Skip to main content
. 2012 Mar 27;3:758. doi: 10.1038/ncomms1755

Figure 6. Inhibition of cellular FAD synthesis blocks LSD1-mediated repression of energy-expenditure genes.

Figure 6

(a) Biosynthesis pathway of FAD in mammalian cells. Flavin mononucleotide (FMN). (b) Effect of the disruption of FAD synthesis on LSD1-target genes. RFK- (blue bars) or FADS- (white bars) knockdown 3T3-L1 cells were induced to differentiate for 24 h, followed by RNA extraction and quantitative RT–PCR. Values are shown as the fold difference against control siRNA-introduced samples (black bars). *P<0.05, **P<0.01 versus control siRNAs by Student's t-test. (c) Venn diagram of the probe sets induced by LSD1-KD and RFK-KD. (d) Unidirectional effects of RFK-KD on LSD1-target genes. (e) Effect of wild-type or FAD-binding mutant-type LSD1 on promoter activity. GAL4-fused LSD1-expressing plasmid (0.1 or 0.5 μg) was transfected into 293T cells, together with the GAL4x5-containing luciferase reporter construct. **P<0.01 versus GAL4 mock by Student's t-test. (f) Effect of RFK-KD on LSD1-mediated transcriptional repression. Control (black bars) or RFK (blue bars) siRNA-introduced 3T3-L1 cells were transfected with indicated GAL4 plasmids 48 h before the luciferase measurement. *P<0.05 between indicated conditions by Student's t-test. (g) Effect of lumiflavin treatment on LSD1-mediated transcriptional repression. 3T3-L1 cells were exposed to vehicle (black bars) or 50 μM lumiflavin (red bars) 48 h before the luciferase measurement. (h) Effect of lumiflavin on endogenous LSD1-target genes. Differentiating 3T3-L1 cells were exposed to vehicle (black bars), 25 μM (orange bars) or 50 μM (red bars) lumiflavin for 24 h and were subjected to RNA analyses. (i) Increase of FAD concentration during adipogenic differentiation of 3T3-L1 cells. **P<0.01 versus day 0 by Student's t-test. (j) Increase of FAD concentration after 24-hour palmitate exposure in mature 3T3-L1 adipocytes (day 7). Values are normalized to the protein concentration. *P<0.05 versus control by Student's t-test. All histogram values are means±s.d. of three independent samples.