Fig. 3.

CSF-1R internalization is mediated predominantly by clathrin-sensitive small vesicle endocytosis with macropinosomes playing a minor role. (A) BMMs were stimulated with CXCL12 or CSF-1 and stained for total CSF-1R following fixation and Triton-X-100-mediated permeabilization. Note the lack of clustering and internalization of CSF-1R when stimulating with CXCL12. Boxed areas are shown at higher magnification in the lower left corner. Scale bar: 5 µm. (B) Quantification of macropinosomes (MP) per cell from A. Data show the mean±s.e.m. (n = 30 cells). Bars not connected by the same letter are significantly different (P<0.05, ANOVA followed by Tukey HSD post hoc analysis. (C) Quantification of the localization of CSF-1R to macropinosomes after treatment with CSF-1 or CXCL12 for 15 minutes. Data show the mean±s.e.m. (n = 240 cells across three experiments); P<0.001 (Student's t-test). (D) Cell surface staining of the CSF-1R after stimulation with CSF-1 or CXCL12 in BMMs. Dashed white lines show cell outlines. Scale bar: 10 µm. (E) Quantification of the CSF-1R cell surface fluorescence intensity during CXCL12 and CSF-1 treatment. A.U., arbitrary units. Data show the mean±s.e.m. (n>50 cells from two experiments); *P<0.05 (two-way ANOVA followed by Sidak's multiple comparison test). (F) BMMs were stained for total CSF-1R and CXCR4 before and after exposure to CSF-1 and CXCL12. CSF-1R was selectively transported to macropinosomes, whereas CXCR4 was not. Scale bar: 10 µm. (G) BMMs were treated with CSF-1 (3 minutes) and chased for the times indicated before visualizing the presence of Oregon-Green–transferrin (TfR) and 10,000 Da TXR–Dex. Scale bar: 10 µm.