Journal: The American Journal of Pathology
Article Title: Inhibiting the Secreted RGDKGE Collagen Peptide Selectively Controls CD8 + T-Cell Migration on Denatured Collagen-IV and Enhances Their Accumulation in Tumors
doi: 10.1016/j.ajpath.2025.09.008
Figure Lengend Snippet: RHOA and MYO9B play roles in the ability of the RGDKGE collagen peptide (P2) to modulate CD8 + T-cell migration on denatured collagen-IV (Den-Coll-IV). A and B: Whole cell lysates were prepared from Jurkat T cells treated with the P2 or control peptide (CP; A ) or dimethyl sulfoxide (DMSO) buffer control or RHOA inhibitor rhosin ( B ) and analyzed by Western blot analysis for total and activated RHOA using an active RHO detection kit. C and D: Quantification of Jurkat T-cell migration on denatured collagen-IV ( C ) or native collagen-IV (Nat-Coll-IV; D ) in the presence of DMSO or RHOA inhibitor rhosin. C: Data represent change in cell migration from four independent experiments with control DMSO treatment set to 100% for comparison. D: Data represent change in cell migration from three independent experiments with control DMSO treatment set to 100% for comparison. E and F: Cell lysates were prepared from Jurkat T cells treated with the P2 or CP ( E ) or DMSO or SRC non-receptor tyrosine kinase (SRC) inhibitor ( F ), and analyzed by Western blot analysis for MYO9B. G: Western blot analysis for MYO9B levels in control or MYO9B-specific siRNA-transfected Jurkat T cells. H and I: Quantification of control and MYO9B-specific knockdown Jurkat T-cell migration on denatured collagen-IV ( H ) or native collagen-IV ( I ). H: Data represent change in cell migration from four independent experiments with control siRNA treatment set to 100% for comparison. I: Data represent change in cell migration from two independent experiments with control siRNA treatment set to 100% for comparison. J: Quantification of MYO9B knockdown Jurkat T-cell migration on denatured collagen-IV in the presence of CP or P2. Data represent change in cell migration from three independent experiments with control CP treatment set to 100% for comparison. K: Example of F-actin polarization ( arrows ) in control or MYO9B-specific siRNA-transfected Jurkat T cells seeded onto denatured collagen-IV. L: Quantification of the mean percentage of F-actin polarized cells per ×400 field in control and MYO9B knockdown Jurkat T cells seeded onto denatured collagen-IV. Data represent percentage of F-actin polarized T cells from two independent experiments calculated from 10 ×400 microscopic fields per condition. Data are given as means ± SEM ( C , D , H – J , and L ). ∗ P < 0.05, ∗∗ P < 0.01. Scale bars = 20 μm ( K ).
Article Snippet: To detect active RHOA, the (Rhotekin-RBD) active RHO detection kit (Cell Signaling) was used.
Techniques: Migration, Control, Western Blot, Comparison, Transfection, Knockdown
