HRT2 Antibody - #AF9092

Figure 5 Regulation of SIRT1/Notch signal pathway contributes to the effect of MLT on inhibiting M1-type Mφ polarization. RAW 264.7 Mφs were exposed to LPS for 6 h and followed by MLT treatment for 24 h, then treated with SIRT1 agonists (SRT1720) or inhibitors (EX527) for another 6 h and followed by different analysis. (A) Reduction of the ratio of M1-type Mφs while enhancement of the ratio of M2-type Mφs after the treatment with MLT, aggravated in combination with SRT1720, and reversed in the presence of EX527, was determined by flow cytometry assay. (B) The relative mRNA levels of M1-type Mφ genes (IL-6 and TNF-α) and M2-type Mφ genes (IL-4 and IL-10) were measured by RT-qPCR analyses (n = 3). The levels of IL-6 and TNF-α mRNA were reduced in the presence of MLT treatment, downregulated in combination with SRT1720, and upregulated in the presence of EX527. (C,D) The relative protein levels of SIRT1 and NICD in Mφs were measured by Western blot analyses (n = 3). (C) Western blotting showed the upregulation of the SIRT1 protein with the downregulation of the NICD protein, aggravated in combination with SRT1720, and reversed in the presence of EX527. (D) Quantitative analysis showed the increased level of the SIRT1 protein, with the reduced level of the NICD protein after the treatment with MLT, aggravated in combination with SRT1720, and reversed in the presence of EX527. (E) IP with the anti-NICD antibody was used to verify the interaction between SIRT1 and NICD. The interaction between SIRT1 and NICD in the LPS + MLT-treated Mφs was markedly increased compared with that in the LPS-treated Mφs. Moreover, treatment with SRT1720 further aggravated this interaction, while treatment with EX527 ameliorated the interaction. (F) The relative mRNA levels of downstream target genes of the SIRT1/Notch signaling pathway were measured by RT-qPCR analyses in Mφs (n = 3). The levels of Notch1, HES1, HEY2 and NRARP mRNA were downregulated after the treatment with MLT, downregulated in combination with SRT1720, and upregulated in the presence of EX527. (G,H) The relative protein levels of downstream factors of the SIRT1/Notch signaling pathway were measured by Western blot analyses in Mφs (n = 3). (G) Western blotting showed the downregulation of downstream factors protein of the SIRT1/Notch signaling pathway in Mφs. (H) Quantitative analysis showed the reduced levels of Notch1, HES1, HEY2 and NRARP protein after the treatment with MLT, downregulated in combination with SRT1720, and upregulated in the presence of EX527. Data are expressed as mean ± standard deviation. The two groups among the five groups are compared by using an independent t-test.