Fig 1: Measuring hCES2 activity in pancreatic cancer cells. Probe 2 (20 µM) was incubated for 3 hours with the pancreatic cancer cell line, SU.86.86. Top row, probe 2 with SU.86.86 cells containing a vector overexpressing CES2 (CES2 OE) displayed strong red over yellow fluorescence, indicating high conversion of probe 2 to product 3. Bottom row, probe 2 with parental SU.86.86 showed weak red fluorescence but high yellow fluorescence indicative of poor conversion of probe 2 to 3. Far top right, Western blot showing hCES2 expression levels. Far bottom right, bar graph quantifying the ratio of red over yellow fluorescence for CES2 OE and parental. Light gray bars indicate pretreatment with the CES2 inhibitor, LPA, dark gray bars are no pretreatment (images in Fig. S9†). Ratios are an average of 3 regions in an 8-chambered well plate. Magnification is 20×. Scale bars = 50 µm. Statistical significance is calculated using unpaired t test compared with CES2 (***p < 0.001).
Fig 2: Detecting hCES2 activity in orthotopic SU.86.86 xenografts. A few drops of probe 2 (20 µM) was added to a SU.86.86 frozen tissue slice (10 µm in thickness) on a microscope slide and imaged. (A) Single channel red fluorescence as a function of time whereby probe 2 added to SU.86.86 CES2 OE tissues (top row) led to larger time dependent increase in fluorescence, compared to control vector SU.86.86 tissues (bottom row). (B) Ratiometric images obtained after 3 hour incubation. Tissues were washed 3 × with PBS to obtain images in the yellow channel. A higher red/yellow ratio was observed for CES2 OE orthotopic xenografts compared to the respective control. Average fluorescence was collected at 12 different regions of the tissues at 3 hours to construct a box plot (far right). Magnification is 20×. Scale bars = 50 µm. Statistical significance is calculated using the Mann–Whitney–Wilcoxon test compared to CES2 OE (****p < 0.0001).
Supplier Page from Sino Biological, Inc. for Human CES2 / Carboxylesterase-2 Protein (His Tag)