Figure 1. Depolarization induces H1... | Rockefeller University Press

Figure 1.

$Figure 1. Depolarization induces H1 PARylation and relaxation in PMNs. (A) FRAP analysis (on at least 20–30 cells) of resting (NaCl) and stimulated (KCl) in PMNs. Neuronal excitation leads to increased dynamics of linker histone H1.0 on chromatin in PMNs. (B) Quantification of H1 immobile fraction in resting (NaCl, left) and stimulated (KCl, right) PMNs. Error bars represent standard deviation. *, P < 0.001, two-tailed Student’s t test. (C) PAR level observed by PAR immunofluorescence in resting PMNs (top) and in KCl-stimulated PMNs (bottom). DAPI, right. Bar, 100 µm. (D) Elevated PAR levels are restored when depolarized PMNs are treated with the PARP inhibitor 3AB. Bar, 100 µm. (E) Quantification of PAR levels. Elevated PAR levels in depolarized PMNs are restored by DHIQ or by 3AB, shown as arbitrary units. *, P < 0.05, two-tailed Student’s t test. Experiments were conducted at least twice. (F) Western blots of poly-ADP ribosylated PARP1 immunoprecipitated by PAR antibodies. Shown are unstimulated (NaCl, left lane), depolarized (KCl, middle lane), and depolarized PMNs treated with the PARP1 inhibitor DHIQ (KCl+DHIQ, right lane). The experiment was performed at least twice (independently), with similar results. (G) Western blots of poly-ADP ribosylated PAR immunoprecipitated with H1 antibodies. Shown are unstimulated (NaCl, left lane) and depolarized (KCl, right lane) PMNs. (H) Quantification of PAR levels over actin (*, P < 0.05, two-tailed U test).$

Depolarization induces H1 PARylation and relaxation in PMNs. (A) FRAP analysis (on at least 20–30 cells) of resting (NaCl) and stimulated (KCl) in PMNs. Neuronal excitation leads to increased dynamics of linker histone H1.0 on chromatin in PMNs. (B) Quantification of H1 immobile fraction in resting (NaCl, left) and stimulated (KCl, right) PMNs. Error bars represent standard deviation. *, P < 0.001, two-tailed Student’s t test. (C) PAR level observed by PAR immunofluorescence in resting PMNs (top) and in KCl-stimulated PMNs (bottom). DAPI, right. Bar, 100 µm. (D) Elevated PAR levels are restored when depolarized PMNs are treated with the PARP inhibitor 3AB. Bar, 100 µm. (E) Quantification of PAR levels. Elevated PAR levels in depolarized PMNs are restored by DHIQ or by 3AB, shown as arbitrary units. *, P < 0.05, two-tailed Student’s t test. Experiments were conducted at least twice. (F) Western blots of poly-ADP ribosylated PARP1 immunoprecipitated by PAR antibodies. Shown are unstimulated (NaCl, left lane), depolarized (KCl, middle lane), and depolarized PMNs treated with the PARP1 inhibitor DHIQ (KCl+DHIQ, right lane). The experiment was performed at least twice (independently), with similar results. (G) Western blots of poly-ADP ribosylated PAR immunoprecipitated with H1 antibodies. Shown are unstimulated (NaCl, left lane) and depolarized (KCl, right lane) PMNs. (H) Quantification of PAR levels over actin (*, P < 0.05, two-tailed U test).

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