treated with DMSO, Cpd. A, or Cpd. B at 4, 0.4, and 0.04 μM separately.
(F) Western blot analysis of histone methylation during brown adipocyte differentiation.
(G) qRT-PCR analysis of LSD1 expression level during brown adipocyte differentiation. The data are represented as means ± SD of triplicates.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2 LSD1 Activity Is Required for Brown Fat Differentiation
(A) qRT-PCR analysis of LSD1 mRNA levels in C3H10T1/2 cells transfected with control siRNA or LSD1 siRNA.
(B) Morphological differentiation at day 8. C3H10T1/2 cells were transfected with control siRNA or LSD1 siRNA, and then induced to differentiate for 8 days. Differentiated brown fat cells were stained with oil red O.
(C) qRT-PCR analysis of BAT makers and adipogenesis transcriptional factors in brown fat cells induced to differentiate for 8 days.
(D) Western blot analysis of H3K4me2 in undifferentiated and differentiated (day 8) brown fat cells.
(E) Morphological differentiation at day 8. Images show the rescue of the differentiated phenotype in the LSD1-shRNA cell line stably overexpressing GFP or mutated LSD1-K661A, and the increase in the differentiated phenotype in the control-shRNA or LSD1-shRNA cell lines stably overexpressing LSD1-WT.
(F) qRT-PCR analysis of BAT maker expression in the differentiated control-shRNA or LSD1-shRNA cell lines stably overexpressing GFP, wild-type LSD1, or LSD1-K661A during brown fat differentiation. The data are represented as means ± SD of triplicates.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 3
Knockdown or Inhibition of LSD1 Activates Wnt/β-Catenin Signaling
(A) Gene set enrichment analysis (GSEA) for the biological process in Gene Ontology (GO) using RNA-seq-based expression profile. The left panel shows that the GO term brown fat cell differentiation (GO: ) was enriched in genes highly expressed in DMSO-treated cells, while the right panel shows that the regulation of the Wnt signaling pathway (GO: ) is overrepresented in genes with high expression in Cpd.-A-treated cells. The green curve illustrates the enrichment score (ES), which is used to indicate the degree to which the gene set is enriched at the top or bottom of the ranked gene list. Black bars show the position of genes belonging to the gene set in the ranked gene list included in this GSEA analysis.
(B) Western blot analysis of protein expression of LSD1, p-β-catenin (Ser675), and total β-catenin during brown fat differentiation. C3H10T1/2 cells were transfected with siCtrl or siLSD1, or treated with LSD1 inhibitors Cpd. A or Cpd. B 12 hr after seeding. After 48 hr, these cells were induced to differentiate to brown fat cells for 0 day, 3 days, or 8 days further.
(C) qRT-PCR analysis of mRNA expression of b-catenin, cyclin D1, and c-myc during brown adipocyte differentiation. The data are represented as means ± SD of triplicates.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4
The Direct Target Genes of LSD1 Include Wnt Signaling Components
(A and B) qRT-PCR analysis of mRNA expression of Wnt signaling components in C3H10T1/2 cells differentiated for 0 day and 6 days (A) and in C3H10T1/2 cells differentiated for 3 days in the presence of DMSO, Cpd. A, or Cpd. B (B). Statistical analysis was done using Student’s t test. *p < 0.05, **p < 0.01.
(C and D) ChIP-PCR analysis of H3K4me2 and LSD1 enrichment on the promoters of Wnt pathway components. The error bars represent ±SD values.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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7. Figure 5
Blocking Wnt/β-Catenin Signaling Rescues Brown Adipogenesis in the Presence of LSD1 Inhibition
(A) Morphological differentiation at day 8. Images show that the brown adipogenesis defects in the Cpd.-A-treated cells can be rescued by the Wnt/β-catenin signaling inhibitor LGK974.
(B) Proliferation curve of C3H10T1/2 cells treated with DMSO, Cpd. A, LGK974, or Cpd. A combined with LGK974.
(C) qRT-PCR analysis of mRNA expression of Ucp1, Cidea, and cyclin D1. The error bars represent ±SD values.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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8. Figure 6
Lsd1 Deletion in Mice Leads to Upregulation of Wnt Pathway Genes and Inhibition of Brown Adipogenesis
Newborn Lsd1flox/flox mice were injected with adenoviral Cre (Lsd1 KO) or control viruses (Control). The mice were killed 14 days later, and interscapular BATs were excised and analyzed.
(A) Representative images of BAT from control and Lsd1 KO mice.
(B) Relative BAT weight after normalization to the body weight of control (n = 7) and Lsd1 KO (n = 8) mice. Data are presented as means ± SEM. Statistical analysis was done using Student's t test. **p < 0.01.
(C) Sections of BAT from control and Lsd1 KO mice were stained with H&E. Small areas are magnified for more detail. Scale bars, 20 μm.
(D and E) qRT-PCR analysis of brown fat from control and Lsd1 KO mice for the expression of Lsd1 and brown fat marker genes (D) or Wnt pathway genes (E). All experiments were performed in triplicate, and the data are presented as means ± SEM. Statistical analysis was done using Student's t test. *p < 0.05, **p < 0.01.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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