Fig. 3
LSD1 is activated in response to senescent stress. (a) U2OS cells transfected with siRNA for LSD1 and treated with 2 µM etoposide for 5 days were subjected to immunoblot analysis. (b) U2OS cells transfected with siRNA for LSD1 and treated with 2 µM etoposide for 7 days were subjected to qPCR (left panel) and immunoblot analysis (right panel). (c) U2OS cells treated with 100 nM ORY-1001 and 2 µM etoposide for 5 days were subjected to immunoblot analysis. (d) U2OS cells treated with 100 nM ORY-1001 and 2 µM etoposide for 7 days were subjected to qPCR (left panel) and immunoblot analysis (right panel). (e, f) U2OS cells treated with 100 nM ORY-1001 and 2 µM etoposide for 6 days were subjected to ChIP-qPCR analysis to measure H3K4me2 and LSD1 bound to the Sirtuin-4 promoter sites. (g) U2OS cells transfected with p3XFLAG-LSD1 and treated with 2 µM etoposide for 5 days were subjected to immunoblot analysis. (h) U2OS cells transfected with p3XFLAG-LSD1 and treated with 2 µM etoposide for 7 days were subjected to qPCR (left panel) and immunoblot analysis (right panel). Protein levels relative to histone H3 levels (a, c, g) or γ-tubulin levels (b, d, h, right panel) were quantified using NIH ImageJ software. Original blots are presented in Supplementary Information. Data are representative of three (a, c, g) or two (b, d, h, right panel) independent experiments and values are shown as mean ± s.e.m. Statistical significance is shown using Student’s t-test analysis; *p < 0.05; **p < 0.01; ***p < 0.005; n.s., not significant (p > 0.05).
