Fig. 6

BAI1/Rac1 signaling facilitates efferocytosis and participates in crosstalk with Notch1 signaling in CAFs. Phagocytic activity of CAFs toward ApoSQ in the absence or presence of annexin V (AnxV, 10 μg/ml; a), transfected with BAI1 siRNA (d) or the BAI1-Flag construct (g), pretreated with a Rac1 inhibitor (NSC23766, 100 μM; k), or transfected with Notch1 siRNA (p). Representative dot plots showing the percentages of phagocytosing CAFs (PKH26⁺/PKH67⁺) and nonphagocytosing CAFs (PKH26⁺/PKH67^-) as indicated in the corresponding quadrants as determined by flow cytometry (left) and quantification of these populations (right). l Phagocytic activity of CAFs toward PKH67⁺ ApoSQ (green) in the absence or presence of NSC23766. The nucleus and actin cytoskeleton of CAFs were stained with DAPI (blue) and TRITC (tetramethylrhodamine)-conjugated phalloidin (red), respectively. Representative image of CAFs phagocytosing ApoSQ (left). The ROI − Merge panels show high-magnification images of regions of interest as indicated by the white squares in the Merge panels. Original magnification: ×200. Scale bars = 20 μm. The arrowheads indicate CAFs with engulfed apoptotic 344SQ cells or fragments. Phagocytosis was quantified by a phagocytic index based on confocal microscopy (right). Immunoblot analysis of the indicated proteins in CAFs exposed to ApoSQ for 20 h (b, e, m) or 2 h (h). c, f, i, n ELISA of WISP-1 in CAF CM and ApoSQ-CAF CM 20 h after exposure to ApoSQ. Relative Rac1 activity in CAFs at the indicated time points after ApoSQ stimulation (j) or transfection with Notch1 siRNA before ApoSQ stimulation for 24 h (o). NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t test. The data are from one experiment representative of three independent experiments with similar results (a, d, g, k, l and p left, b, e, h, m) or from three independent experiments (mean ± standard error; a, d, g, k, l and p right, c, f, i, j, n, o)