Journal: Advanced Science

Article Title: Inhibition of SIRT7 Overcomes Radioresistance in Pancreatic Neuroendocrine Tumors by Reactivating MEN1 Expression

doi: 10.1002/advs.202519824

Figure Lengend Snippet: SIRT7 inhibition suppresses DNA damage repair pathways and sensitizes PanNET cells to irradiation. (A) Volcano plot from RNA‐seq comparing SIRT7‐knockdown (SIRT7‐KD) versus control (NC) in QGP‐1cells, highlighting representative DNA damage response (DDR) genes. n = 3 biologically independent samples per group. (B) Gene set enrichment analysis (GSEA) showing pathway‐level changes in SIRT7‐KD versus control cells. n = 3 biologically independent samples per group. (C) GO enrichment analysis of downregulated biological processes in SIRT7‐KD cells versus controls, with DDR‐related terms highlighted. n = 3 biologically independent samples per group. (D,E) RT–qPCR validation of DDR gene expression in BON‐1 (D) and QGP‐1 (E) cells treated with vehicle (Ctrl), SIRT7 inhibitor (SIRT7i, 10 µM), irradiation (IR, 4 Gy), or the combination (IR / 4 Gy + SIRT7i / 10 µM). Cells were collected 24 h after irradiation. n = 3 biologically independent experiments. Statistics: one‐way ANOVA. (F, G) Time‐course immunoblot analysis of γ‐H2AX, RAD50, and MRE11 in BON‐1 (F) and QGP‐1 (G) cells treated with IR (4 Gy) in the absence or presence of SIRT7i (10 µM) at the indicated times (0, 1, 4, 12, 24 h). n = 3 independent experiments. (H,I) Dose‐response immunoblot analysis in BON‐1 (H) and QGP‐1 (I) cells treated with increasing concentrations of SIRT7i (0, 5, 10, 15 µM) in Control (Cas9/vector) cells or MEN1‐knockout (MEN1‐KO) cells generated by CRISPR–Cas9. n = 3 independent experiments. Data are presented as mean ± SEM unless otherwise indicated. Significance is shown in the figure; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were probed with primary antibodies specific to the following proteins: MEN1 (BBI, Cat# D263072), SIRT7 (ABclonal, Cat# A21731), DNMT1 (ABclonal, Cat# A16729), MRE11 (ABclonal, Cat# A4222), RAD50 (ABclonal, Cat# A3078), γ ‐ H2AX (ABclonal, Cat# AP0687), Caspase‐3 and Cleaved Caspase‐3 (Proteintech, Cat# 66470‐2‐Ig), and Flag epitope tag (ABclonal, Cat# AE005).

Techniques: Inhibition, Irradiation, RNA Sequencing, Knockdown, Control, Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Western Blot, Plasmid Preparation, Knock-Out, Generated, CRISPR

Journal: Advanced Science

Article Title: Inhibition of SIRT7 Overcomes Radioresistance in Pancreatic Neuroendocrine Tumors by Reactivating MEN1 Expression

doi: 10.1002/advs.202519824

Figure Lengend Snippet: Establishment of a radioresistant BON‐1 model identifies SIRT7 as a mediator of acquired radioresistance. (A) Workflow schematic showing stepwise fractionated irradiation used to generate radioresistant BON‐1 cells (BON‐1/RR) from parental BON‐1 cells, followed by RNA‐seq profiling. (B) Representative phase‐contrast images of parental and resistant BON‐1 cells at 72 h after 4 Gy irradiation. Scale bar, 200 µm. (C) Volcano plot of differentially expressed genes (resistant vs parental BON‐1), with double‐strand break (DSB) repair genes highlighted. n = 3 biologically independent samples per group. (D) Immunoblot validation of selected DSB repair factors (RAD50, MRE11, BRCA2, POLQ, RAD51, XRCC2, FEN1, BLM, FOXM1) in parental and resistant BON‐1 cells. n = 3 independent experiments. (E) Heatmap of selected differentially expressed genes showing transcriptional upregulation in resistant cells, including SIRT7. n = 3 biologically independent samples per group. (F) Cell viability after increasing irradiation doses (0, 1, 4, 8 Gy) comparing parental vs resistant BON‐1 cells. n = 5 biologically independent experiments. Statistics: unpaired two‐sided t‐test. (G) Cell viability of parental versus resistant BON‐1 cells treated with SIRT7 inhibitor (SIRT7i; 0, 5, 10, 15 µM) in combination with 4 Gy irradiation. n = 5 biologically independent experiments. Statistics: unpaired two‐sided t‐test. (H) Representative colony formation images of parental and resistant BON‐1 cells after graded irradiation (0, 1, 4, 8 Gy). (I) Quantification of clonogenic growth shown as colony area (% of non‐irradiated control). n = 3 biologically independent experiments. Statistics: unpaired two‐sided t‐test. (J) Representative colony formation images of parental and resistant BON‐1 cells after 4 Gy irradiation combined with increasing SIRT7i doses (0, 5, 10, 15 µM). (K) Quantification of colony area (% of 0 µM control). n = 3 biologically independent experiments. Statistics: unpaired two‐sided t‐test. Data are presented as mean ± SEM unless otherwise indicated; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were probed with primary antibodies specific to the following proteins: MEN1 (BBI, Cat# D263072), SIRT7 (ABclonal, Cat# A21731), DNMT1 (ABclonal, Cat# A16729), MRE11 (ABclonal, Cat# A4222), RAD50 (ABclonal, Cat# A3078), γ ‐ H2AX (ABclonal, Cat# AP0687), Caspase‐3 and Cleaved Caspase‐3 (Proteintech, Cat# 66470‐2‐Ig), and Flag epitope tag (ABclonal, Cat# AE005).

Techniques: Irradiation, RNA Sequencing, Western Blot, Biomarker Discovery, Control

Journal: Advanced Science

Article Title: Inhibition of SIRT7 Overcomes Radioresistance in Pancreatic Neuroendocrine Tumors by Reactivating MEN1 Expression

doi: 10.1002/advs.202519824

Figure Lengend Snippet: SIRT7 inhibition enhances radiation‐induced DNA damage, oxidative stress, and apoptosis in PanNET cells in a MEN1‐dependent manner. (A) Immunoblot analysis of γ‐H2AX, cleaved caspase‐3, RAD50, MRE11, SIRT7, and MEN1 in BON‐1 and QGP‐1 cells treated with SIRT7 inhibitor (SIRT7i, 10 µM) and/or ionizing radiation (IR, 4 Gy). Cells were harvested 12 h post‐IR. n = 3 independent experiments. (B,C) Annexin V/PI flow cytometry apoptosis assays performed 12 h post‐IR in BON‐1 and QGP‐1 cells under the indicated treatments. n = 3 biologically independent experiments. Statistics: one‐way ANOVA. (D,E) γ‐H2AX immunofluorescence staining (D) and quantification of γ‐H2AX foci (E) in BON‐1 and QGP‐1 cells at 12 h and 24 h post‐IR under the indicated treatments. Scale bar, 10 µm. n = 3 biologically independent experiments; Statistics: one‐way ANOVA at each time point. (F, G) Comet assays in BON‐1 and QGP‐1 cells at 12 h post‐IR under the indicated treatments. Left, representative comet images; right, quantification of tail moment. n = 3 biologically independent experiments. Statistics: one‐way ANOVA. (H, I) Intracellular ROS measured by DCFH‐DA staining and flow cytometry in BON‐1 and QGP‐1 cells at 12 h post‐IR under the indicated treatments; representative histograms (H) and quantification (I) are shown. n = 3 biologically independent experiments. Statistics: one‐way ANOVA. (J,K) Colony formation assays in BON‐1 and QGP‐1 cells treated with increasing concentrations of SIRT7i (0–10 µM) with or without IR; representative colony images (J) and quantification (K) of colony area are shown. n = 3 biologically independent experiments. Statistics: one‐way ANOVA. Data are presented as mean ± SEM unless otherwise indicated. Significance is shown in the figure; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were probed with primary antibodies specific to the following proteins: MEN1 (BBI, Cat# D263072), SIRT7 (ABclonal, Cat# A21731), DNMT1 (ABclonal, Cat# A16729), MRE11 (ABclonal, Cat# A4222), RAD50 (ABclonal, Cat# A3078), γ ‐ H2AX (ABclonal, Cat# AP0687), Caspase‐3 and Cleaved Caspase‐3 (Proteintech, Cat# 66470‐2‐Ig), and Flag epitope tag (ABclonal, Cat# AE005).

Techniques: Inhibition, Western Blot, Flow Cytometry, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Inhibition of SIRT7 Overcomes Radioresistance in Pancreatic Neuroendocrine Tumors by Reactivating MEN1 Expression

doi: 10.1002/advs.202519824

Figure Lengend Snippet: The SIRT7 inhibitor 97491 enhances radiation‐induced antitumor efficacy in PanNET patient‐derived xenograft (PDX) models. (A) Schematic of the PDX study design and treatment schedule. n = 5 mice per group. (B) Representative images of excised tumors at endpoint from each group. n = 5 mice per group. (C) Tumor growth curves during the treatment period. n = 5 mice per group. Statistics: one‐way ANOVA at each timepoint. (D) Body weight monitored throughout the study. n = 5 mice per group. Statistics: one‐way ANOVA at each timepoint. (E) Representative H&E and IHC staining of tumor sections for Ki‐67, MEN1, γ‐H2AX, cleaved caspase‐3 (cleaved CASP3), MRE11, and RAD50. Images are shown at 200×; scale bar, 100 µm. (F–K) Quantification of IHC positivity (% positive cells per field) for (F) Ki‐67, (G) MEN1, (H) γ‐H2AX, (I) cleaved CASP3, (J) MRE11, and (K) RAD50. n = 5 mice per group; Statistics: one‐way ANOVA. Data are presented as mean ± SEM unless otherwise indicated. Significance is shown in the figure; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were probed with primary antibodies specific to the following proteins: MEN1 (BBI, Cat# D263072), SIRT7 (ABclonal, Cat# A21731), DNMT1 (ABclonal, Cat# A16729), MRE11 (ABclonal, Cat# A4222), RAD50 (ABclonal, Cat# A3078), γ ‐ H2AX (ABclonal, Cat# AP0687), Caspase‐3 and Cleaved Caspase‐3 (Proteintech, Cat# 66470‐2‐Ig), and Flag epitope tag (ABclonal, Cat# AE005).

Techniques: Derivative Assay, Immunohistochemistry