Fig. 4
Analysis of CK20 and Ki67 expressions in colonoids in response to tofacitinib or budesonide. (a) Representative images of immunohistochemical staining of Ki67 and CK20 protein expression in colonoids from N = 6 donors (3HC and 3 UC) in response to vehicle control (DMSO 0.033%), tofacitinib and budesonide cultured at either 2% or 20% oxygen. Graphs showing quantification of the percentage of (b) Ki67 expressing cells and (c) CK20 positive area in colonoids after treatment with tofacitinib and budesonide compared to vehicle control (DMSO 0.033%) cultured at 2% and 20% oxygen. (d) Representative image from the QuPath analysis of CK20 and Ki67 double fluorescence staining. We programmed QuPath to identify the number of single positive Ki67 cells annotated with pink lines (white arrow), single positive CK20 cells annotated with green lines (white squarehead) and Ki67 and CK20 double positive cells annotated with yellow lines (white circlehead). (e) Representative images of double fluorescence staining showing co-expression of CK20 and Ki67 in colonoids from N = 6 donors (3HC and 3UC) in response to vehicle control (DMSO 0.033%), tofacitinib and budesonide cultured at 2% oxygen. (f) Graphs showing quantification of the percentage of cells co-expressing Ki67 and CK20 in colonoids after treatment with tofacitinib and budesonide compared to vehicle control (DMSO 0.033%) cultured at 2% and 20% oxygen. QuPath23 (version 0.3.2) software was used for the quantification. Differences evaluated using paired t-test. **P < 0.01.
