Fig 1: Binding of FL1 to uPAR initiates internalization and lysosomal routing in PDAC cells through an LRP1-independent mechanism.(A) Left, representative flow cytometry histograms showing specific binding of A647-coupled FL1 to uPAR-expressing cells. Right, number of receptor binding sites per cell, estimated using QSC beads (n = 3). Numbers below 1 × 103 reflect a lack of uPAR expression. (B) Confocal microscopy analysis showing uPAR-dependent FL1 uptake and lysosomal trafficking in murine receptor–positive KPC cells. Cells were incubated for 4 hours at 37°C with A647-labeled mAb in the absence or presence of E64d. No uptake is observed in uPARKO cells. Cell membranes, in green, and nuclei, in blue, were stained with Alexa 488-labeled wheat germ agglutinin and DAPI, respectively. Scale bar, (pink) 20 μm and (white) 10 μm. (C) Real-time monitoring of FL1 uptake using the Incucyte S3 live-cell analysis system. The red fluorescent intensity shows a clear time-dependent increase in the uPAR-positive cells (n = 3). (D) Scatter plot of total uptake at 20 hours normalized to the phase area (cell confluence) (n = 3). (E) Flow cytometry analysis of A647-labeled FL1 internalization in the entire PDAC cell panel after 20 hours of incubation at 37°C with A647-FL1 and isotype control mAb, A647-IgG1. After incubation, surface-bound mAbs were removed by proteolytic treatment with a trypsin-EDTA, proteinase K solution (n = 2). (F) Correlation between flow cytometry and Incucyte live-imaging data. The regression line (blue) and the R-squared value (red) are displayed. (G) Scatterplot illustrating the relationship between detected cellular uPAR receptor number (y axis) and corresponding FL1 internalization (x axis). (H) Histograms showing internalized 125I labeled FL1 in selected human (left) and murine (right) uPAR-expressing cells in the presence and absence of 500 nM RAP (n = 3). All data are representative of at least two independent experiments.
Fig 2: In vitro cytotoxicity induced by FL1-PNU.(A) Overview of GlyClick conjugation producing homogeneous DAR 2 PNU–conjugated ADCs (detailed in Materials and Methods). On the right is the chemical structure of the Fc-conjugated linker payload (shown as a red octagonal star on the left side of the mAb). (B) Dose-response curves showing the in vitro cytotoxic activity of PNU-FL1 ADC compared to NC ADC, aTNP-PNU as assessed by MTS assay. The percentage of cell viability relative to untreated control cells (n = 3, ±SD) is shown, while the fitted EC50 values are provided in Table 1. (C) Viability curves of various uPAR-positive cells following preincubation with the lysosomal protease inhibitor, (100 μM E64d), and LRP1-antagonist, (500 nM RAP), before a 72-hour exposure to FL1-PNU and NC ADC (n = 3, ±SD). All assays were performed as three independent replicates.
Supplier Page from Abcam for Recombinant Human LRPAP1 protein (His tag N-Terminus)