Fig 2: LSD1 is highly related to the prognosis of ovarian cancer patients. (A,B) KM curves comparing overall survival of ovarian cancer patients of low and high LSD1 mRNA levels in the cohorts of TCGA_OV (A) and GSE9891 (B). (C,D) Forest plots of Cox regression analyses of TCGA_OV and GSE9891 datasets show univariate (C) and multivariate (D) analyses to determine risk factors associated with overall survival of patients with ovarian cancers. * represents p < 0.05. (E−G) KM curves comparing overall survival of ovarian cancer patients of low and high LSD1 mRNA levels in the cohorts of GSE26193 (E), GSE63885 (F), and GSE18520 (G).

Fig 3: LSD1 interacts with LC3B through its SWIRM domain. (A) Bacterially produced GST-LC3B was used to pull down HA-LSD protein expressed in HEK293T cells. (B) Immunofluorescence staining analysis of LSD1 (red) and LC3B (green) in HEK293T cells following co-transfection with expression vectors for HA-LSD1 and Myc-LC3B. DAPI was used to label the nuclei. Scale bar, 5 μm. (C) Schematic illustration of N-terminal HA-tagged full-length (FL) LSD1 and its truncation constructs (ranges indicate chain of amino acids present in construct). The numbers labeled in LSD1 structure sketches indicate the amino acid residue positions. (D) The FL or various truncation mutants of HA-LSD1 shown in (C) were incubated with GST-LC3B. The immunoprecipitation and Western blotting analyses were carried out to detect interaction with anti-HA antibody. Original Western blot images are available in Supplementary Materials.

Fig 4: LC3B is demethylated by LSD1, and destabilized by LSD1 expression. (A) Dox-induced knockdown of LSD1 in HO8910 cells was generated. Then, the samples expressing Myc-LC3B protein or not were precipitated using an anti-Myc antibody and analyzed using an anti-pan-me-K antibody to detect methylation of LC3B. (B) Inhibition of LSD1 demethylation activity in HO8910 cells was achieved by treatment with TCP. Then the samples expressing Myc-LC3B protein or not were precipitated using an anti-Myc antibody and analyzed using an anti-pan-me-K antibody to detect methylation of LC3B. (C,D) Western blotting analysis of changes in total LC3B expression in SKOV3 (C) or HO8910 (D) treated (+) or untreated (−) with Dox to induce LSD1 knockdown with CHX treated for indicated times. The band intensity of total LC3B (LC3B-I and LC3B-II) was quantified, and the corresponding values were labeled below the LC3B Western blot panel. Original Western blot images are available in Supplementary Materials.

Fig 5: LSD1 is highly and aggressively expressed in ovarian tumors of different types. (A) Representative IHC images for LSD1 staining in different histotypes of ovarian cancers. Scale bar, 100 μm. The rectangle boxes shown in each IHC image in the upper panel indicate the areas that have been magnified in the lower panel for clearer visualization of the antibody staining. (B) Quantification of LSD1 staining score of normal ovary and ovarian tumors in IHC assay of tissue microarray. (C) Quantification of percentage of LSD1-positive staining of M0 and M1 metastasis stage in IHC assay of ovarian tissue microarray. (D–F) The comparison of LSD1 mRNA and protein expression between ovarian cancer tissues and normal controls in TCGA ovarian cancer cohort (TCGA_OV) (D), a series of GEO datasets (F), and CPTAC protein data (E). (G,H) mRNA expression levels of LSD1 at different stages of ovarian cancers of different datasets: TCGA_OV (G) and the Etemadmoghadam-studied dataset (H). * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.