Journal: PLoS ONE
Article Title: Decreased Camptothecin Sensitivity of the Stem-Cell-Like Fraction of Caco2 Cells Correlates with an Altered Phosphorylation Pattern of Topoisomerase I
Figure Lengend Snippet: CPT response of CSC or non-CSC cell subpopulations of Caco2. ( A ) FACS analysis of Caco2 cells stained with PE-conjugated CD44 (top panel) or CD133 (lower panel) specific antibodies. ( B ) FACS analysis of Caco2 cells before or after FACS sorting based on CD44. (I) Unstained cells, (II) Caco2 cells stained for CD44 before sorting, (III) Caco2 cells after sorting for CD44+ cells, (IV) Caco2 cells after sorting for CD44− cells. The cells were analyzed after sorting by staining for CD44 ( C ) Survival assay of the FACS sorted CD44+ or CD44− cells. The cells were incubated with DMSO (the solvent of CPT) or 0.1, 0.2, or 0.4, µM of CPT for 60 hours before the percentage of viable cells was measured by the MTT method. ( D ) Graphical depiction of the REEAD assay. S(hTopI) folds into a dumbbell shaped structure, which supports cleavage and ligation activity mediated by TopI. Hereby the substrate is converted to a closed circle, which is hybridized to a surface-attached RCA-primer matching the specific primer annealing sequence (p) on the substrate. Subsequently, phi-polymerase is added to support RCA. The resulting RCA products are visualized by hybridization of fluorescently labeled probes matching the complementary identifier sequence (i) of the circles and the products analyzed using a fluorescence microscope. TopI is illustrated as a gray pacman with a “T”, the phi-polymerase as a dark gray circle. The RCA-primers and detection probes are indicated on the figure. ( E ) Measurement of TopI activity by REEAD in the CD44+ and CD44− FACS sorted cell populations, respectively. Representative microscopic images obtained by analyzing the TopI activity present in whole cell extracts from 10-10 4 cells are shown in the top panel. Signals obtained from circularized S(hTopI) are shown in red, while signals obtained from control circles are shown in green. The lower panel shows a quantitative depiction of three independent experiments. ( F ) CPT sensitivity of TopI in the sorted CD44+ and CD44− cells, respectively. S(hTopI) was incubated with whole cell extract from 10 4 cells in presence of 15 µM, 30 µM, 60 µM CPT or DMSO as indicated in the figure. One example out of 12 individual microscopic images of each reaction sample is shown in the top panel, where red signals correspond to circularized S(hTopI) while green signals were generated from control circles. The lower panel shows a quantitative depiction of three independent experiments. For all REEAD experiments, to avoid misinterpretation due to potential uneven distribution of RCA primers printed on the surface, the number of red and green signals were counted on 12 microscopic images and the results depicted as the ratio between the number of red (R) TopI specific- and green (G) control circle generated signals (R/G) as described  .
Article Snippet: FACS analysis and cell sorting Caco2 cell subpopulations were counted or separated into two fractions by FACS using a Gallios Flow Cytometer (Beckman Coulter Danmark ApS) after staining with CD44-PE or CD133-PE antibodies.
Techniques: Cycling Probe Technology, FACS, Staining, Clonogenic Cell Survival Assay, Incubation, MTT Assay, Ligation, Activity Assay, Sequencing, Hybridization, Labeling, Fluorescence, Microscopy, Generated