1. A B C D ID8 vector scr sh cx2 cx3 cx4 cx6 CX3CR1 β-tubulin * ID8
Supplementary Figure 2. Creation of sublines of ID8 with downregulated CX3CR1. Downregulation of CX3CR1 in clones of ID8 stably transfected with a pool of shRNA constructs, scrambled shRNA, and vector control was determined using Western blot (A) and immunofluorescence staining (B). For Western blot the antibodies were used at the following dilutions: 1:200 rabbit anti-human-CX3CR1 in 1:200 animal-free blocker overnight at 4 C, and 1:500 mouse anti-human-α-tubulin in 3% BSA in deionized water for 1 h at room temperature (RT). Immunoreactive bands were visualized with an anti-(rabbit (or mouse) IgG)-peroxidase (1:1000 in 3% BSA in a solution of 50 mM tris-buffered saline, pH 7.4, 150 mM NaCl, and 0.05% Tween-20 (TBST), and enhanced chemiluminescence was read using Chemidoc (Bio-Rad) and Bio-Rad Chemidoc ImageReader software. For immunofluorescence staining the cells were cultured on poly-D-lysine-coated glass coverslips, fixed, permeabilized with 0.1% Triton X100, and blocked in 10% goat serum for 1 h at RT. Mouse anti-human- CX3CR1 antibodies were used at 1 μg/ml and incubated with cells for 2 h at RT. Secondary anti-mouse Alexa433-conjugated antibodies were used at 1:500 and incubated with cells for 1 h at RT in the dark. 4',6-Diamidino-2-phenylindole (DAPI) was added to the secondary antibody solution to a final concentration of 10 g/ml 10 min prior to the end of the incubation period. The cells were washed, air dried, and mounted on glass slides using ProlongGold. Fluorescent imaging was performed using a Zeiss AxioObserverD.1 fluorescence microscope. Cell migration (C) and proliferation (D) assays were performed as described before [19]. Briefly, cell migration was assessed by plating 5,000 of ID8 or clone cells on Transwell cell migration chambers with 8 micron pores bottom-coated with Matrigel for 5 hours in the presence of 5 nM CX3CL1 followed by removal of non-migrated cells by cotton swabs, visualization of migrated cells by hematoxylin staining, and quantification of migrated cells. Cell proliferation was conducted in the presence of 15 nM CX3CL1 for 24 h followed by WST-1 assay (BioVision); WST-1 reagent was incubated with cells for 1 h followed by measurement of OD440 as a readout of the assay. *p<0.05, Students’ t-test.