Fig. 6
LSD1 is activated by SLC52A1 overexpression. (a) U2OS cells transfected with pcDNA3-HA-SLC52A1 and treated with 2 µM etoposide for 24 h were subjected to measurement of intracellular FAD levels. Data are mean ± s.d. (n = 4 independent cultures). (b) U2OS cells transfected with pcDNA3-HA-SLC52A1 and treated with 2 µM etoposide for 24 h were subjected to cell fractionation and measurement of FAD levels in each fraction. (c) SLC52A1-overexpressing U2OS cells treated with 2 µM etoposide and 100 nM ORY-1001 for 24 h were subjected to immunoblot analysis. Protein levels relative to histone H3 or p53 levels were quantified using NIH ImageJ software. (d–f) SLC52A1-overexpressing U2OS cells treated with 2 µM etoposide without (d and e, left panels) or with (d and e, right panels) 100 nM ORY-1001 for 24 h was examined by qPCR and immunoblot analysis (f). (g, h) SLC52A1-overexpressing U2OS cells treated with 2 µM etoposide and 100 nM ORY-1001 for 7 days were subjected to SA-β-gal (g) and EdU (h) assays. The percentage of SA-β-gal positive cells (g) and EdU positive cells (h) are shown. Data are mean ± s.d. (n = 3 independent cultures). Representative microscopic images are shown as Fig. S2c,d. (i) U2OS cells transfected with siRNA for SLC52A1 and treated with 2 µM etoposide for 2 days were subjected to qPCR. (j) SLC52A1-depleted U2OS cells treated with 2 µM etoposide for 2 days were subjected to immunoblot analysis. Protein levels relative to histone H3 or p53 levels were quantified using NIH ImageJ software. Original blots are presented in Supplementary Information. (k–m) SLC52A1-depleted U2OS cells treated with 2 µM etoposide for 2 days were subjected to qPCR (k and l) and immunoblot analysis (m). (c–f) qPCR and immunoblot data are representative of two independent experiments and values are shown as mean ± s.e.m. (c, f, j, m) Protein levels relative to histone H3 levels (c, j), p53 levels (c, j) or γ-tubulin levels (f, m) were quantified using NIH ImageJ software. Original blots are presented in Supplementary Information. Statistical significance is shown using Student’s t-test analysis; *p < 0.05; ***p < 0.005; n.s., not significant (p > 0.05).
