Selective imaging of cathepsin L in breast cancer by fluorescent activity-based probes

Highly-selective fluorogenic substrate and activity-based probe for monitoring cathepsin L activity in the breast cancer cell line MDA-MB-231.


Figure S6
Colocalization between cathepsin L ABP and cathepsin L antibody. Panel A MDA-MB-231 cells were incubated with MP-cL3 (red) probe (1M) for 8 hours, and then were fixed with methanol, stained with cathepsin L antibody (green) and subjected for confocal fluorescence microscopy (DAPI is colored blue). Panel B Graphs present pixel intensity from the five lines drawn on the image. The intensity in both channels (red for ABP, and green for cathepsin antibody) was adjusted to 100% (y axis). On the x axis the length of line (in nanometers) is presented. The high weighted colocalization between red and green channels is indicated by overlapping traces (high intensity red signals correlate with high intensity green signals, and the same is true for low intensity signals).

Figure S7
Cathepsin L labeling by MP-cL3 probe in MDA-MB-231 cells. The figure represents seven slides from two independent experiments. To ensure cathepsin L specific labeling, cells were incubated with MP-cL3 (red) probe (1 M) for 8 hours, and then were fixed with methanol, stained with cathepsin B antibody (green) and subjected for confocal fluorescence microscopy. Results demonstrate that the red spots (active cathepsin L) partially overlays with cathepsin B, indicating that these two enzymes are located in the same lysosomes. However, there are also some vesicles where only cathepsin B is present. The aggregated weighted colocalization coefficient from eight slides was calculated. Circles around cells represent the area taken to calculate weighted colocalization coefficient within single cells. Scale bar 20 m.

Figure S8
Colocalization between cathepsin L ABP and cathepsin B antibody. Panel A MDA-MB-231 cells were incubated with MP-cL3 (red) probe (1M) for 8 hours, and then were fixed with methanol, stained with cathepsin B antibody (green) and subjected for confocal fluorescence microscopy (DAPI is colored blue). Panel B Graphs present pixel intensity from the five lines drawn on the image. The intensity in both channels (red for ABP, and green for cathepsin antibody) was adjusted to 100% (y axis). On the x axis the length of line (in nanometers) is presented. The poor weighted colocalization between red and green channels is indicated by nonoverlapping traces (high intensity red signals do not correlate with high intensity green signals, and the same is true for low intensity signals).

Figure S9
Cathepsin L labeling by MP-cL3 probe in MDA-MB-231 cells. The figure represents eight slides from two independent experiments. Cells were incubated with MP-cL3 (red) probe (1 M) for 24 hours, and then were fixed with methanol, stained with cathepsin L antibody (green) and subjected for confocal fluorescence microscopy. Results demonstrate that after prolonged incubation MP-cL3 probes loses the selectivity and labels also cathepsin B (as indicated by immunoblotting). The aggregated weighted colocalization coefficient from eight slides was calculated. Circles around cells demonstrate the area taken to calculate weighted colocalization coefficient within single cells. Scale bar 20 m.

Figure S10
Cathepsin L labeling by MP-cL3 probe in MDA-MB-231 cells. In the experiment, cells were incubated with MP-cL3 (red) probe (1 M) for 24 hours, and then were fixed with methanol, stained with cathepsin L antibody (green) and subjected for confocal fluorescence microscopy. Results demonstrate that after prolonged incubation MP-cL3 probes loses the selectivity and labels also cathepsin B (as indicated by immunoblotting). Scale bar 20 m.              Table S13 The structures of amino acids that were also used In the HyCoSuL, but were not presented In Table 7.