Figure 9.
SHP-1 mediates efferocytosis paralysis. (A) Representative immunofluorescence images from direct co-cultures of senescent Panc1 cells and human MDMs. Cells were specifically stained for SHP-1 (green), macrophage marker CD45 (red), Phalloidin (magenta), and Hoechst (blue); scale bar: 20 µm. n = 3. (B) BMDMs from tdTomato+(orange) mice were co-incubated with senescent WT or Cd47-deficient 3T3 cells for the times shown on the left of the images. Fixed samples were stained for SHP-1 (green) and Phalloidin (blue). Scale bars indicate 50 µm (main) or 12.5µm (inset). n = 2. (C) Schematic overview of the intensity profile measurement of SHP-1 in BMDMs. Intensity was measured across the widest point of each cell. Intensity profiles were generated by measuring the distance in µm and the gray value intensity. (D) Quantification of intensity profile measurement of SHP-1 in BMDMs. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; ****P < 0.0001. n = 20 biological replicates. (E) Quantification of PHK26-labelled corpse efferocytosis in the presence of absence of NSC-87877 (SHP-1 and SHP-1 inhibitor). Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; *P < 0.05, ***P < 0.001, ****P < 0.0001; n = 3 biological replicates. (F and G) Quantification of efferocytosis of apoptotic corpses by MDMs in the presence of senescent NHLF (F) or Panc1 cells (G). Macrophages were pre-incubated for 1 h with NSC-87877, and then co-cultures were assembled prior to exposure to pHrodo labeled apoptotic corpses. Efferocytotic capability of macrophages with (blue line) or without inhibitor (black line) was monitored over time. Data are representative of three independent experiments. All values are means ± SEM.

SHP-1 mediates efferocytosis paralysis. (A) Representative immunofluorescence images from direct co-cultures of senescent Panc1 cells and human MDMs. Cells were specifically stained for SHP-1 (green), macrophage marker CD45 (red), Phalloidin (magenta), and Hoechst (blue); scale bar: 20 µm. n = 3. (B) BMDMs from tdTomato+(orange) mice were co-incubated with senescent WT or Cd47-deficient 3T3 cells for the times shown on the left of the images. Fixed samples were stained for SHP-1 (green) and Phalloidin (blue). Scale bars indicate 50 µm (main) or 12.5µm (inset). n = 2. (C) Schematic overview of the intensity profile measurement of SHP-1 in BMDMs. Intensity was measured across the widest point of each cell. Intensity profiles were generated by measuring the distance in µm and the gray value intensity. (D) Quantification of intensity profile measurement of SHP-1 in BMDMs. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; ****P < 0.0001. n = 20 biological replicates. (E) Quantification of PHK26-labelled corpse efferocytosis in the presence of absence of NSC-87877 (SHP-1 and SHP-1 inhibitor). Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; *P < 0.05, ***P < 0.001, ****P < 0.0001; n = 3 biological replicates. (F and G) Quantification of efferocytosis of apoptotic corpses by MDMs in the presence of senescent NHLF (F) or Panc1 cells (G). Macrophages were pre-incubated for 1 h with NSC-87877, and then co-cultures were assembled prior to exposure to pHrodo labeled apoptotic corpses. Efferocytotic capability of macrophages with (blue line) or without inhibitor (black line) was monitored over time. Data are representative of three independent experiments. All values are means ± SEM.

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