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: SIRT3 is transcriptionally down-regulated by KRAS G12D . A, differential gene expression of nicotinate and nicotinamide metabolism pathways. B, SIRT3 mRNA levels of HPNE cells with KRAS/Off or KRAS/On for 3 h to 96 h. The data are presented as mean ± SD ( n = 3). p values were determined by Student’s t test. ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. C, SIRT3 and KRAS protein levels in HPNE cells with KRAS/Off or KRAS/On for 3 h to 96 h. Cell extracts were analyzed by Western blotting using tubulin as the loading control. Note that the images of KRAS and tubulin bands were derived from the same source images shown in , C and D , as they were from the same experiment. D, effect of SCH772984 on expression of ERK1/2, phosphorylated ERK1/2, and SIRT3 protein levels in KRAS/On HPNE cells. Cells were treated with SCH772984 at indicated concentrations for 24 h. Cell extracts were analyzed by Western blotting using vinculin as the loading control. HPNE KRAS/Off cell extracts were used for comparison. E, effect of SCH772984 on expression of ERK1/2, phosphorylated ERK1/2, and SIRT3 protein levels in KRAS/On HEK293 cells. Cells were treated with SCH772984 at indicated concentrations for 24 h. Cell extracts were analyzed by Western blotting using vinculin as the loading control. HEK293 KRAS/Off cell extracts were used for comparison. F, schematic illustration of the promoter activity assay to identify potential KRAS regulated region in the SIRT3 promoter. G, activity of truncated SIRT3 promoters in HPNE KRAS/Off and HPNE KRAS/On (12 h) cells. The data are presented as mean ± SD ( n = 3). p values were determined by Student’s t test. ∗, p < 0.05;∗∗, p < 0.01. HPNE: human pancreatic normal epithelial cell.

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, Western Blot, Control, Derivative Assay, Expressing, Comparison, Activity Assay

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: SIRT3 suppresses pancreatic cancer growth in vitro and in vivo . A, kaplan-meier survival plot of pancreatic cancer patients with high and low SIRT3 expression at 75%/25% cutoff from The Cancer Genome Atlas database. B, SIRT3 protein levels in SIRT3 overexpressed (SIRT3 OE) and control (Vector) PANC-1 cells. C, growth curves of PANC-1 Vector cells and PANC-1 SIRT3 OE cells in vitro . The data are presented as mean ± SD ( n = 3). p values were determined by Student’s t test. ∗∗, p < 0.01. D, SIRT3 protein levels in SIRT3 knockdown (SIRT3 sh1, sh2) and control (NC) PANC-1 cells. E, growth curves of PANC-1 NC, SIRT3 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; ∗∗∗∗, p < 0.0001. F, schematic illustration of study design to evaluate the impact of SIRT3 on PANC1 tumor growth in vivo . G, growth curve of SIRT3 overexpressed (SIRT3 OE) and control (Vector) PANC1 subcutaneous tumors. The data are presented as mean ± SD ( n = 5). p values were determined by Student’s t test. ∗∗, p < 0.01. H , PANC1 subcutaneous tumors of the SIRT3 overexpression and control group. The characters at the top of the photo represent the mouse number. The length of the scale bar represents 1 cm. I, SIRT3 overexpressed and the control PANC1 tumor weight. The red and blue points connected by the line represent tumors inoculated on each side of the same mouse. p values were determined by Student’s t test. ∗∗, p < 0.01. J, SIRT3 immunohistochemistry of the SIRT3 overexpressed and the control PANC1 subcutaneous tumors. The images are representative sections of tumor #2 shown in A , which contains the IHC images of all tumors including the same source images shown here. The length of the scale bar represents 100 μm. K, Growth curve of SIRT3 knockdown (SIRT3 sh1, 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 < 0.01; ∗∗∗, p < 0.001. L, PANC1 subcutaneous tumors of the SIRT3 knockdown and control group. M, SIRT3 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 < 0.01; ∗∗∗∗, p < 0.0001. N, SIRT3 immunohistochemistry of the SIRT3 knockdown and the control PANC1 subcutaneous tumors. The images are representative sections of tumors #2 and #3 in B , which contains the IHC images of all tumors including the same source images shown here. The length of the scale bar represents 100 μm.

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: In Vitro, In Vivo, Expressing, Control, Plasmid Preparation, Knockdown, Over Expression, Immunohistochemistry

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