Increased survival of monocytes through a CYTL1/CCR2/MEK pathway. (A) ChIP-seq analysis of the CYTL1 gene in healthy donor monocytes. H3K27me3, H3K4me1, H3K4me3, and H3K27ac ChIP-seq data were obtained in silico. ChIP-seq of EGR1 was performed in monocyte samples from 2 healthy donors and 2 patients with CMML. Dotted rectangle indicates a suspected gene enhancer, based on histone marks repartition. (B) ChIP-qPCR analysis of EGR1 enrichment on CYTL1 enhancer using 2 sets of primers (Enh Set 1 and Set 2) in CMML monocytes with low (similar to controls) or high (compared with control; see Figure 3E) CYTL1 gene expression; CMML#1 and CMML#2 shown in panel A. (C) Immunoblot analysis of indicated proteins (p-ERK: ERK phosphorylated on thr202 and tyr204) in cells treated with 100 ng/mL rhCYTL1 for 15 minutes or 8 nM CAS 445 for 30 minutes or both. Actin was used as a loading control. Lower panel: quantification of the above immunoblot. (D) Healthy donor monocytes were incubated for 24 hours with or without 100 ng/mL CYTL1 before measuring the surviving fraction of cells by flow cytometry (AnV^–/PI^–, results normalized to control monocytes). Mann-Whitney U test: ****P < .0001. (E) Healthy donor monocytes were incubated for 24 hours with 100 ng/mL CYTL1 in the absence or presence of CAS 445479-97-0 (8 nM) or RS 504393 (10 µM) before measuring the surviving fraction of cells by flow cytometry (AnV^–/PI^–, results normalized to CYTL1 alone). Error bars are mean ± SEM. Kruskal-Wallis test, Dunn’s multiple comparison: *P < .05; **P < .01. AU, arbitrary units; HD_bed, healthy donor browser extensible data; IgG, immunoglobulin G.