JCS03437 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression of Myc-RhoB T19N or Q63L mutants does not alter F-actin staining with phalloidin or actin content in Triton-soluble and −insoluble fractions. (A) Confocal images of F-actin staining with Oregon Green 488 phalloidin in HEK293 cells stably expressing CXCR2 and transfected with empty vector, Myc-RhoB T19N or Myc-RhoB Q63L. Cells were stimulated with vehicle (0 min) or 100 ng/ml CXCL8 for 30 minutes. Transfected cells were identified by staining with anti-Myc antibody. Bars, 5 μm (B) Western blot analysis of Triton-soluble (S) and −insoluble (I) actin isolated from cells expressing empty pcDNA 3 vector, Myc-RhoB T19N, or Myc-RhoB Q63L mutants that were stimulated with vehicle (Unt) or 100 ng/ml CXCL8 for 30 minutes.

• Supplemental Figure 2 -

  Fig. S2. RhoB siRNA impairs CXCR2-mediated chemotaxis. (A)Boyden chamber chemotaxis assay used to assess chemotaxis of HEK293 cells stably expressing CXCR2 and transiently transfected with either control (Ctrl) siRNA or RhoB-specific siRNA. The graphs display number of cells ± s.e.m. from 20 separate fields using the 20× objective lens Significant differences between Ctrl siRNA transfected cells versus RhoB-specific siRNA transfected cells are indicated (*P<0.005, Student’s t-test). (B) Western blot analysis of RhoB protein levels from Ctrl siRNA or RhoB-specific siRNA transfected cells used in Boyden chamber assay. β-tubulin western blot is shown as a control to monitor equal loading of protein. Data shown are representative from three separate experiments.

• Supplemental Figure 3 -

  Fig. S3. Confocal images of immunofluorescence staining of HEK293 cells stably expressing CXCR2 and transfected with Myc-RhoB WT. CXCR2 and LAMP-1 staining (A) or Rab11a staining (B) in cells stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 3 hours. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored where red is CXCR2, green is LAMP-1 (A) or Rab11a (B), and blue is Myc-RhoB WT. Bars, 10 μm

• Supplemental Figure 4 -

  Fig. S4. Transfection of cells with RhoB-specific siRNA decreases co-localization of CXCR2 with LAMP-1 following 3 hours of CXCL8 stimulation. Confocal images of immunofluorescence staining of HEK293 cells stably expressing CXCR2. CXCR2 and LAMP-1 staining in cells transfected with Ctrl siRNA (A) or RhoB-specific siRNA (B) and stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 3 hours. Transfected cells were identified by Cy3-siRNA. Overlay images are pseudocolored where red is CXCR2, green is LAMP-1, and blue Cy3-siRNA. Images were processed using Photoshop (Adobe Systems, San Jose, CA). Bars, 10 μm (C) Quantitation of co-localization of CXCR2 with LAMP-1 in cells stimulated with vehicle and cells stimulated with CXCL8 for 3 hours. Values shown are the mean ± s.e.m. Quantitation of the percentage of CXCR2 co-localized with LAMP-1 in 20 fields was performed using the MetaMorph Imaging system (Universal Imaging). Significant differences of cells transfected with Ctrl siRNA versus cells transfected with RhoB-specific siRNA are indicated (*P<0.005, Student's t-test). (D) Western blot analysis of RhoB protein levels from Ctrl siRNA or RhoB-specific siRNA transfected cells used for immunofluorescence staining. β-tubulin western blot is shown as a control to monitor equal loading of protein. Data shown are representative from three separate experiments.

• Supplemental Figure 5 -

  Fig. S5. Actin disrupting agents Latrunculin B and Cytochalasin D cause CXCR2 accumulation in the Rab11a compartment. Confocal images of immunofluorescence staining of CXCR2 and Rab11a in HEK293 cells stably expressing CXCR2. Overlay images are pseudocolored where red is CXCR2 and green is Rab11a. (A) Cells stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes and treated with 0.1 μM latrunculin B for 10 minutes. (B) Cells stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes and treated with 2 μM cytochalasin D for 60 minutes. Images were processed using Photoshop (Adobe Systems, San Jose, CA). Bars, 10 μm. Images shown are representative of at least 20 cells from three separate experiments.

• Supplemental Figure 6 -

  Fig. S6. CXCL8-induced CXCR2 degradation following 3 hours of CXCL8 stimulation in HEK293 cells stably expressing CXCR2 and transfected with Myc-RhoB WT. Representative western blot analysis of CXCR2 expression in vector and Myc-RhoB WT transfected cells in the presence of 20 μg/ml cycloheximide following stimulation with vehicle (Unt) or 100 ng/ml CXCL8 for 30 minutes, 180 minutes, or 360 minutes. Actin is shown as a control to monitor for equal loading of protein. Data shown are representative from three separate experiments.

• Supplemental Figure 7 -

  Fig. S7. Confocal images of immunofluorescence staining of HEK293 cells stably expressing CXCR2 and transfected with Myc-RhoB WT. CXCR2 and Rab11a staining in cells stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored where red is CXCR2, green is Rab11a (B) and blue is Myc-RhoB WT. Bars, 10 μm

• Supplemental Figure 8 -

  Fig. S8. Expression of Myc-RhoB Q63L does not cause transferrin receptor to co-localize with EGFP-Rab7. Confocal images of immunofluorescence stained transferrin receptor and EGFP-Rab7 in HEK293 cells stably expressing CXCR2 and transiently transfected with EGFP-Rab7 and empty vector or Myc-RhoB Q63L. Overlay images are pseudocolored where transferrin receptor is red and EGFP-Rab7 is green. Images were processed using Photoshop (Adobe Systems, San Jose, CA) and are representative of 30 fields. Bars, 10 μm. Data shown are representative from three separate experiments.

• Movie 1 -

  Movie 1. Time-lapse confocal images of HEK293 cells stably expressing CXCR2 and transiently transfected with EGFP-Rab11a and empty vector. A series of 60 images was taken at 1 second intervals following 30 minutes of CXCL8 stimulation. Data shown are representative for 60 cells observed in three separate experiments.

• Movie 2 -

  Movie 2. Time-lapse confocal images of HEK293 cells stably expressing CXCR2 and transiently transfected with EGFP-Rab11a and Myc-RhoB T19N. A series of 60 images was taken at 1-second intervals following 30 minutes of CXCL8 stimulation. Data shown are representative for 60 cells observed in three separate experiments.