FIG. 5.

A histone deacetylase inhibitor and a mutation in the RCL domain change the MENT location in nuclear chromatin. (A) Immunofluorescence microscopy of proliferating 3T3/MENT-63 cells (panels 1 to 3), the same cells incubated in the presence of 10 mM sodium butyrate for 48 h (panels 5 to 7), and 3T3/MENTov-16 cells stably expressing a MENT-ovalbumin swap mutant (panels 9 to 11) fixed in acetone-methanol and stained with α-MENT (panels 1, 5, and 9) and Hoechst 33258 (panels 2, 6, and 10). Panels 3, 7, and 11, overlays of images stained with α-MENT and Hoechst. Panels 4, 8, and 12, immunofluorescence microscopy of unfixed, unstained proliferating NIH 3T3 cells transiently expressing GFP fusions with MENTwt (panels 4 and 8) and MENT-ovalbumin swap (panel 12). The cells in panel 8 were incubated for 48 h with 10 mM sodium butyrate. Bar, 10 μm. (B) Protein samples from the nuclei of 3T3/MENT-63 cells stably expressing MENTwt (MENT-63) and control NIH 3T3-lacI cell clones stably transfected with nonexpressing vector (NIH 3T3) incubated in the absence (−) or presence (+) of 10 mM sodium butyrate (But) for 48 h were analyzed by SDS-PAGE and Western blotting with α-MENT, α-H3me2K9, α-H3me3K9, α-H3acK9, and α-H4acK12 as indicated. (C) Protein samples from the nuclei of proliferating (p) and quiescent (q) 3T3/MENT-63 cells and control NIH 3T3-lacI cells were analyzed by SDS-PAGE and Western blotting with α-MENT, α-H3me2K9, α-H3me3K9, α-H3acK9, and α-H4acK12 as indicated.