Journal: The Journal of Biological Chemistry

Article Title: KRAS G12D mutation promotes pancreatic tumorigenesis by suppressing sirtuin three via the guanine nucleotide exchange factor RCC1

doi: 10.1016/j.jbc.2025.111057

Figure Lengend Snippet: Down-regulation of SIRT3 by KRAS G12D is mediated via RCC1. A, schematic illustration of the study design to identify potential KRAS regulated SIRT3 promoter binding proteins. B, silver staining of the proteins binding to the SIRT3 promoter DNA. The biotin-labeled DNA probe spanning −720 to −600 bp of the SIRT3 promoter and the nuclear protein extracts from the KRAS/Off or KRAS/On (12 h) cells were used in the DNA pull-down assay. The pulldown products were run on an SDS-PAGE. After electrophoresis, the proteins in the gel were revealed by silver staining. C, RCC1 protein levels pulled-down by the SIRT3 promoter DNA probe from the nuclear protein extracts of the KRAS/Off or KRAS/On (12 h) cells. D, RCC1 and SIRT3 mRNA levels in RCC1 overexpressed and control HPNE cells. The data are presented as mean ± SD ( n = 3). p values were determined by Student’s t test. ∗∗, p < 0.01; ∗∗∗, p < 0.001. E, RCC1 and SIRT3 protein levels in RCC1 overexpressed and control HPNE cells. F, activity of the −800 bp and −600 bp SIRT3 promoter in the RCC1 overexpressed and control HPNE cells. The data are presented as mean ± SD ( n = 3). p values were determined by Student’s t test. ∗∗∗, p < 0.001. G, enrichment level of −700 to −600 bp SIRT3 promoter in the RCC1 chromatin co-immunoprecipitation. Flag-tagged RCC1 was transfected to pull-down the bond DNA, followed by qPCR using the primers spanning −700 to −600 bp of the SIRT3 promoter for the determination of the enrichment level. The data are presented as mean ± SD ( n = 3). p values were determined by Student’s t test. ∗∗∗∗, p < 0.0001. HPNE: human pancreatic normal epithelial cell; RCC1, regulator of chromosome condensation 1.

Article Snippet: Antibodies specific for the following markers were purchased from the indicated suppliers: SIRT3 (10099-1-AP, Proteintech; 1:1000), RCC1 (22142-1-AP, Proteintech; 1:1000), ERK (4695, Cell Signaling Technology; 1:1000), p-ERK (4370, Cell Signaling Technology; 1:1000), Vinculin (4650, Cell Signaling Technology; 1:1000), α-Tubulin (2144, Cell Signaling Technology; 1:1000), KRAS (sc-30, Santa Cruz; 1:1000).

Techniques: Binding Assay, Silver Staining, Labeling, Pull Down Assay, SDS Page, Electrophoresis, Control, Activity Assay, Immunoprecipitation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: KRAS G12D mutation promotes pancreatic tumorigenesis by suppressing sirtuin three via the guanine nucleotide exchange factor RCC1

doi: 10.1016/j.jbc.2025.111057

Figure Lengend Snippet: Abrogation of RCC1 suppresses pancreatic cancer cell proliferation and tumor growth through up-regulation of SIRT3. A, RCC1 gene expression levels in normal pancreas and pancreatic cancer tissues. RCC1 expression in normal pancreas was acquired from the GTEx database, and the expression in pancreatic cancer tissue was acquired from The Cancer Genome Atlas database. p values were determined by Welch's t test. ∗, p < 0.05. B, kaplan-meier survival plot of pancreatic cancer patients with high and low RCC1 expression at a 75%/25% cutoff from The Cancer Genome Atlas database. C, RCC1 mRNA level and RCC1 and SIRT3 protein levels of PANC1 NC, RCC1 sh1 and sh2 cells. The data are presented as mean ± SD ( n = 3). p values were determined by Student’s t test. ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. D, growth curves of PANC1 NC, RCC1 sh1, and sh2 cells in vitro . The data are presented as mean ± SD ( n = 3). p values were determined by Student’s t test. ∗∗∗, p < 0.001. E, growth curve of RCC1 knockdown (RCC1 sh1, RCC1 sh2) and control (NC) PANC1 subcutaneous tumors. The data are presented as mean ± SD ( n = 5). p values were determined by Student’s t test. ∗, p < 0.05; ∗∗∗∗, p < 0.0001. F, PANC1 subcutaneous tumors of RCC1 knockdown group and control group. The length of the scale bar represents 1 cm. G, RCC1 knockdown and the control PANC1 tumor weight. The data are presented as mean ± SD ( n = 5). p values were determined by Student’s t test. ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. H, SIRT3 immunohistochemistry of RCC1 knockdown and control PANC1 subcutaneous tumors. The images are representative sections of tumor #1 in C , which contains the full IHC images of all tumors including the same source images shown here. The length of the scale bar represents 100 μm. RCC1, regulator of chromosome condensation 1.

Article Snippet: Antibodies specific for the following markers were purchased from the indicated suppliers: SIRT3 (10099-1-AP, Proteintech; 1:1000), RCC1 (22142-1-AP, Proteintech; 1:1000), ERK (4695, Cell Signaling Technology; 1:1000), p-ERK (4370, Cell Signaling Technology; 1:1000), Vinculin (4650, Cell Signaling Technology; 1:1000), α-Tubulin (2144, Cell Signaling Technology; 1:1000), KRAS (sc-30, Santa Cruz; 1:1000).

Techniques: Gene Expression, Expressing, In Vitro, Knockdown, Control, Immunohistochemistry