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. 2021 Oct 18;10(10):2786. doi: 10.3390/cells10102786

Figure 5.

Figure 5

M can associate with chromatin in living cells dependent on residues R170 and K172. (A–C) Nuclear association of GFP alone or the indicated GFP fusion proteins (H3 histone, Mwt and mutants M-R170A and R170A/K172A) was assayed 24 h after transfection of HeLa cells by treating with 1% NP-40 for 15 min at room temperature. (A) Cells were subjected to CLSM; the green channel (GFP) is shown, with cellular dsDNA shown in blue (Hoechst). Note that not all cells in the panel are transfected. (B,C) Image analysis was performed using the NIH ImageJ software on CLSM files such as those shown (A) to determine the level of protein associated with the host cell chromatin. Results represent the mean +/− SEM (n = 15), expressed as a percentage relative to the value for GFP-H3 (B) or for the mutant derivatives of M relative to GFP-Mwt (C). (D–F). Cells were treated with NP40 as in (A) followed by incubation with DNase I (0.2 mg/mL) or RNase A (0.2 mg/mL) for 1 h at room temperature. (D) Cells were imaged as per (A). (E,F) Images such as those shown in (D) were analyzed as per (B,C). Results represent the mean +/− SEM (n = 15) as a percentage relative to nuclei treated with NP-40 alone, for GFP-H3 (E) and GFP-Mwt (F), respectively, as indicated. ** p < 0.01, *** p < 0.001.