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. 2019 Jul 17;10:1661. doi: 10.3389/fimmu.2019.01661

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Copyright © 2019 Gao, Shang, Zhang, Zhang, Zhang, Zhang and Yang.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Binding of lnc-C/EBPβ with C/EBPβ isoform LIP and WDR5 is required for Lnc-C/EBPβ mediated Differentiation of MDSC. (A) RIP in V5-tagged LAP or LIP and lnc-C/EBPβ cotransfected HEK293T cells. RIP was performed using anti-V5 antibody and then PCR for lnc-C/EBPβ. pcDNA3.1, control plasmid; PC, positive control; NC, water. (B) Immunoblot of LAP and LIP in HEK293T cells. LIP/pcDNA3.1 (without V5-tagged), V5-tagged LAP/pcDNA3.1, and different concentrations of lnc-C/EBPβ pcDNA3.1 plasmids were cotransfected into 293T cells. IP was performed using V5 antibodies, and then immunoblot using anti-C/EBPβ (specific for both LAP and LIP). (C,D) CHIP-PCR on the promoter of IL4il of MDSCs. (E) IP-MASS of C/EBPβ interacting proteins (upper); Five C/EBPβ interacting partners associated with WDR5 were listed (lower). (F) Immunoblot of C/EBPβ and WDR5 interaction in GM-CSF alone and GM-CSFplusIL-6 mediated MDSCs. (G) RIP-PCR for lnc-C/EBPβ used WDR5 antibody in differently generated MDSCs. (H) RNA-protein pull-down in lnc-C/EBPβ and WDR5-V5 co-transfected HEK293T cells. No RNA and antisense RNA as control. (I) Immunostaining and RNA-FISH in mouse MDSC (M-MDSC, left) and human monocytes (Hu-MDSC, right). Red, WDR5; Green, lnc-C/EBPβ; Blue, nuclei. (J) CHIP-PCR in the H3K4me3 enrichment region on the promoter of IL4il. (K) Immunoblotting of LAP and LIP in transfected HEK293T cells using anti-C/EBPβ antibody. 293T cells did not express LAP and LIP. (L) Flow cytometry of Gr1⁺CD11b⁺, Gr1^hiCD11b⁺, and Gr1^lowCD11b⁺ MDSCs in WDR5 knockdown or exogenous WDR5 treated MDSCs. The percentages were compared (n = 3). (M) Flow cytometry of CD11b⁺Ly6G⁺Ly6C⁻ and CD11b⁺Ly6G⁻Ly6C⁺ MDSC in WDR5 knockdown or exogenous WDR5 treated MDSCs. The percentages were compared (n = 3). (N) Flow cytometry of MDSCs in mouse spleen after injecting WDR5 knockdown or exogenous WDR5 treated CD45.1⁺ BMCs. Percentage changes of MDSC subsets was compared (n = 6). (O) QRT-PCR of mouse WDR5 in cells transfected with different concentrations siRNA and flow cytometry of CD11b⁺ly6G⁺Ly6C⁻, CD11b⁺ly6G⁻Ly6C⁺ MDSCs in WDR5 knockdown treated MDSCs. kdNC, siRNA control; kdWDR5, WDR5 siRNA; oeNC, control empty plasmid; oeWDR5, WDR5 plasmid. Error bars in (C,D,J,L–O) represent standard deviations from 3 independent measurements. Two-tailed, paired T-test was used in (C,D,J,L–O). ^*p < 0.05; ^**p < 0.05; NS, no significant.