Figure 2

LSD1 is critical for the invasive phenotype of A549 cells by regulating the integrin pathway. (A) Knockdown efficiency of LSD1 in cells stably expressing LSD1 shRNA (KD9-clone 9, KD15-clone 15 in A549 cells, KD21-clone 21, KD22-clone 22 in H460 cells and KD2-clone 2 in H1975 cells) relative to shGFP control cells was shown by western blot. LSD1 protein levels were shown by western blot and the β-actin protein was used as loading controls. Data are representative of three independent experiments. (B) The RNA-seq of A549 cells. Differentially regulated genes after LSD1 shRNA knockdown (shGFP vs KD15) were shown by the Volcano blot. 917 up-regulated genes (p < 0.05 & log₂- fold change > 2) are shown in red, while 423 down-regulated genes are shown in green (p < 0.05 & log₂-fold change < −2). (C) The bar graph showing the representative canonical pathways affected by LSD1 knockdown. The Ingenuity activation z-score is a statistical measure of the match between expected relationship direction from literature and observed gene expression in RNA-seq. The z-score of Integrin Signaling (z-score <−2) predicts the significant inhibition state of the integrin signaling pathway in A549 shLSD1 (KD15) relative to A549 shGFP. The differentially regulated genes in the pathways are shown in a table (right panel). (D) Representative images of invasion assay of A549 cells expressing GFP shRNA control or LSD1 shRNA (left panel). Bar graphs showing the quantification of Crystal Violet staining of Boyden Chamber transwell filters (right panel). The bar graphs represents the mean ± SEM for n = 4 (A549) and n = 5 (H1975). *P < 0.05 (Student’s t-test). Scale bars, 50 µm.