Fig. 3. WDFY3 deficiency led to defects in LC3-associated phagocytosis (LAP) and the degradation of engulfed ACs.

a Cre⁻ and Cre⁺ BMDMs were incubated with PKH26-labeled ACs for 1 h. After washing away the unengulfed ACs, BMDMs were placed back to the incubator for another 3 h. BMDMs were then fixed and imaged. The percentage of BMDMs showing non-fragmented PKH26 signals in the total number of PKH26⁺ BMDMs was quantified (n = 4 and 5 biological replicates for Cre− and Cre+ respectively, each from the average of 3 technical replicates). b Cre⁻ and Cre⁺ BMDMs were incubated with TAMRA-labeled ACs for 1 h. After washing away the unengulfed ACs, BMDMs were either collected for flow cytometry to quantify the MFI of TAMRA or placed back to the incubator for another 16 h and then collected for flow cytometry. The rate of degradation was calculated, as shown in the schematics (n = 8 and 9 biological replicates for Cre− and Cre+ respectively). c Cre⁻ and Cre⁺ BMDMs were incubated with ACs labeled by Hoechst, which stains DNA and is pH-insensitive, and pHrodo, which is pH-sensitive and shows fluorescent signal only under an acidified environment in the phagolysosome. The percentage of Hoechst⁺ BMDMs indicates uptake. The percentage of Hoechst⁺/pHrodo⁺ BMDMs in Hoechst⁺ BMDMs indicates acidification of the engulfed cargos (n = 8 biological replicates, each from the average of 2 technical replicates). d Schematics of known functional domains and binding partners of human WDFY3. e The interaction between WDFY3 and GABARAP was assessed by co-immunoprecipitation. Cre⁻ and Cre⁺ BMDM cell lysates were incubated with anti-GABARAP antibody and protein A/G agarose beads. Beads-bound proteins were detected with anti-WDFY3 antibodies (n = 3 independent experiments with similar results). HC refers to heavy-chain. f Cre⁻ and Cre⁺ BMDMs were incubated with ACs for 1 h. Unbound ACs were washed away and BMDMs were collected for measurement of LC3-II by western blot (n = 5 biological replicates. The image shows the representative blot. * denotes non-specific band). g BMDMs were incubated with Hoechst-labeled ACs to allow efferocytosis. After removal of unbound ACs, BMDMs were collected and treated with digitonin to remove non-membrane bound LC3, and then immunostained for LC3 that is lipidated and membrane-bound. LC3-II staining was then quantified by flow cytometry for BMDMs that had engulfed Hoechst-labeled ACs (n = 5 biological replicates). Data are presented as mean ± SEM in (b, c, f), or as median ± 95% CI in (a, g). Two-sided P values were determined by a two-way ANOVA with Tukey’s multiple comparisons test in (b, c, f), or by Mann–Whitney test in (a, g).