Figure 7.

Ectopic expression of active PKD1 in PanIN cells increases the DCLK1⁺ stem cell population

(A) Representative plot of flow cytometry analysis for cells positive for DCLK1 and PKD1 in SM3 PanIN cells (left); quantification of the percentage of SM3 cells that are DCLK1+ and PKD1⁺ (right). “Other” indicates single positive cells or double-negative cells. Error bars indicate the standard deviation.

(B) Immunofluorescence for DCLK1 (green in overlay) and PKD1 (pink in overlay) in SM3 organoids with DAPI nuclear stain. Scale bars indicate 10 μm.

(C and D) Human constitutively active PKD1 (PKD1.CA) was overexpressed in mouse SM3 PanIN cells and flow cytometry analyses of cells for DCLK1 were performed. C shows representative flow cytometry plots. D shows quantification of flow cytometry analyses, where the graph indicates the fold change in DCLK1⁺ cells from five independent experiments. Statistical significance, indicated by ∗, was determined by the paired t test; p value = 0.0205. Error bars represent the standard deviation.

(E) Quantitative PCR analysis for huPRKD1, to indicate overexpression of human constitutively active PKD1. Statistical significance is represented by ∗ (t test; p value = 0.0003). Error bars indicate the standard deviation.

(F) Quantitative PCR analysis for expression of indicated genes and Gapdh (normalization) in SM3 cells overexpressing PKD1.CA versus control (n = 3 per condition). Statistical significance, indicated by ∗, was determined by the t test; DCLK1, p value = 0.0043; Pdx1, p value = 0.0003; Notch1, p value = 0.0002; Oct4, p value = 0.0279; Satb2, p value = 0.0040; Hes1, p value = 0.0135; CD133, p value = 0.0015. Error bars indicate the standard deviation.