Fig 2: Rab20 association with early phagosomes in IFN-?-activated macrophages. (A) Analysis of the association of endogenous Rab20 with phagosomes in RAW264.7 macrophages. RAW264.7 macrophages were incubated with 3-µm IgG-coated beads for 1 h and 15 min of chasing, and were subsequently fixed and stained for Rab20. Nuclei were stained with Hoechst 33258 and are shown in blue. Magnified images show the association of Rab20 with phagosomes. Scale bar: 10 µm. (B) Quantitative analysis of the association of endogenous Rab20 with phagosomes at the indicated time-points. Data show the mean±s.e.m. of one representative experiment out of three independent experiments. At least 100 phagosomes were analyzed in each experiment. ***P=0.001 (Student's two-tailed unpaired t-test). (C) Analysis of the association of endogenous Rab20 with phagosomes in bone marrow macrophages (BMM) as in A. Scale bar: 10 µm. (D) Quantitative analysis of the association of endogenous Rab20 with phagosomes in BMM as in B. (E) Western blot analysis of the association of endogenous Rab20 with phagosomes in RAW264.7 macrophages that were untreated or treated with 200 U/ml IFN-? for 16 h. Latex-bead phagosomes were isolated from untreated or IFN-?-treated RAW264.7 macrophages after 15, 30 and 60 min of chase. Rab20 and Rab5a levels were detected and actin was used as the loading control. (F) Quantification of the association of Rab20 with phagosomes from E, calculated relative to actin and normalized to the value of untreated macrophages at 15 min. Data show the mean±s.e.m. of three independent experiments. *P=0.05 (Mann-Whitney test). (G) IFN-?-treated RAW264.7 macrophages were incubated with 1-µm IgG-coated beads for 1 h and then processed for cryosectioning. Rab20 was detected by using a rabbit anti-Rab20 antibody followed by labeling with 10-nm Protein-A–gold. Arrowheads indicate the presence of Rab20 at the phagosomal membrane. (H) Quantitative analysis of the number of phagosomes positive for Rab20 in cells treated with IFN-? or left untreated.

Fig 3: The association of Rab20 with phagosomes extends the residence time of Rab5a. (A) A snapshot of RAW264.7 macrophages coexpressing mCherry and EGFP–Rab5a at time 0, analyzed by live-cell imaging. (B) Time series showing the association of EGFP–Rab5a with the phagosome of interest (asterisk) in macrophages expressing mCherry and EGFP–Rab5a. (C) Quantitative analysis of the association of EGFP-Rab5a (left panel) and mCherry (right panel) with phagosomes. (D) A snapshot of RAW264.7 macrophages coexpressing mCherry–Rab20 and EGFP–Rab5a at time 0, analyzed by live-cell imaging. (E) Time series showing the association of EGFP–Rab5a with the phagosome of interest (asterisk) in macrophages expressing EGFP–Rab5a and mCherry–Rab20. (F) Quantitative analysis of the association of EGFP–Rab5a (left panel) and mCherry–Rab20 (right panel) with phagosomes. (G) A snapshot at time 0 of RAW264.7 macrophages coexpressing mCherry–Rab20T19N and EGFP–Rab5a at time 0, analyzed by live-cell imaging. (H) Time series showing the association of EGFP–Rab5a with the phagosome of interest (asterisk) in macrophages expressing EGFP–Rab5a and mCherry–Rab20T19N. (I) Quantitative analysis of the association of EGFP–Rab5a (left panel) and mCherry–Rab20T19N (right panel) with phagosomes. The intensity of both mCherry and EGFP signals were normalized to the fluorescence intensity of the cytoplasm. The data show the mean±s.d. from three (mCherry and mCherry–Rab20) or two (mCherry–Rab20T19N) independent experiments. n, the number of phagosomes analyzed in at least three different cells. Grey lines in panels C, F and I represent the background levels. Scale bars: 10 µm.

Fig 4: EGFP-Rab20 expression induces the enlargement of early and late endosomes in macrophages. Cells were transfected with EGFP-Rab20 or EGFP-Rab20T19N and subsequently immunolabeled for (A) the early endosomal marker EEA-1 or (B) the late endosomal marker LAMP-2. Nuclei were stained with Hoechst 33258 and are shown in blue. Insets show regions of interest indicated by the white rectangles. Scale bar, 10 µm. Quantitative analysis of the size of (C) early and (D) late endosomes in EGFP–, EGFP-Rab20–, or EGFP-Rab20T19N–expressing RAW264.7 macrophages. For each group, at least 20 cells were analyzed. Data show mean ± SD of three independent experiments. The p values were calculated using Student's two-tailed t test. *p = 0.05. (E) Left, snapshot of RAW264.7 macrophages expressing EGFP-Rab20 analyzed by live-cell imaging. Scale bar, 10 µm. Right, the process of homotypic fusions between Rab20-positive endosomes. White arrows indicate fusion events. (F) Left, snapshots at time 0 of RAW264.7 macrophages coexpressing mCherry- or EGFP-Rab20 with EGFP-Rab5, EGFP-Rab7 (pseudocolored in red), and tdTomato-LAMP-1; white squares show zoomed-in areas on the right. Right, zoomed snapshots at the indicated time points of vesicles (asterisks). Scale bar, 10 µm. (G) The kinetics of Rab20 and Rab5, Rab7 or LAMP-1 association with endosomes shown in F.

Fig 5: Rab20 mediates the recruitment of Rabex-5 into phagosomes in IFN-?-activated macrophages. (A) Western blot analysis of Rab20, Rab5a and Rabex-5 levels in macrophages stably expressing Rab20 shRNAs (2640, 2643 and 2644). Actin was used as a loading control. Con, control. (B) Quantitative analysis of the expression level of Rab20 in macrophages stably expressing Rab20 shRNAs. The intensity was calculated relative to the actin loading control and normalized to the relative intensity in control samples. The data show mean±s.e.m. of three independent experiments. *P=0.05 (Student's two-tailed t-test). (C) Western blot analysis of Rab20, Rab5a and Rabex-5 levels in isolated phagosomes from control and Rab20-knockdown (KD) macrophages with or without IFN-? treatment. (D) Quantitative analysis of the association of Rabex-5 with phagosomes in control and Rab20-knockdown macrophages with or without IFN-? treatment. The relative Rabex-5 intensity was compared with the actin loading control and normalized against the 15 min time-point.