Figure 4. BMAL1-KD increased senescence in SW480 BMAL1-KD but not in HCT116 BMAL1-KD and SW620 BMAL1-KD cells. (A) Senescence-associated β-galactosidase (SA-β-gal) activity was obviously increased in SW480 BMAL1-KD cells, but not in HCT116 BMAL1-KD nor in SW620 BMAL1-KD cells. SA-β-gal activity was measured by β-galactosidase staining (blue). Scale Bar represents 10 μM. Representative staining of three independent experiments was shown. (B) Immunofluorescence identified phosphorylated H2AX (pH2AX, red) in cell nuclei (Hoechst 33342, blue) of BMAL1-KD and control CRC cell lines. Representative staining of three independent experiments were shown. Scale bar, 20 μm. (C) Western-blot revealed significant increase of pH2AX mainly in SW480 BMAL1-KD cells. Left, a representative immunoblot of three independent experiments was shown. Right, Bar charts represented pH2AX expression level normalized to HSC70 (n=7; *p<0.05; ***p<0.001). All data are shown as means ± SEM.