Fig 2: Overexpression of Sirt6 in macrophages regulates the expression of apoptosis-related genes and Sirt6 in podocytes under high-glucose conditions. The diabetic internal environment was mimicked by a Transwell co-culture system under high-glucose conditions to co-culture the macrophages and podocytes. The cells in this experiment were divided into 8 groups as follows: i) The NM + HG + Con group (control; 50 mmol/l of high-glucose medium in the upper chamber, and podocytes with high-glucose medium in the lower chamber); ii) the HG + Con group (macrophages with high-glucose medium in the upper chamber, and podocyte with high-glucose medium in the lower chamber); iii) the HG + NC + Con group (macrophages transfected with NC plasmid with high-glucose medium in the upper chamber, and podocytes with high-glucose medium in the lower chamber); iv) the HG + Sirt6 + Con group (macrophages transfected with Sirt6 overexpression plasmid with high-glucose medium in the upper chamber, and podocytes with high-glucose medium in the lower chamber); v) the M-Sirt6 group (high-glucose medium in the upper chamber, and podocytes transfected with Sirt6 overexpression plasmid with high-glucose medium in the lower chamber); vi) the HG + M-Sirt6 group (macrophages with high-glucose medium in the upper chamber, and podocytes transfected with Sirt6 overexpression plasmid with high-glucose medium in the lower chamber); vii) the HG + NC + M-Sirt6 group (macrophages transfected with NC plasmid with high-glucose medium in the upper chamber, and podocytes transfected with Sirt6 overexpression plasmid with high-glucose medium in the lower chamber); and viii) the HG + Sirt6 + M-Sirt6 (macrophages transfected with Sirt6 overexpression plasmid with high-glucose medium in the upper chamber, and podocytes transfected with Sirt6 overexpression plasmid with high-glucose medium in the lower chamber). RT-qPCR was performed to examine the effects of the overexpression of Sirt6 on the mRNA expression levels of (A) Bcl-2, (B) Bax and (C) Sirt6 in the Transwell co-culture system. (D) Western blot analysis was performed to examine the effects of the overexpression of Sirt6 on the protein expression levels of Bcl-2, Bax and Sirt6 in the Transwell co-culture system. (E) Relative protein levels are presented as bar diagrams. GAPDH was used as an internal control. Data are presented as the means ± SD from 3 independent experiments (*P<0.05 and **P<0.01, compared with the NM + HG + Con group; ^^P<0.01, compared with the HG+NC+Con group; ##P<0.01, compared with the M-Sirt6 group; &&P<0.01, compared with the HG + M-Sirt6 group).

Fig 3: Overexpression of Sirt6 in macrophages regulates the expression of markers of macrophages and Sirt6 under high-glucose conditions. The diabetic internal environment was mimicked by a Transwell co-culture system under high-glucose conditions to co-culture the macrophages and podocytes. The cells in this experiment were divided into 8 groups as follows: i) The NM + HG + Con group (control; 50 mmol/l of high-glucose medium in the upper chamber, and podocytes with high-glucose medium in the lower chamber); ii) the HG + Con group (macrophages with high-glucose medium in the upper chamber, and podocyte with high-glucose medium in the lower chamber); iii) the HG + NC + Con group (macrophages transfected with NC plasmid with high-glucose medium in the upper chamber, and podocytes with high-glucose medium in the lower chamber); iv) the HG + Sirt6 + Con group (macrophages transfected with Sirt6 overexpression plasmid with high-glucose medium in the upper chamber, and podocytes with high-glucose medium in the lower chamber); v) the M-Sirt6 group (high-glucose medium in the upper chamber, and podocytes transfected with Sirt6 overexpression plasmid with high-glucose medium in the lower chamber); vi) the HG + M-Sirt6 group (macrophages with high-glucose medium in the upper chamber, and podocytes transfected with Sirt6 overexpression plasmid with high-glucose medium in the lower chamber); vii) the HG + NC + M-Sirt6 group (macrophages transfected with NC plasmid with high-glucose medium in the upper chamber, and podocytes transfected with Sirt6 overexpression plasmid with high-glucose medium in the lower chamber); and viii) the HG + Sirt6 + M-Sirt6 (macrophages transfected with Sirt6 overexpression plasmid with high-glucose medium in the upper chamber, and podocytes transfected with Sirt6 overexpression plasmid with high-glucose medium in the lower chamber). RT-qPCR was performed to examine the effects of the overexpression of Sirt6 on the mRNA expression levels of (A) CD86, (B) CD206, and (C) Sirt6 in the Transwell co-culture system. (D) Western blot analysis was performed to examine the effects of the overexpression of Sirt6 on the protein expression levels of CD86, CD206 and Sirt6 in the Transwell co-culture system. (E) Relative protein levels are presented as bar diagrams. GAPDH was used as an internal control. Data are presented as the means ± SD from 3 independent experiments (^^P<0.01, compared with the HG + Con group; &&P<0.01, compared with the HG + M-Sirt6 group).

Fig 4: Real-time reverse-transcription polymerase chain reaction revealed nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase sirtuin-6 (SIRT6) mRNA in cancer and normal lung tissue from 20 Chinese Han patients with non-small cell lung cancer (NSCLC). SIRT6 mRNA levels were significantly lower in cancer tissue versus normal lung tissue, and SIRT6 mRNA levels decreased with tumour stage. Data presented as mean ± SD (*P < 0.05 versus normal lung specimens; #P < 0.05 versus TNM I–II specimens; One-way analysis of variance)

Fig 5: Proliferation, apoptosis and cloning efficiency of Huh-7 cells following overexpression of SIRT6. (A) mRNA expression of SIRT6 as determined via reverse transcription-quantitative PCR analysis. (B) Protein levels of SIRT6 as determined via western blotting. (C) Cell proliferation following transfection for 12, 24 and 48 h as determined using a Cell Counting Kit-8 assay. (D) Apoptosis of transfected cells as determined via flow cytometry. (E) Clone formation assay and cloning efficiency relative to control. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. control. SIRT6, sirtuin 6; NC, negative control.