Sirtuin 2 (SIRT2) is a member of sirtuin protein family. cell

Sirtuin 2 (SIRT2) is a member of sirtuin protein family. cell survival of the cells. Collectively, our study has suggested an important role of SIRT2 in regulating both the cell cycle and basal survival of microglia. test. values less than 0.05 were considered statistically significant. Results We applied SIRT2 siRNA to decrease the SIRT2 levels in BV2 cells. At 48 hrs or 72 hrs after the treatment of 100 nM SIRT2 siRNA, SIRT2 levels were assessed by Western blot (Physique 1). Quantifications of the Western blots showed that SIRT2 silencing led to significant decreases in the SIRT2 levels (Physique 1). Intracellular LDH assay was conducted to determine the effects of SIRT2 silencing on the survival of the cells, which showed that treatment of the cells with SIRT2 siRNA for 48 or 72 hrs led to a significant decrease in the number of making it through BV2 cells (Physique 2). Physique 1 European blot assay showed that the SIRT2 siRNA treatment led to a significant decrease in the SIRT2 levels of BV2 cells. The cells were transfected with SIRT2 siRNA for either 48 or 72 hrs, and subsequentlythe SIRT2 levels of the cells were decided … Physique 2 Treatment of BV2 cells with SIRT2 siRNA led to a significant decrease in the number of making it through BV2 cells, as assessed by intracellular LDH assay. The cells were treated with 100 nM SIRT2 siRNA for either 48 or 72 hrs, and subsequently intracellular … Cell cycle analysis was conducted to determine if the SIRT2 reductions led to the decreased in the number of making it through cells by generating inhibition of cell cycle of the cells. Our study has suggested that SIRT2 silencing produced cell cycle arrest of BV2 cells at G0/G1 phase: The SIRT2 silencing led to a significant increase in the percentage of cells in G0/G1 phase from 61.2% to 79.6%, as well as a significant decrease in the percentage of cells in S phase from 31.1% to 14.7% (Figure 3A, ?,3B3B). Physique 3 SIRT2 silencing led to significant modifications of the cell cycle of BV2 cells. A. Associate histograms depicting cell cycle information of control BV2 cells and the BV2 cells transfected with 100 nM siRNA. W. Quantifications of the histograms suggested … We further decided if SIRT2 silencing may also impact the apoptosis and necrosis of the cells by conducting FACS-based Annexin V/7-AAD staining assay. The SIRT2 silencing was shown to produce an increase in the late-stage apoptosis cells, as indicated by the increase in Annexin V+/7-AAD+ cells (Physique 4A, ?,4B).4B). In contrast,the SIRT2 silencing did not affect the number of necrotic cells (Annexin V-/7-AAD+ cells) (Physique 4A, ?,4B4B). Physique 4 Treatment of Rabbit Polyclonal to MRPS16 microglial BV2 cells with SIRT2 siRNA led to a significant increase in late-stage apoptosis of the cells, as assessed by FACS-based Annexin V/7-AAD staining. A. The FACS diagrams showed that SIRT2 siRNA induced an increase in the number of … Conversation The major findings of our current studies include: First, SIRT2 reductions by SIRT2 siRNA can produce cell cycle arrest of BV2 cells MLN8237 (Alisertib) manufacture at G0/G1 phase, by both significantly increasing percentage of the cells in G0/G1 phase and significantly decreasing percentage of the cells in S phase; second, the SIRT2 reductions can also increase late-stage apoptosis of the cells; and third, the SIRT2 reductions can lead to a decrease in the number of making it through cells, which may result from the effects of SIRT2 reductions on both cell cycle and cell survival of BV2 cells. Collectively, our study has suggested important functions of SIRT2 in regulating both the cell cycle and the basal survival of microglial BV2 cells. SIRT2 has been shown to play seemingly paradoxical functions in both cell cycle and cell survival: Several studies did not find any significant functions of SIRT2 in the cell cycle rules of U251MG cells [13], HeLa cells and HEK293 cells [14], while SIRT2 has been shown to prevent the leave from the mitosis of osteoblastic cell collection Saos2 [11] and myelomonocytic cell collection U937 MLN8237 (Alisertib) manufacture [12]. Multiple studies have also suggested contrasting functions of SIRT2 inhibition in MLN8237 (Alisertib) manufacture cell death under numerous conditions: SIRT2 inhibition has been shown to produce beneficial effects in models of PD, HD [7,8] and ischemic myocardial damage [6]. However, SIRT2 inhibition has also been shown to produce apoptosis of C6 glioma cells and.