Differential HDAC1 and 2 Recruitment by Members of the MIER Family
Fig 2
Confocal analysis of MIER2 and MIER3 subcellular localization.
(A-C) MCF7 cells were transfected with a plasmid encoding myc tag alone or myc-tagged -MIER1α, -MIER2 or -MIER3 and localization was analyzed by confocal microscopy using DAPI (panels b, e, h, k) and the 9E10 myc tag antibody (AlexaFluor 488) (panels a, d, g, j, m, n). The merged DAPI and Alexafluor 488 channels are shown in panels c, f, i &l. (A) Illustrative examples of cells showing whole cell localization of the myc-tag (panels a-c), nuclear localization of MIER1α (panels d-f) and MIER3 (panels j-l) and mainly nuclear localization MIER2 (panels g-i). Panels m & n show and enlargement of the cells indicated by arrows in panels g & j; the brightness was increased in these panels to better illustrate the cyotplasmic localization of MIER2 (m), compared to exclusively nuclear localization of MIER3 (n). For all panels, white arrows indicate cells that display whole cell staining, with nuclear staining more intense than cytoplasmic (N>C); white arrowheads indicate cells showing whole cell staining with nuclear staining equal in intensity to cytoplasmic staining (N = C); red arrows with white outlines indicate cells with exclusively nuclear staining. (B) Histogram showing the percentage of cells, ± S.D., displaying each staining pattern: N, exclusively nuclear; N>C, nucleus more intensely stained than cytoplasm; N = C, nucleus and cytoplasm display equal staining intensity. Fields were selected at random and the staining pattern of all expressing cells in the field were scored visually from the compiled z-stacks. Only cells expressing myc-tagged proteins were included in the total counts and used to calculate percentages; 50–80 cells were scored for each construct. (C) Bar graph showing the intracellular distribution of each protein. Fields were selected at random and all cells expressing myc-tagged proteins in the field were analyzed, as described in the Materials and Methods. Pixel values for the nuclear and cytoplasmic compartments were measured in compiled confocal z-stacks using Image J v1.50 g. Plotted is the average value ± S.D. in each compartment, using measurements from 30–40 cells for each construct. (D-E) HEK293 cells were transfected with a plasmid encoding myc-tag alone or myc-tagged -MIER1α, -MIER2 or -MIER3 and localization was analyzed by confocal microscopy, as described for MCF7. (D) Histogram showing the percentage of cells ± S.D. in each of the categories described above. Localization was determined as described above for panel B; 50–80 cells were scored for each construct. (E) Bar graph showing the intracellular distribution of each protein, determined as described above in panel C. Plotted is the average value ± S.D. in each compartment, using measurements from 30–40 cells for each construct. For panels B-E, asterisks indicate values that are significantly different (p<0.05); there was no significant difference between the MIER1α and MIER3 values in any of the panels.