Single cell suspensions COS-7 cells were incubated with 1 mg/ml biotinylated HABP for 90 min at room temperature with gentle agitation. Cells were centrifuged ...
Supplemental material
Fig.S1. No hyaluronan expression was detected on COS-7 cell membrane. Single cell suspensions COS-7 cells were incubated with 1 mg/ml biotinylated HABP for 90 min at room temperature with gentle agitation. Cells were centrifuged, washed, and resuspended in PBS with 0.1% BSA. Streptavidin-conjugated FITC in PBS/BSA was added to a final dilution of 1:200 and incubated for 45 min. Cells were washed, resuspended in PBS/BSA, and subjected to flow cytometry analysis. Hyaluronan expression was determined by digestion with 16 units/ml streptomyces hyaluronidase prior to the addition of HABP. No hyaluronan expression was observed on COS-7 cell membrane. 1, hyaluronan expression in negative control cells treated by streptavidin-conjugated FITC. 2, hyaluronan expression in native COS-7 cells. 3, hyaluronan expression in hyaluronidase-treated COS-7 cells.
Fig.S2. oHA treatment had no influence on the amount of CD44 on cell surface Cell surface expression of CD44 receptor in cells with or without oHA treatment was detected by flow cytometry. Histograms indicated log fluorescence intensity (x axis) versus relative cell number (y axis). (A) HK-2 cells; (B) BT-549 cells. 1, isotype-matched antibody as negative control; 2, naïve cells; 3, oHA treated cells.
Table S1 oHA treatment has no effect on endogenous HA generation in COS-7 cells Quantitation of hyaluronan production after oHA treatment for 2 h (fg) Control (DMEM) 62.5 µg/ml oHA 125 µg/ml oHA 250 µg/ml oHA
23.0±2.5 20.0±3.4 18.0±2.6 24.0±3.2
The amount of hyaluronan was determined as described previously (35).
