Fig. 1: NEK6 modulates LSD1 activity in cells.

a Illustration depicting the principle of the fluorescent reporter system. Cells of interest are transduced with a vector expressing the fluorescent reporter under control of the synthetic promoter. Subsequent transduction with a construct expressing the fusion protein of human LSD1 and the reverse tetracycline repressor protein (rTetR), both with selection cassettes and expression under a constitutive promotor enables the observation of LSD1 activity in living cells. Upon doxycycline (DOX) treatment, the rTetR-LSD1 fusion protein is recruited to the synthetic promoter, together with endogenous co-regulators, leading to the suppression of mCherry expression. Interference with the expression of LSD1 co-regulators (e.g., NEK6) influences LSD1 silencing activity. b Left: Validation of the coregulatory effect of NEK6 on LSD1. Reporter fluorophore expression was monitored over the course of 11 days in NIH/3T3 cells expressing the following shRNAs: shNek6.1, shNek6.2, shLsd1 (positive control), or shCtrl (neutral control). The median reporter expression of cells with Dox-induced rTetR-LSD1 recruitment was normalized to reporter cells without rTetR-LSD1 recruitment. The x-axis shows the time course. Right: Statistical analysis was performed by a one-way ANOVA of the area under curve followed by Dunnett´s multiple comparisons test (n = 3, mean ± SEM; ****:p < 0.0001, **:p < 0.001). c Representative immunofluorescence microscopy images of NIH/3T3 cells (top) and HEK293 cells (bottom) stained for endogenous LSD1 and NEK6 show clear colocalization in spherical nuclear sub-compartments. Nuclei were stained with DAPI. Zoom-in pictures of respective colocalized spots marked with boxes are shown for LSD1, NEK6, and for merged channels. Scale bars = 10 µm.