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. 2015 Oct 27;14(22):3613–3623. doi: 10.1080/15384101.2015.1100777

Figure 3.

Figure 3.

NML knockdown prevents heterochromatin formation on rDNA. (A) A549 cells were treated with 0.3 µM CX5461 for 2 hrs and 24 hrs. Endogenous NML-SirT1 binding was analyzed by SirT1 IP and NML protein gel blot. (B) Pre-rRNA level in senescent cells induced by 0.1 µM doxorubicin or 0.3 µM CX5461 for 7 d was determined by RT-qPCR. Values are mean ± SD of triplicates. (C) A549 cells were incubated with indicated drugs for 4 d The endogenous SirT1-NML complex was detected by IP-western blot. (D) Pre-rRNA level in A549 NML knockdown cells treated with 0.1 µM doxorubicin for 24 and 72 hrs was analyzed by RT-qPCR. Values are mean ± SD of triplicates. (E, F) A549 NML knockdown cells were treated with 0.3 µM CX5461 for 1 and 7 d H3K9Me3 and H3K27Me3 levels on rDNA promoter were detected by ChIP and qPCR. (G) A549 expressing 2 different NML shRNA were treated with CX5461 for 7 d and analyzed for histone methylation at the rDNA promoter by ChIP.