Physciosporin significantly suppressed spheroid formation in a dose-dependent manner in cells overexpressing either protein individually (Figure 5A–C) but exerted no inhibitory activity on cells co-overexpressing both Gli1/2 and ΔEN1 (Figure 5A,D), providing further confirmation that physciosporin suppresses colon cancer cell stemness through the SHH and Notch signaling pathways. This evidence concerns the gene SHH and colonic neoplasm.