Fig 1: Lactate levels are elevated in mice with liver fibrosis. (a) The 8-week-old mice were treated with CCl4 for 6 weeks, and the fibrosis was assessed by H&E, Masson, and Sirius red staining. Scale bars are 100 µm. (b) Kit to detect lactate level in the liver tissue. At least 3 liver sections were analyzed per mouse (number of mice: n = 12). (c, d) Western blot showing expression of acetylation and a-SMA in the livers of mice treated with CCl4. (e) Detection of the expression levels of M2 macrophage surface markers CD163, Arg1, and IL-10 in the liver by ELISA. **P < 0.01, ***P < 0.001.
Fig 2: HSCs regulate the transformation of M1/M2 macrophages. (a) Western blot showing expression of a-SMA in the aLX-2. Activated HSCs (aLX-2) were obtained by treating LX-2 cells with TGF-ß. (b) Lactate level in aLX-2. (c) The level of glycolysis in aLX-2. (d) Detection of the expression levels of M1 macrophage surface markers IL-1ß, IL-6, iNOS, and TNF-a in transformed THP-1 cells cultured with aLX or its supernatant and their supernatants by ELISA. (e) Detection of the expression levels of M2 macrophage surface markers Arg1, CD163, IL-10, and TGFß in co-aLX-2 co-LX-a cells and their supernatants by ELISA. (f) Western blot showing expression of acetylation n co-aLX-2 co-LX-a cells and their supernatants. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 3: Lactic acid stimulates the conversion of M1 to M2 macrophages. (a) The mRNA expression levels of Arg-1, CD163, IL-10, and TGF-ß1 in M1 macrophages cultured with and without lactate by qPCR. (b) The levels of histone acetylation modifications at the promoter regions of genes (Arg-1, CD163, IL-10, and TGF-ß1) were examined by ChIP-qPCR. (c) IP-western blot detected the level of histone acetylation inM1 macrophages after treated with 20 mM lactate or vehicle for 24H. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 4: Regulation of primary activated HSCs on the M1/M2 phenotypic transition of macrophages. (a) Western blot showing expression of a-SMA in the HSC and a-HSC (activated HSCs). (b) Lactate level in HSCs and a-HSCs. (c) The level of glycolysis in HSCs and a-HSCs. (d) Detection of the expression levels of M1 macrophage surface markers IL-1ß, IL-6, iNOS, and TNF-a in co-aLX-2 co-LX-a cells and their supernatants by ELISA. (e) Detection of the expression levels of M2 macrophage surface markers Arg1, CD163, IL-10, and TGFß in the supernatants of HSCs and a-HSCs by ELISA. (f) Western blot shows the expression of histone acetylation in the supernatants of HSCs and a-HSCs. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 5: NLRP3 significantly increases in bladder macrophages in diabetic mice that undergo cystitis. (A, B) Mouse bladders were sorted for bladder macrophages based on positivity for F4/80, and further for M1 (CD163-) and M2 (CD163+) subtypes among all F4/80+ macrophages, shown by representative flow charts (A) and by quantification (B). (C) ELISA for some critical factors for macrophage phenotype and functionality. *p<0.05. ns: no significance.
Supplier Page from Abcam for Mouse CD163 ELISA Kit