Figure 2.
Figure 2. RhoA-dependent NMII isoform localization. (A) Representative confocal microscopy images of in vitro cultured control and inhibitor-treated MKs sorted on day 5 of culture on the expression of CD41a+CD42+ followed by treatment with the indicated inhibitors: (from left) control, C3 transferase (RHO inhibitor), Y27632 (ROCK inhibitor), or P18 (MLC kinase inhibitor). Cells were stained for NMIIB and costained with DAPI to indicate the mitotic cells. At least 3 independent samples were analyzed and the number of mitotic cells was counted for NMII localization. The corresponding histogram plot indicates the percentage of cells with NMIIB localization at the cleavage furrow. (B) A representative confocal image of MKs expressing constitutively active (CA) RhoA (RhoAL63) or dominant negative (DN) RhoA (RhoAN19). Cells were fixed and stained for NMIIA, NMIIB, and DAPI. At least 3 independent samples were analyzed and the number of mitotic cells was counted for NMII localization. The corresponding histogram plot indicates the percentage of cells with NMIIA and NMIIB localization at the cleavage furrow. (C) Representative confocal microscopy image of in vitro cultured MKs treated with exozyme C3 transferase. Cells were stained for NMIIB and costained with DAPI to indicate the nucleus. (D) Representative confocal microscopy images of day 9 in vitro cultured control and inhibitor treated ERYs: (from left) control, Y27632, P18, and C3 transferase. Cells were stained for NMIIA and costained with DAPI to indicate the mitotic cells. At least 3 independent samples were analyzed and the number of mitotic cells was counted for NMIIA localization. The corresponding histogram plot indicates the percentage of cells with NMIIA localizing at the cleavage furrow. (E) A representative confocal image of erythroblasts expressing CA RhoA (RhoAL63) or DN RhoA (RhoAN19). Cells were fixed and stained for NMIIA, NMIIB, and DAPI. At least 3 independent samples were analyzed and the number of mitotic cells was counted for NMII localization. The corresponding histogram plot indicates the percentage of cells with NMIIA and NMIIB localization at the cleavage furrow. ND, not detected. All bars represent 10 μm.

RhoA-dependent NMII isoform localization. (A) Representative confocal microscopy images of in vitro cultured control and inhibitor-treated MKs sorted on day 5 of culture on the expression of CD41a+CD42+ followed by treatment with the indicated inhibitors: (from left) control, C3 transferase (RHO inhibitor), Y27632 (ROCK inhibitor), or P18 (MLC kinase inhibitor). Cells were stained for NMIIB and costained with DAPI to indicate the mitotic cells. At least 3 independent samples were analyzed and the number of mitotic cells was counted for NMII localization. The corresponding histogram plot indicates the percentage of cells with NMIIB localization at the cleavage furrow. (B) A representative confocal image of MKs expressing constitutively active (CA) RhoA (RhoAL63) or dominant negative (DN) RhoA (RhoAN19). Cells were fixed and stained for NMIIA, NMIIB, and DAPI. At least 3 independent samples were analyzed and the number of mitotic cells was counted for NMII localization. The corresponding histogram plot indicates the percentage of cells with NMIIA and NMIIB localization at the cleavage furrow. (C) Representative confocal microscopy image of in vitro cultured MKs treated with exozyme C3 transferase. Cells were stained for NMIIB and costained with DAPI to indicate the nucleus. (D) Representative confocal microscopy images of day 9 in vitro cultured control and inhibitor treated ERYs: (from left) control, Y27632, P18, and C3 transferase. Cells were stained for NMIIA and costained with DAPI to indicate the mitotic cells. At least 3 independent samples were analyzed and the number of mitotic cells was counted for NMIIA localization. The corresponding histogram plot indicates the percentage of cells with NMIIA localizing at the cleavage furrow. (E) A representative confocal image of erythroblasts expressing CA RhoA (RhoAL63) or DN RhoA (RhoAN19). Cells were fixed and stained for NMIIA, NMIIB, and DAPI. At least 3 independent samples were analyzed and the number of mitotic cells was counted for NMII localization. The corresponding histogram plot indicates the percentage of cells with NMIIA and NMIIB localization at the cleavage furrow. ND, not detected. All bars represent 10 μm.

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