Figure 1.
Expression of CD47, β1-integrin and α5-integrin receptors on various Jurkat cell lines used.
6A is the wildtype Jurkat T-cells and parental strain to A1, which is deficient in β1-integrin expression. JinB8 is a CD47-deficient derivative of Jurkat cells, while JinB8-CD47 is stably reconstituted with expression of CD47 isoform 2. Cells were harvested, washed with PBS and incubated with the indicated primary antibodies. Following washes and incubation with fluorophore-conjugated secondary antibodies, bound antibodies was assessed by flow cytometry as an indicator of receptor expression levels. Normal mouse IgG was used as a non-specific primary antibody control.
Figure 2.
Incubation of Jurkat cells with 4N1K results in non-specific binding of various antibodies.
Jurkat 6A or A1 cells were incubated without or with treatment (1 mM MnCl2, 50 µM 4N1K or 50 µM 4NGG) for 30 min at 37°C, along with one of three antibodies: A) HUTS-21 to detect activated epitopes of β1-integrins, B) TS2/16 to detect total β1-integrins, C) a non-specific IgG control antibody, and D) no primary antibody control treatment. Cells were then washed, incubated with fluorophore-conjugated secondary antibody, and antibody binding was assessed using flow cytometry. E) Flow cytometry data of A1 cells incubated with the indicated concentrations of 4NGG or 4N1K peptide and fluorophore-conjugated secondary antibody. Histogram data as shown is for one experiment that is representative of three independently conducted experiments.
Figure 3.
Immobilized 4N1K binds non-specifically to several IgG antibodies.
A) Wells of a 96-well dish were coated with the indicated concentrations of peptides and washed with PBS. The amount of adsorbed peptides was quantified using the CBQCA protein assay kit as indicated in methods and materials. The plotted data represent the means ± standard deviation of three replicate wells of one experiment that is representative of three independently conducted ones. B) HUTS-21 or an IgG isotype control antibody were added to wells pre-coated with 50 µM 4NGG, 50 µM 4N1K, 50 µM poly-L-lysine, or 1%BSA/PBS. Following washes and incubation with a secondary antibody, bound antibodies were detected using a microplate spectrofluorometer. Data as plotted are the means ± standard deviation for three replicate samples of one experiment (*, p<0.05) that is representative of three independently conducted experiments. C) Same as in B) but titrating amounts of 4NGG and 4N1K were adsorbed to wells and only secondary antibodies were added.
Figure 4.
Cells bind to immobilized 4N1K independently of CD47 expression.
A) Jurkat, JinB8, or JinB8-CD47 cells pre-labeled with Celltracker Green were added to wells pre-coated with 50 µM 4NGG, 50 µM 4N1K, or 50 µM poly-L-lysine, and incubated for 20 min. In-well fluorescence was measured using a spectrofluorometer both before and after washes to remove non-adherent cells, and % adhesion was calculated as stated in methods and materials. Data as plotted are the means ± standard deviation for three replicate samples of one experiment (*, p<0.05; **, p<0.002) that is representative of three independently conducted experiments. B) Same as in A) but with titrating amounts of 4NGG and 4N1K pre-adsorbed to wells. Data as plotted are the means for samples conducted in duplicates.
Figure 5.
Cell adhesion to fibronectin is decreased with 50 µM 4N1K treatment.
Jurkat 6A and A1 cells were labeled with Celltracker Green, then treated with buffer, 50 µM 4NGG, 50 µM 4N1K, or 1 mM MnCl2 for 20 min prior to seeding onto fibronectin-coated wells. In-well fluorescence was measured using a spectrofluorometer both before and after washes to remove non-adherent cells, and % adhesion was calculated as stated in methods and materials. Data as plotted are the means ± standard deviation for three replicate samples of one experiment (*, p<0.001).
Figure 6.
4N1K-mediated inhibition of adhesion occurs in a CD47-independent manner.
A) Jurkat, JinB8 and JinB8-CD47 cells were labeled with Celltracker Green, then treated with buffer, 50 µM 4N1K, or 1 mM MnCl2 for 20 min prior to seeding onto fibronectin-coated wells and % adhesion calculated as stated before. Data as plotted are the means ± standard deviation of three replicate samples of one experiment (*, p<0.05) that is representative of three independently conducted ones. B) Same as in A) but with titrating amounts of 4NGG and 4N1K treatment of cells.