Fig 1: LSD2 expression levels in human colorectal cancer (CRC) tissue and cells. (A) The ONCOMINE database was used to examine mRNA expression of LSD2 in CRC tissue. (B) Real-time Polymerase chain reaction (PCR) to evaluate mRNA expression of LSD2 in clinical CRC specimens relative to normal tissues (n=36). Data were expressed as means ± SEM. (C, D) Western Blot evaluation of LSD2 protein expression in CRC and normal tissue. (E) LSD2 protein expression in normal and CRC tissue was analysed by IHC. (F) Pearson’s χ2 tests to assess the association between colon tissue type and LSD2 expression by IHC(n=36). (G) Western blotting to assess LSD2 expression in CRC cell lines.
Fig 2: LSD2 down-regulation inhibits CRC cell proliferation. (A) Western blot showing protein expression of LSD2 and GAPDH (loading control) in HCT-116 and LoVo cells with control or sh-LSD2. (B, C) BrdU/PI assay to detect and quantification of DNA synthesis in HCT-116 and LoVo cells with control or sh-LSD2. **p < 0.01. (D) Cell Counting Kit-8 (CCK8) assay to detect the viability of HCT-116 and LoVo cells with control or sh-LSD2. (E, F) Colony formation assays and quantification in approximately 500 HCT-116 and LoVo cells with control or sh-LSD2. Data were expressed as means ± SEM, **p < 0.01.
Fig 3: LSD2 promoted CRC cells proliferation in vivo. (A) Effects of LSD2 down-expression on tumor growth in a xenograft mouse model. Empty vector or sh-LSD2 was transfected into LoVo cells, which were injected in the nude mice (n = 6), and the tumors were obtained at day 35 and was determined by measuring their tumor volume (B) and tumor weight (C). Data were expressed as means ± SEM. (D) The tumor sections were under IHC staining using antibodies against LSD2, p53, p21,ki-67, *P < 0.05.
Fig 4: LSD2 regulates colorectal cancer (CRC) cell proliferation and apoptosis through p53. ShRNA mediates p53 knockdown in HCT-116 LSD2-downregulated cell lines, while p53 over-expression in HCT-116 LSD2-upregulated cell lines was observed. (A) Protein expression of p53, p21, pRb and LSD2 measured by Western blot among the CRC cell lines mentioned above. (B) BrdU/PI assays were performed to measure DNA synthesis in the indicated CRC cell lines. (C) Quantitative analysis of DNA synthesis. (D) Western blot showing p53 and LSD2 protein expression in the p53 LSD2 double-downregulated HCT-116 and LoVo cell lines. (E) Flow cytometry detection of the apoptotic rates of the indicated CRC cell lines. (F) Quantitative analysis of DNA apoptosis. Data were expressed as means ± SEM. **p < 0.01.
Fig 5: LSD2 over-expression promotes CRC cell proliferation. (A) Western blot detection of LSD2 and GAPDH (loading control) protein expression in Caco2 and SW1116 cells with control or LSD2. (B, C) BrdU/PI assay to detect and quantify DNA synthesis in Caco2 and SW1116 cells with control or LSD2. **p < 0.01. (D) Cell Counting Kit-8 (CCK8) assay to measure the viability of Caco2 and SW1116 cells with control or LSD2. (E, F) Colony formation assays and quantification in approximately 500 Caco2 and SW1116 cells with control or LSD2. Data were expressed as means ± SEM, **p < 0.01.
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