Figure 3
Phosphorylation of LSD1 at serine-111 by PKC-θ regulates its pro-EMT function. (a) Immunofluorescence microscopy was performed on indicated cells fixed and probed with anti-LSD1, anti-PKC-θ and DAPI. Representative images for each dataset are shown. Graph depicts the PCC for LSD1 and PKC-θ (n = 30 individual cells). −1 = inverse of colocalization; 0 = no colocalization; +1 = perfect colocalization. (b) Partial LSD1 amino acid sequence indicating the location of peptides positive for phosphorylation. Red amino acids = NLS region; blue amino acid = serine-111; green bars = location of peptides and are numbered in order of mean signal intensity. (c) Model of LSD1 generated using Phyre2, highlighting the close proximity of the serine-111 phosphorylation site to the positively charged NLS domain (inner box). LSD1 structure is based on PDB code 2V1D and cartoons were created in Pymol. (d) Partial LSD1 amino acid sequence indicating potential PKC-θ phosphorylation motifs near the NLS region (described in45). Red amino acids = NLS region; blue amino acids = potential PKC-θ phosphorylation sites. (e) LSD1-s111p TNFI in indicated cell lines (n > 30 individual cells). Representative images for each dataset are shown. (f) Graph indicates LSD1-s111p TNFI after treatment with vehicle alone, BIM or C27 as determined by immunofluorescence microscopy (n > 20 individual cells). (g) LSD1 H3K4 demethylation assay was performed on nuclear extracts from indicated cells after treatment with BIM, C27, or vehicle alone. Graph depicts percentage LSD1 demethylase activity relative to control (n = 2). (h) PCC for LSD1 and Snail after treatment with either BIM, C27, or vehicle alone (n ≥ 10 individual cells). −1 = inverse of colocalization; 0 = no colocalization; +1 = perfect colocalization. (i) LSD1-WT and LSD1-Mut plasmid construct sequences. Blue amino acid = mutation site. (j) Graphs indicate LSD1-s111p TNFI, vimentin TCFI, and Snail TNFI after incubation with LSD1-WT, LSD1-Mut, or vector only as determined by immunofluorescence microscopy (n = 40 individual cells). Scale bars = 10 µM. All data represents the mean ±SE. ns = p > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Mann-Whitney test.
