Figure 2

LSD1 increases transcriptional activity of RORα2. (A) Transcriptional activation of the RORα2E-luciferase reporter by RORα2 WT or E542K mutant with LSD1 WT or K661A mutant in HEK293T cells. Cells were transfected with either 100 ng of RORa2E luciferase reporter along with 100 ng of RORα2 WT/RORα2 E542K or 100 ng of LSD1 WT/LSD1 K661A. Results are expressed as fold activation compared to empty vector. Data are represented as mean ± S.E.M. for three independent experiments. P value is shown from Student’s t test analysis. *p < 0.001. (B) Binding affinity of RORα2 WT or E542K mutant with LSD1 was assessed in HEK293 cells expressing indicated constructs. (C) Binding affinity of LSD1 WT or K661A mutant with RORα2 was assessed in HEK293 cells expressing indicated constructs. (D) Effect of LSD1 knockdown on RORα2E-luciferase reporter with overexpression of RORα2 in HEK293T cells. Cells were transfected with either 100 ng of RORa2E luciferase reporter along with 100 ng of RORα2 or 400 ng of mock/LSD1 shRNA. Results are expressed as fold activation compared to empty vector. Data are represented as mean ± S.E.M. for three independent experiments. P value is shown from Student’s t test analysis. *p < 0.001. (E) HEK293T cells were cotransfected with RORα2 and LSD1 and treated with or without pargyline (3 mM). Pargyline treatment attenuates transcriptional activation of the RORα2E promoter-luciferase reporter by LSD1. Cells were transfected with either 100 ng of RORa2E luciferase reporter along with 100 ng of RORα2 or 100/400 ng of LSD1. Results are expressed as fold activation compared to empty vector. Data are represented as mean ± S.E.M. for three independent experiments. P value is shown from Student’s t test analysis. *p < 0.05. (F) ChIP assay on the RORα2E promoter-luciferase reporter in HEK293T cells with mock or LSD1 shRNA. Occupancy of the promoter by LSD1, RORα2 and RNA polymerase II was analyzed.