Extended Data Fig. 4: In vitro functional analysis of THP-1 macrophage cell line overexpressing LILRB3-4SNPs variant (‘Risk’) or reference (‘Non-risk’) allele.
From: LILRB3 genetic variation is associated with kidney transplant failure in African American recipients
qPCR on expression changes for immune response (a) and ferroptosis-negatively-associated genes (b) upon LPS stimulation from 0 to 6 h (left) or from 6 to 24 h (right). The heatmap colors (blue color for positive values and brown for negative values, respectively) along with the numbers indicate the average of log2(fold changes) from duplicated biological experiments. Cell viability analysis within 48 h upon LPS stimulation (c) and in conjunction with ferroptosis-inhibitor (lipro-1) treatment (d) in THP-1 cells overexpressing LILRB3-4SNPs variant (n = 4) or reference allele (n = 4) (Student’s t-Test, two sided). The bar plots represent the mean values of the cell viability measurements from four biological replicates and error bars represent one standard deviation. Following a 6 h LPS stimulation, all cell lines exhibited increased expression of crucial inflammatory response markers linked to the LILRB family, including TNFα and IL1β, and the expression of these genes decreased from 6 to 24 h. The cell line overexpressing the variant allele produced greater quantities of TNFα, TNFAIP3 and IL1β upon 6-h LPS treatment, with less attenuation between 6 and 24 h than the reference allele, implicating increased inflammation associated with the SNPs (a). Expression of 5/6 ferroptosis negatively-associated genes in cell line with the SNPs decreased more between 6 and 24 h post LPS treatment, consistent with enhanced ferroptosis at 24 h linked to the SNPs (b). Cell viability analysis demonstrated a reduced viability upon LPS stimulation in cells with the SNPs (c, upper, orange vs green) but not those without the SNPs (c, lower, orange vs green). This phenomenon for the SNPs was reversed by ferroptosis inhibitor, Liproxstatin-1 (Lipro-1, at 0.625 umol) that targets lipid peroxidation (d, orange bar, upper). Lipor-1 had no effect on the cells without SNPs (d, orange bar, lower).
