Fig. 6: SIRT1 and CUL4B promote pancreatic cancer stem cell properties and SIRT1 promotes pancreatic tumor xenograft growth in NOD/SCID mice by promoting the development of cancer stemness.

A Western blot analysis of stem cell marker expression in PANC-1 cells with stably overexpressed or knocked down SIRT1 and CUL4B. β-actin served as a loading control. Protein expression was quantified by gray scanning. S1, SIRT1; C4, CUL4B. B PANC-1 cells with stably overexpressed or knocked down SIRT1 and CUL4B. Representative images of spheres grown in suspension culture for 15 days. Cells were plated in an ultra-low attachment six-well plate (5000/well). C CD133 staining of PANC-1 cells was assessed using flow cytometry. The box shows the percentage of CD133⁺ cells. D PANC-1 cells were engineered to stably express firefly luciferase, carrying an empty vector or SIRT1. These cells were injected subcutaneously into the groin (n = 7) of 6-week-old female NOD/SCID mice under limiting dilutions 1 × 10⁶, 1 × 10⁵, 1 × 10⁴, 1 × 10³, 5 × 10², 2 × 10², or 50 cells. Tumors were quantified using bioluminescence imaging 4 weeks after initial implantation. Representative in vivo bioluminescent images are shown. E Tumor specimens were examined using in vitro measurements. F CSC frequency was calculated using Extreme Limiting Dilution Analysis (ELDA) software (http://bioinf.wehi.edu.au/software/elda/index.html). G The volume of tumor specimens was detected in vitro. The data shown are the mean ± SD. ∗p < 0.05, ∗∗p < 0.01; two-tailed unpaired t test. H Ki67 staining was performed via immunohistochemistry on fixed sections. All tumor cells were counted in at least five random fields. I Heatmap of differentially expressed genes (fold-change > 1.2, p < 0.001) in control (Vector-1, Vector-2, and Vector-3) and SIRT1-overexpressing (SIRT1-1, SIRT1-2, and SIRT1-3) tumor samples. (Blue) downregulated genes; (red) upregulated genes. J Ten enriched KEGG pathways comprised of upregulated or downregulated genes mediated by SIRT1 overexpression. The Rich Factor represents the ratio of differentially expressed genes to the total genes annotated in a pathway. A greater Rich Factor indicates greater intensity. The Q-value represents the corrected p-value ranging 0–1; a lower Q-value indicates greater intensity. K Total RNA was extracted from tumor samples, and RT-qPCR analysis of mRNAs performed in control vector or SIRT1-overexpressing. A–C, H, K Error bars represent the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; two-tailed unpaired t test.