Fig. 4. The C-terminal WDFY3 is sufficient for regulating degradation yet the full-length WDFY3 is required for the uptake of ACs during efferocytosis.

a Schematics of lentiviral overexpression of C-terminal WDFY3 in BMDMs of Cre^- and Cre⁺ mice. b C-terminal WDFY3 did not restore uptake, yet partially rescued the defects in cargo acidification in Cre⁺ mice (n = 7 biological replicates, each from the average of 2 technical replicates). c C-terminal WDFY3 restored LC3-II levels in Cre⁺ mice as determined by western blot (n = 5 biological replicates. * indicates non-specific bands) and in (d) by flow cytometry (n = 3 biological replicates). e BMDMs from GFP-LC3 mice were transfected with tdTomato-fused C-terminal WDFY3_(2981-3526) plasmid via electroporation. BMDMs were fed with Hoechst-labeled ACs. Unengulfed ACs were washed away and BMDMs were imaged to visualize GFP-LC3 phagosome association, C-WDFY3 intracellular localization, and GFP-LC3/tdTomato-WDFY3 colocalization with and without AC engulfment (n = 21 cells from 5 biological replicates. Images are representatives of 5 independent experiments each using one GPF-LC3 mouse). Data are presented as mean ± SEM. Two-sided P values were determined by a two-way ANOVA with Tukey’s multiple comparisons test in (b–d) or by unpaired t test in (e).