Fig 6. Effects of SIRT1 knockdown by RNAi on the mRNA level and the release of IL-1β and TNF-α in RAW264.7 cell culture treated with 30 mM high glucose.

(A) Cells cultured in NG medium were transfected with 33 nM scramble or SIRT1-specific siRNA duplex (serial number: 2195, 1003, and 576) for 6h, incubated for another 42h, and then harvested for the detection of SIRT1 suppression by western blot (df = 3, f = 38.663, p = 0.002). (B-C) Cells receiving RNAi treatment were continued to be cultured in 30 mM D-glucose-containing medium for additional 8h, mRNA levels of IL-1β (B, df = 4, f = 250.541, p < 0.001) and TNF-α (C, df = 4, f = 24.145, p < 0.001) were assessed by real-time PCR. Each data point is the mean ± SEM of n = 3 and normalized against corresponding β-ACTIN protein level (A) or GAPDH mRNA level (B-C) with the value in NG with scramble siRNA group arbitrarily set as 1. (D-E) The culture medium was also collected and used for ELISA for detecting the release of IL-1β (D, df = 4, f = 110.68, p < 0.001) and TNF-α (E, df = 4, f = 80.041, p < 0.001). Each data point is the mean ± SEM of n = 3. *p < 0.05; **p < 0.01; ***p < 0.001. 30 mM of D-mannitol served as an osmotic control. ‘NG’, 5.6 mM normal glucose; ‘HG’, 30 mM high glucose; ‘DM’, 30 mM D-mannitol.