Fig. 4: NELF is globally and transiently released from chromatin upon macrophage activation. | Nature Communications

Fig. 4: NELF is globally and transiently released from chromatin upon macrophage activation.

From: Negative elongation factor complex enables macrophage inflammatory responses by controlling anti-inflammatory gene expression

Fig. 4

a, b Heat map (a) and average signals (b) of NELF-E ChIP-seq around TSS of all BMDM-expressed genes (n = 10076 genes) for BMDM treated with LPS as indicated. The rows in a were sorted by decreasing NELF-E occupancy in TSS regions in resting BMDM. c Immunoblotting of chromatin-associated NELF-E, NELF-B, and actin in BMDM treated as indicated (n = 4). NELF-E and NELF-B bands were quantified by densitometry, normalized to internal control (actin), and expressed relative to untreated (0 h) sample (=1) (bottom) (NELF-E LPS 0 h/0.5 h P = 0.0063, LPS 0.5 h/1 h P = 0.0138; NELF-B LPS 0 h/0.5 h P = 0.005, and LPS 0.5 h/1 h P = 0.0314). d Immunoblotting of chromatin-associated NELF-B, p65, Pol II S2P, and actin in mock (PBS) and flavopiridol (300 nM)-pretreated (0.5 h) BMDM followed by LPS (0.5 h) (n = 3). NELF-B bands in (d, e, f) were quantified and normalized as in (c) (bottom) (LPS 0 h/0.5 h P = 0.0077, flavopiridol pretreated LPS 0 h/0.5 h P = 0.0322). e Immunoblotting of chromatin-associated NELF-B, p65, and actin in WT and MyD88 KO BMDM with or without Pam3CSK4 stimulation (10 ng/ml for 0.5 h) (n = 3). Statistics of NELF-B bands (bottom) (WT/WT + Pam3csk4, P = 0.0081; MyD88 KO/MyD88 KO + Pam3csk4, P = 0.2311). f Immunoblotting of chromatin-associated NELF-B and actin in mock (DMSO) and Bay 11–7082 (5 μM)-pretreated (0.5 h) BMDM followed by LPS (0.5 h). p-p65 blot is control for Bay 11-7082 activity (n = 3). Statistics of NELF-B bands (bottom) (LPS 0 h/0.5 h, P = 0.0016; Bay 11–7082 pretreated LPS 0 h/0.5 h, P = 0.1042). g NELF-E occupancy in Tnf, Jun, Cited2, Fos TSS regions was assessed by ChIP-qPCR in mock (DMSO) and Bay 11–7082 (5 μM)-pretreated (0.5 h) BMDM followed by LPS (0.5 h) (n = 3) (P values for different comparative conditions in Tnf, Jun, Cited2, and Fos TSS are listed respectively: LPS 0 h/0.5 h P = 0.0137, 0.0094, 0.0006, 0.0006; LPS 0.5 h/LPS 0.5 h+Bay 11–7082, P = 0.0345, 0.0245, 0.023, 0.1082; LPS 0 h/LPS 0.5 h+Bay 11–7082, P = 0.5634, 0.5074, 0.5464, 0.3562). Mean + SD. *P < 0.05, **P < 0.01, ***P < 0.001, N.S. P > 0.05 by two-sided paired Student’s t test. n represents biologically independent experiments. Source data are provided as a Source Data file.

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