Fig 2: SIRT6 regulates the malignant phenotype of pancreatic cancer cells by activating the NF-κB pathway. (A) Nuclear NF-κB p65 and cytoplasmic IκBα protein levels were detected by western blot analysis. (B) Nuclear NF-κB p65 expression was evaluated by immunofluorescence. (C) PANC-1 cells were treated with vector, SIRT6, and SIRT6 + RANKL, and then nuclear NF-κB p65 and cytoplasmic IκBα protein levels were assessed by western blotting. (D) The viability of PANC-1 cells was evaluated by Cell Counting Kit-8 assay. (E) ROS level was detected by DCFH-DA method. (F) The expression levels of GPX4, SLC7A11, HK2 and LDHA were determined by western blotting. **P<0.01 vs. vector group and &P<0.05 vs. SIRT6 group. SIRT6, sirtuin6; ROS, reactive oxygen species; GPX4, glutathione peroxidase 4; HK, hexokinase; LDHA, lactate dehydrogenase A.

Fig 3: Overexpression of SIRT6 induces ferroptosis and inhibits glycolysis in pancreatic cancer cells. (A and B) After transfection with pcDNA-SIRT6, the expression of SIRT6 in PANC-1 cells was detected by (A) reverse transcription-quantitative PCR and (B) western blotting. (C) The viability of PANC-1 cells was determined by Cell Counting Kit-8 assay. (D) Fe2+ content was evaluated by Fe2+ detection kit. (E) PANC-1 cells transfected with pcDNA-SIRT6 were treated with erastin or ferrostatin-1, and then Fe2+ content was evaluated by Fe2+ detection kit. (F) ROS level was detected by DCFH-DA method. (G) GPX4 and SLC7A11 expression levels were assessed by western blot analysis. (H-J) Glucose consumption, lactic acid and ATP production were evaluated by the corresponding kits. (K) HK2 and LDHA expression levels were detected by western blotting. **P<0.01 vs. control and vector group and #P<0.05 vs. SIRT6 group. SIRT6, sirtuin6; ROS, reactive oxygen species; GPX4, glutathione peroxidase 4; HK, hexokinase; LDHA, lactate dehydrogenase A.

Fig 4: Upregulation of SIRT6 inhibits growth of pancreatic cancer xenografts in vivo. (A) Different groups of PANC-1 cells (vector and SIRT6-overexpressing) were inoculated subcutaneously (200 ml) in nude mice at a density of 1x106/ml and the xenografted tumor size was measured every 7 days. After 28 days, the tumor was stripped from euthanized mice, and the tumor growth curve was drawn. (B and C) The body weight and xenografted tumor mass of nude mice were calculated. (D) The expression levels of GPX4, SLC7A11, HK2 and LDHA were assessed by immunohistochemistry. (E) Nuclear NF-κB p65 and cytoplasmic IkBα protein levels were detected by western blot analysis. **P<0.01 vs. vector group. SIRT6, sirtuin6; GPX4, glutathione peroxidase 4; HK, hexokinase; LDHA, lactate dehydrogenase A.

Fig 5: SIRT6 is lowly expressed in pancreatic cancer tissues and cells. (A) The expression of SIRT6 was detected in 68 pairs of PC and paracancerous tissues using reverse transcription-quantitative PCR. (B) Kaplan-Meier survival curve was used to evaluate the overall survival time of patients with PC who had low expression of SIRT6 (n=34) and high expression of SIRT6 (n=34). (C) The survival rate was determined by Kaplan-Meier-plotter online analysis. (D) The expression of SIRT6 in 3 random pairs of PC and adjacent tissues was determined by western blot analysis. (E) Expression of SIRT6 protein in HPDE cells and PC cell lines (SW1990, PC-2, BxPC-3 and PANC-1) was detected by western blotting. ***P<0.001 vs. NC group, **P<0.01 vs. normal and HPDE cells and *P<0.05 vs. HPDE cells. SIRT6, sirtuin6; PC, pancreatic cancer; HPDE, human normal pancreatic epithelial; NC, negative control.