Androgen-induced gene 1 correlates with extracellular vesicle-associated cholesterol metabolism and hepatic metastasis of pancreatic cancer

Figure 3 Androgen-induced gene 1 knockdown suppresses pancreatic ductal adenocarcinoma liver metastatic growth in vivo. A: Sanger sequencing chromatograms of the androgen-induced gene 1 (AIG1) target region. Overlapping peaks downstream of the single-guide RNA target site (highlighted in yellow) indicate successful clustered regularly interspaced short palindromic repeats/Cas9-mediated indel mutations; B: Western blot analysis of AIG1 expression in KPC-1-wild type, non-targeting control single-guide RNA (sgControl) and AIG1 knockdown (AIG1-KD) cells. Quantification of AIG1 protein levels normalized to β-actin from three independent biological replicates is presented on the right. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ^aP < 0.01; C: In vivo bioluminescence imaging of liver metastases two weeks after portal vein injection of sgControl or AIG1-KD cells into male C57BL/6J mice (n = 5 per group). Images from all individual mice are shown. Quantification of average radiance (× 10⁷ photons/second/cm²/steradian) is shown beneath. Each dot represents an individual mouse. Comparisons between AIG1-KD group and sgControl group were performed using unpaired t-test, ^bP < 0.0001; D: Gross morphology of livers harvested two weeks after portal vein injection of sgControl or AIG1-KD cells (n = 5 per group). Metastatic lesions are outlined with red dashed lines. Images from all individual mice are shown. Scale bar = 1 cm. AIG1: Androgen-induced gene 1; AIG1-KD: Androgen-induced gene 1 knockdown; wt: Wild type; sgControl: Non-targeting control single-guide RNA.