Fig 1: In vitro EMT system used to study HOTAIR function Epi and Mes cell lines corresponding to HA5-early and HA5-late, respectively (Castro-Vega et al, 2013), stained for F-actin fibers by Phalloidin-TRITC and for DNA by DAPI (scale bar, 16 µm).HOTAIR levels quantified in Epi and Mes cells by random-primed RT–qPCR, relative to RPL11 mRNA (gray dots); bars represent mean ± standard deviation for three replicates.Protein levels of EMT markers in whole protein extracts of parental Epi and Mes cells assessed by Western blot.HOTAIR quantification by random-primed RT–qPCR in Epi cell lines normalized to RPL11 mRNA and represented as a fold change to CTR. Each dot corresponds to independent replicate; bars represent mean ± standard deviation values.Quantification of HOTAIR variants associated with LSD1 and EZH2 by RT–qPCR in two independent RIP experiments (R1 and R2), represented as a % of input, with fold-enrichment.Distribution of full-length and truncated variants of HOTAIR between cytoplasm (cyt), nucleoplasm (nuc), and chromatin (chr) fractions in Mes and transduced Epi cells (HOT, HOT?P, HOT?L) assessed by RT–qPCR relative to GAPDH mature mRNA.Subcellular distribution and protein levels of LSD1, RNA Pol II, H3K4me3, and GAPDH in cytoplasm, nucleoplasm, and chromatin fractions in Epi cells expressing CTR (C), HOT (H), HOT?P (P), and HOT?L (L).Protein levels of LSD1, EZH2, and GAPDH in whole protein extracts assessed by Western blot in Epi-CTR, -HOT, -HOT?P, and -HOT?L cells. * The Lsd1 signal at 75 kD was detected only in total and cytoplasm protein fractions and may correspond to either degradation product or Lsd1 variant. Source data are available online for this figure.