![Figure 8. Exogenous IL-17A promotes colorectal sphere maintenance, proliferation, and migration. (A) CICs and non-CICs from two independent patient specimens were plated in limiting dilution (50, 10, or 1 cell[s] per well) in 96-well plates and tested for the effect of exogenous IL-17A (100 ng/ml). The presence of spheres was evaluated after 14 d. Data are mean ± SD (n = 20); ***, P < 0.05. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (B) The tumorspheres area from one specimen (064; sphere frequency/sphere footprint using ImageJ software) was evaluated after 14 d. The mean area of spheres treated with IL-17A was 4,438.5 ± 960 µm2 compared to 2,458.6 ± 481 µm2 in the untreated group. P = 0.073. Student’s t test was used to assess the significance. The experiment was performed once. (C and D) Cells were sorted for CD44high/+ and CD44low/− and treated with IL-17A (100 ng/µl) for 12 h. (D) Cells were stained for β-catenin with DAPI nuclear background. Bar, 25 µm. (C) Beta-catenin localization was quantified. The experiment was performed once. (E–G) CICs cell viability, apoptosis, and proliferation of three independent specimens were assessed by (E) CellTiter-Glo, (F) caspase 3/7 activation, and (G) thymidine incorporation after stimulation with vehicle and exogenous IL-17A (100 ng/ml). Non-CICs from three independent specimens were plated and tested for the effect of exogenous IL-17A (H–J). Cell viability, apoptosis, and proliferation were assessed by CellTiter-Glo (H), caspase 3/7 activation (I), and thymidine incorporation (J) after stimulation with vehicle and exogenous IL-17A (100 ng/ml). Data are mean ± SD (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001; unlabeled, P > 0.05. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (K) Label retention of CICs from three independent patient specimens was evaluated by the staining with PKH-26-PE dye and the FACS analysis at day 0 (control) and after 8 d of stimulation with the vehicle and IL-17A. The experiment was performed once. (L) Migration of CICs from a human sample stimulated with exogenous IL-17A or a vehicle was evaluated by the scratch assay. The area of the scratch was monitored by time-lapse microscopy for 64 h. The experiment was performed twice and representative data are shown.](https://cdn.rupress.org/rup/content_public/journal/jem/210/13/10.1084_jem.20131195/4/jem_20131195_fig8.jpeg?Expires=1767655204&Signature=iXZ8EMbYlqNZ2EZG89UFIM57SfyMpeMjw~pHb1s~nK3TlhSf-XcaF0lmjDzucr2~7iXTDZEi5RpMAMhV1~bGGns2fBiziHzrH4FFucOhNdegjtK9foFqk38WhpKOzMbkJZd6MTqASczelLDRv7Fu-TNm8Tq1RlNNjwnppzkx9y9x5sHgPptVm9gUU6k3g5Hlg0nH9wY817fkyrSCM3c7otJ8Th1cbZ4eXhNrYH7MbgdN~elaaxrkcaZYu~8rxMZjla51erGK4WEdWh~EpkrlumXnruTZpgPpWbmgVQhVZnzsBvQnMOySPbcS0rVMtBhXoTzyTRpY7MVnYWi~V2sJ3g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Exogenous IL-17A promotes colorectal sphere maintenance, proliferation, and migration. (A) CICs and non-CICs from two independent patient specimens were plated in limiting dilution (50, 10, or 1 cell[s] per well) in 96-well plates and tested for the effect of exogenous IL-17A (100 ng/ml). The presence of spheres was evaluated after 14 d. Data are mean ± SD (n = 20); ***, P < 0.05. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (B) The tumorspheres area from one specimen (064; sphere frequency/sphere footprint using ImageJ software) was evaluated after 14 d. The mean area of spheres treated with IL-17A was 4,438.5 ± 960 µm2 compared to 2,458.6 ± 481 µm2 in the untreated group. P = 0.073. Student’s t test was used to assess the significance. The experiment was performed once. (C and D) Cells were sorted for CD44high/+ and CD44low/− and treated with IL-17A (100 ng/µl) for 12 h. (D) Cells were stained for β-catenin with DAPI nuclear background. Bar, 25 µm. (C) Beta-catenin localization was quantified. The experiment was performed once. (E–G) CICs cell viability, apoptosis, and proliferation of three independent specimens were assessed by (E) CellTiter-Glo, (F) caspase 3/7 activation, and (G) thymidine incorporation after stimulation with vehicle and exogenous IL-17A (100 ng/ml). Non-CICs from three independent specimens were plated and tested for the effect of exogenous IL-17A (H–J). Cell viability, apoptosis, and proliferation were assessed by CellTiter-Glo (H), caspase 3/7 activation (I), and thymidine incorporation (J) after stimulation with vehicle and exogenous IL-17A (100 ng/ml). Data are mean ± SD (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001; unlabeled, P > 0.05. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (K) Label retention of CICs from three independent patient specimens was evaluated by the staining with PKH-26-PE dye and the FACS analysis at day 0 (control) and after 8 d of stimulation with the vehicle and IL-17A. The experiment was performed once. (L) Migration of CICs from a human sample stimulated with exogenous IL-17A or a vehicle was evaluated by the scratch assay. The area of the scratch was monitored by time-lapse microscopy for 64 h. The experiment was performed twice and representative data are shown.
