Fig 3. bfc in macrophages and S2 cells is required for efficient efferocytosis.

A. Confocal images showing AC efferocytosis by control-, crq RNAi-, and bfc RNAi-treated S2 cells (the efferocytosis efficiency is quantified in Fig 2C). Live cells are shown in blue, while cells that engulfed FITC-labeled ACs are shown in green. Scale Bars = 50 μm. B. Schematic diagram of the bfc^ko mutant created by CRISPR/Cas9. The exon is denoted as a blue box, and the transcriptional direction is highlighted with an arrow. The double slash represents the Cas9 cutting site. The red marked amino acids represent the mutated sequence. “*” represents the introduced termination codon. C. The bfc mRNA levels were quantified by qPCR in w¹¹¹⁸ and bfc^ko mutant samples. D. The Bfc protein levels were also quantified via western blotting in w¹¹¹⁸ and bfc^ko mutant samples. Actin was used as the loading control. E. AO-stained wild-type, bfc^ko mutant, and bfc^ko mutant re-expressing UAS-bfc (under the control of a crq-Gal4 driver) stage 13 embryos. The insets represent the higher-magnification views of the demarked AO-stained corpses. Scale bar = 20 μm. F. Confocal stacks of the head region of stage 13 embryos on the lateral view. Macrophages and apoptotic bodies were stained with anti-Crq (green) and anti-Dcp1 (magenta) antibodies, respectively in wild-type, bfc^ko mutant, and bfc^ko; crqGal4>UAS-bfc embryos, which were imaged using the enhanced similarly to show macrophages. Scale bar = 20 μm. G. Graph showing the mean PI ± SEM for each genotype showed in (F). H. Western blot analysis of the Crq and Bfc protein levels in stage 13 embryos of w¹¹¹⁸, bfc^ko mutant, and bfc^ko mutant re-expressing UAS-bfc (under the control of a crq-Gal4 driver). Actin was used as the loading control. I-J. Quantification of the Crq and Bfc protein levels in (H) after normalization to the Actin levels; n = 3. Statistical analysis was performed using the student’s t-test(C) and one-way ANOVA(G, I, J).