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. 2012 Aug 21;31(20):3961–3975. doi: 10.1038/emboj.2012.236

Figure 2.

Figure 2

The AATF:MRLC3 cytoplasmic complex is disrupted upon DNA damage. (A) Hypotonic lysis was used to generate nuclear and cytoplasmic fractions. The purity of the fractions was documented with immunoblotting for fibrillarin (nuclear) and tubulin (cytoplasmic). MRLC was exclusively detected in the cytoplasmic compartment. The subcellular distribution of MRLC was not affected by 20 J/m2 UV-C-induced genotoxic stress applied 30 min prior to lysis. (B) AATF resides in the cytoplasm of resting cells and translocates to the nucleus upon genotoxic stress. Nuclear and cytoplasmic fractions of mock- or UV-C-treated (20 J/m2) MEFs were generated as in (A). In the absence of UV, AATF is primarily cytoplasmic. Upon UV-C-induced DNA damage, substantial amounts of AATF are detectable in the nucleus. (C) Indirect immunofluorescence (IF) was performed to verify the subcellular localization of MRLC3 in MEFs 30 min following UV-C (20 J/m2). DAPI staining was used as a counterstain to provide a nuclear reference point. MRLC displayed a granular cytoplasmic staining pattern that was not affected by UV-C irradiation. No MRLC staining could be detected in the nuclei. (D) Indirect IF was performed to verify the subcellular distribution and spatial dynamics of AATF in MEFs 30 min following UV-C (20 J/m2). In resting cells, AATF staining revealed a dominant granular cytoplasmic pattern with some nuclear staining. 30 min following UV-C, only miniscule amounts of AATF remained detectable in the cytoplasm, while the bulk of AATF staining was now nuclear. (E) The DNA damage-induced nuclear re-localization of endogenous AATF was quantified using fluorescence microscopy. Error bars represent s.d. (*P<0.05). (F) Global Ser/Thr phosphatase inhibition promotes the nuclear accumulation of AATF. MEFs were mock-treated or pre-treated with 1 μM OA 60 min prior to lysis. Phosphatase inhibition resulted in nuclear accumulation of AATF. (G) Osmotic stress promotes the nuclear accumulation of AATF. MEFs were mock-treated or exposed to hypertonic Ringer solution 10 min prior to lysis. This non-genotoxic MK2-activating stimulus resulted in nuclear AATF accumulation similar to that seen after DNA damage. Figure source data can be found with the Supplementary data.