Fig. 3: NCOR2-013 inhibits macrophage inflammatory response and antifungal activity.
From: Talaromyces marneffei suppresses macrophage inflammation by regulating host alternative splicing
The human NCOR2-013 overexpressing THP-1 macrophages or NCOR2-013 overexpressing hMDMs were infected with T. marneffei conidia (MOI = 10) for 24 h. (a-c) NCOR2-013 overexpression in THP-1 macrophages was confirmed by RT-qPCR (a) (n = 4 biological replicates, data are presented as mean values ± SD), RNA-seq analysis (b) (n = 3 biological replicates, data are presented as mean values ± SD), and WB (c) (n = 3 biological replicates). d Prediction of subcellular localization for NCOR2-013 using the Hum-mPLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/hum-multi-2/). e The expression levels of NCOR2-013 in the cytoplasm and nucleus in NCOR2-013 overexpressing THP-1 macrophages were detected by WB. β-actin and PCNA were used as the loading control for cytoplasm and nucleus, respectively. f The subcellular localization and quantification of NCOR2-013 in NCOR2-013 overexpressing THP-1 macrophages were demonstrated by immunofluorescence. Quantification of the data using software ImageJ (n = 3 biological replicates). T. marneffei colony forming units (CFU) in NCOR2-013 overexpressing THP-1 macrophages (g) (n = 10 biological replicates, data are presented as mean values ± SD) or hMDMs (h) n = 3 biological replicates, data are presented as mean values ± SD, respectively. Four gradient serial dilutions (100, 10−1, 10−2, 10−3) were performed. i The mRNA expression levels of TNF-α and IL-1β were detected by RT-qPCR in NCOR2-013 overexpressing THP-1 macrophages and control cells infected or uninfected with T. marneffei for 24 h and 48 h. GAPDH was used as the loading control (n = 3 biological replicates, data are presented as mean values ± SD). j The protein levels of TNF-α and IL-1β were measured using CBA human inflammatory cytokines kit in NCOR2-013 overexpressing THP-1 macrophages and control cells infected or uninfected with T. marneffei at 24 h and 48 h (n = 3 biological replicates, data are presented as mean values ± SD). WB were shown were the representative blot. Two-tailed Student’s t test was used to determine significance, denoted by * (P < 0.05), ** (P < 0.01), and ns (not significant). Supplementary material is available (Supplementary Fig. 6; Supplementary Data 1). hMDMs human monocyte-derived macrophages, MOI multiplicity of infection, NC negative control, NCOR2-013 OE NCRO2-013 overexpression, Tm T. marneffei.
