Figure S1.
Impairment of efferocytosis by senescent cells is independent of macrophage polarization. (A) Senescent 3T3 cells (labeled with Green Cytolight Rapid Dye) were co-cultured with naïve BMDMs. Co-cultures were either stimulated with LPS (upper panel) or IL4 and IL13 (lower panel). Images were taken over 24 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 200 µm. (B) Senescent Panc1 cells (labeled with Green Cytolight Rapid Dye) were co-cultured with naïve MDMs. Co-cultures were either stimulated with LPS (upper panel) or IL4 and IL13 (lower panel). Images were taken over 24 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 200 µm. (C) Quantification of efferocytosis of apoptotic corpses by MDMs in the presence of proliferating (black line) or senescent (red line) A549 cells over time. First, co-cultured macrophages were exposed to unlabeled apoptotic corpses. After 24 h, pHrodo labeled apoptotic corpses were added to the co-culture and efferocytotic activity of the macrophages was monitored over time by the IncuCyte S3 system. (D) Efferocytosis of apoptotic corpses by differently stimulated macrophages in the presence of senescent cells (induced by palbociclib). Unstimulated macrophages were polarized to a M1 phenotype by the addition of LPS or to a M2 phenotype by the addition of IL4 and IL13. Data are representative of three independent experiments. All values are means ± SEM; ***P < 0.001; ****P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; n = 3 biological replicates. (E) Quantification of efferocytosis of apoptotic corpses by MDMs that were co-cultured with NHLF, IPF or Panc1 cells and then further stimulated with either LPS (upper row) or IL4 and IL13 (lower row) Efferocytotic capability of macrophages in single (yellow line) or co-culture with proliferating (black line) or senescent (red line) was monitored over time.

Impairment of efferocytosis by senescent cells is independent of macrophage polarization. (A) Senescent 3T3 cells (labeled with Green Cytolight Rapid Dye) were co-cultured with naïve BMDMs. Co-cultures were either stimulated with LPS (upper panel) or IL4 and IL13 (lower panel). Images were taken over 24 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 200 µm. (B) Senescent Panc1 cells (labeled with Green Cytolight Rapid Dye) were co-cultured with naïve MDMs. Co-cultures were either stimulated with LPS (upper panel) or IL4 and IL13 (lower panel). Images were taken over 24 h. In each image group, the same field of view was shown across time. Data are representative of three independent experiments; scale bars indicate 200 µm. (C) Quantification of efferocytosis of apoptotic corpses by MDMs in the presence of proliferating (black line) or senescent (red line) A549 cells over time. First, co-cultured macrophages were exposed to unlabeled apoptotic corpses. After 24 h, pHrodo labeled apoptotic corpses were added to the co-culture and efferocytotic activity of the macrophages was monitored over time by the IncuCyte S3 system. (D) Efferocytosis of apoptotic corpses by differently stimulated macrophages in the presence of senescent cells (induced by palbociclib). Unstimulated macrophages were polarized to a M1 phenotype by the addition of LPS or to a M2 phenotype by the addition of IL4 and IL13. Data are representative of three independent experiments. All values are means ± SEM; ***P < 0.001; ****P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; n = 3 biological replicates. (E) Quantification of efferocytosis of apoptotic corpses by MDMs that were co-cultured with NHLF, IPF or Panc1 cells and then further stimulated with either LPS (upper row) or IL4 and IL13 (lower row) Efferocytotic capability of macrophages in single (yellow line) or co-culture with proliferating (black line) or senescent (red line) was monitored over time.

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