Figure 8.

Impairment of efferocytosis by senescent cells is mediated by CD47 and CD24 expression. (A) CD47 loss-of-function in 3T3 cells was verified by CD47 staining in flow cytometry. (B) Representative immunofluorescence images from senescent 3T3 cells (WT or CD47 KO) showing CD47 (green) and Phalloidin (blue) staining (scale bar: 50 µm). n = 2. (C) Quantification of efferocytosis of apoptotic corpses by BMDMs in the presence of CD47 KO senescent cells (induced by palbociclib). Samples were analyzed by flow cytometry. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; n = 3 biological replicates. (D) Schematic overview of the experimental design. Senescent human fibroblasts were incubated with neutralizing anti-CD47 FAB fragments for 1 h, then direct co-cultures with primary macrophages were assembled for 6 h, followed by exposure to pHrodo labeled apoptotic corpses. Corpse removal was monitored by live cell imaging (IncuCyte). (E and F) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and senescent primary NHLF (E) or IPF-derived fibroblast (IPF; F). Efferocytotic capability of macrophages in co-culture with senescent fibroblast in the presence (blue bar) or absence of FAB fragments (gray bar) was monitored over time using the IncuCyte S3 system. Then area under curve (AUC) from 2 to 20 h was calculated and plotted. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by unpaired Student’s t test. (G) Whole-cell lysates derived from Panc1 cells (WT and CD47 KO) were analyzed by Western blotting for the indicated proteins. GAPDH was used as loading control. Senescence was induced by chemical treatment (Palbociclib). Blots are representative of three independent experiments. GAPDH image is derived from the same blot as Fig. 7 and Fig. S3. CD24 image is derived from the same blot as in Fig. S3. (H) Quantification of efferocytosis of apoptotic corpses by MDMs in the presence of senescent Panc1 cells. Efferocytotic capability of macrophages in co-culture with WT (red line) or CD47 KO (green line) Panc1 cells was monitored over time. Data are representative of at least three independent experiments. All values are means ± SEM. (I) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and proliferating (black bar) or senescent CD47 KO Panc1 cells. Senescent CD47 KO cells were treated with (orange bar) or without anti-CD47 FAB fragments (green bar) prior to the assembly of the co-culture with MDMs. Efferocytotic capability of macrophages was monitored over time using the IncuCyte S3 system. Then area under curve (AUC) from 2 to 22 h was calculated and plotted. Shown values are means of at least technical triplicates ± SEM in one representative experiment. Data are representative of three independent experiments. **P < 0.01. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction. (J) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and proliferating (gray bar) or senescent primary small airway cells (SAEC). Senescent SEAC cells were either untreated (green bar) or treated with anti-CD47 (blue bar) or anti-CD24 FAB fragments (orange bar) or the combination of both FAB fragments (violet bar) prior to the assembly of the co-culture with MDMs. Efferocytotic capability of macrophages was monitored over time using the IncuCyte S3 system. Then area under curve (AUC) from 2 to 22 h was calculated and plotted. Shown values are means of technical quadruplicates ± SEM in one representative experiment. Data are representative of two independent experiments *P < 0.05, **P < 0.005. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction. Source data are available for this figure: SourceData F8.