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. 2024 Sep 30;15:1444558. doi: 10.3389/fimmu.2024.1444558

Figure 4.

Figure 4

Targeting the MHC class II enhanceosome in human neutrophils. (A) Representative histogram for CREB1 phosphorylation (S133) in healthy donor neutrophils after GM-CSF stimulation (gray) compared with unstimulated (white) for 30 min by using Phosflow antibody for flow cytometry. (B) CREB1 phosphorylation at S133 (Creb-P) after GM-CSF stimulation, measured by phospho flow cytometry, relative fold changes to unstimulated control. Medians, Kruskal–Wallis test. Inhibitors ruxolitinib (ΔJAK1/2 inhibitor, final conc. = 3 µM), SB 747651 A (ΔMSK1 inhibitor, final conc. = 10 µM), BI-D1870 (ΔRSK1-4 inhibitor, final conc. = 10 µM), and MK-2206 (ΔAkt1/2/3 inhibitor, final conc. = 10 µM), CAS 92-78-4 (ΔCREB-CBP interaction inhibitor, final conc. = 50 µM) were used. (C) CIITA mRNA expression after stimulation with human recombinant GM-CSF (10 ng/ml) for 4 h, 24 h, and 48h. Values are shown as relative fold change to unstimulated control and internal control (housekeeping genes, HK). Medians with interquartile range, n=6. (D) CIITA mRNA expression after stimulation with human recombinant GM-CSF for 4 h, pretreated with or without ruxolitinib for 1h. Relative control to unstimulated and housekeeping genes (HK). Medians, comparison of GM-CSF-stimulated via Wilcoxon signed rank test. (E) Human neutrophil signaling cascade leading to de novo MHC class II induction after stimulation with GM-CSF. JAK1/2, Janus kinase 1 and 2; STAT5, signal transducer and activator of transcription 5; Y694, tyrosine phosphorylation site at residue 694; CIITA, MHC class II transactivator; p38, p38 mitogen-activated protein kinase; MSK1, mitogen- and stress-activated protein kinase-1; RFX, regulatory factor X; CREB1, cAMP-responsive element binding protein 1; Pol II; polymerase II; CBP, CREB-binding protein; HLA-DR, HLA-DM, Li, MHC class II genes. * P ≤ 0.05; **** P ≤ 0.0001.