Fig. 6. WDFY3 regulates efferocytosis in human macrophages.

a Schematics of human monocyte differentiation to macrophages (HMDMs) and knockdown of WDFY3 with Lipofectamine RNAiMAX-mediated transfection of siRNAs targeting WDFY3, or non-targeting siRNAs as the control. b Validation of knockdown efficiency at mRNA level by qRT-PCR (n = 4 independent experiments, each from the average of 3 technical replicates). c Validation of knockdown efficiency at protein level by western blot (n = 2 biological replicates, data are representative of 3 independent experiments). d Efferocytosis of apoptotic Jurkat cells labeled by both Hoechst and pHrodo. The percentage of HMDMs with Hoechst-labeled ACs (indicating uptake), and the percentage of Hoechst⁺/pHrodo⁺ HMDMs in Hoechst⁺ HMDMs (indicating acidification upon uptake) were quantified by flow cytometry. Both uptake and acidification of ACs were impaired in HMDMs with siRNA-mediated WDFY3 knockdown (n = 4 independent experiments, each from the average of 2 technical replicates). e Fragmentation of engulfed ACs was assessed 3 h after washing away the unengulfed ACs. The percentage of HMDMs with non-fragmented PKH26 staining in all PKH26⁺ HMDMs was determined (n = 4 independent experiments). f Flow cytometry-based degradation assay was performed in HMDMs with procedures as described in Fig. 3b (n = 5 independent experiments). g RNA-seq was performed for HMDMs either unstimulated (M0) or treated with LPS and IFNγ for 18-20 h (M1-like). The expression of WDFY3 was visualized (n = 48 biological replicates). The box shows Q1, median, and Q3; the whiskers show 1.5 x interquartile range, though the lower whiskers in this plot only extend to the minimum as the minimum values are greater than the values corresponding to the lower whiskers. There is one outlier in M0 with FPKM 7.404. There are two outliers in M1-like with FPKMs 5.943 and 4.345. Data are presented as median ± 95% CI. Two-sided P values were determined by Mann–Whitney test.