anti tkt  (Proteintech)

Journal: Life Metabolism

Article Title: The brown fat-enriched exosomal miR-206-3p attenuates hepatic lipogenesis by decreasing pentose phosphate pathway

doi: 10.1093/lifemeta/loaf028

Figure Lengend Snippet: miR-206-3p attenuates lipogenesis by targeting the PPP. (a) Heatmap of liver metabolomics analysis. The 8-week-old male C57BL/6J mice were fed HFD for 16 weeks and injected via tail vein with Ad-GFP or Ad-miR-206 once weekly beginning at week 13 ( n = 4). (b) Metabolomics identified up-regulated (red) and down-regulated (blue) metabolites in Ad-miR-206-injected mice. (c) NADPH/NADP + ratios in primary hepatocytes transfected with NC mimic or miR-206 mimic ( n = 3). (d) Relative levels of acetyl-CoA and glycerol-3-phosphate identified by metabolomics ( n = 4). (e) RT-qPCR analysis of relative miR-206-3p levels in primary hepatocytes ( n = 3). (f) Relative mRNA expression of PPP genes ( G6pd, Pgd, and Tkt ) in primary hepatocytes ( n = 3). (g) Western blot analysis of PPP proteins (G6PD, PGD, and TKT) in primary hepatocytes. Primary hepatocytes were transfected with NC mimic, miR-206 mimic, or miR-206-3p mimic plus miR-206-3p inhibitor. After 48 h, cells were harvested for RNA and protein analyses (e−g). (h) Relative mRNA expression of PPP genes ( G6pd, Pgd, and Tkt ) analyzed in hepatocytes incubated with conditioned medium from mature adipocytes ( n = 3). Brown adipocytes were transfected with miR-206 mimic or NC mimic on day 3. The culture medium was harvested until day 8 after induction for further studies. (i) Relative mRNA expression of PPP genes ( G6pd, Pgd, and Tkt ) in hepatocytes pretreated with BAT-derived exosomes (5 μg) from 8-week-old WT and ob/ob mice ( n = 3). (j) Relative mRNA expression of PPP genes ( G6pd, Pgd, and Tkt ) in the livers of the WT and AKO male mice at 8 weeks of age ( n = 7−9). Values are means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by Student’s t test or ANOVA test.

Article Snippet: Primary antibodies against the following proteins were used: CD9 (Santa Cruz, sc-13118, 1:200), TSG101 (ProteinTech Group, 28283-1-AP, 1:200), ALIX (ProteinTech Group, 12422-1-AP, 1:1000), peroxisome proliferator-activated receptor gamma (PPARγ, CST, #2443, 1:1000), G6PD (Abcam, ab210702, 1:1000), PGD (Abcam, ab12199, 1:1000), TKT (Santa Cruz, sc-390179, 1:500), and HSP90 (Santa Cruz, sc-13119, 1:1000).

Techniques: Injection, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Incubation, Derivative Assay

Journal: Life Metabolism

Article Title: The brown fat-enriched exosomal miR-206-3p attenuates hepatic lipogenesis by decreasing pentose phosphate pathway

doi: 10.1093/lifemeta/loaf028

Figure Lengend Snippet: miR-206-3p is reduced in human obesity. (a) qPCR analysis of relative miR-206-3p levels in serum-derived exosomes from healthy controls (Non-MAFLD, n = 24) and MAFLD patients ( n = 32). (b−g) Correlations between (b) BMI, (c) waist, (d) TG, (e) TC, (f) LDL, and (g) APOB and the expression levels of miR-206-3p in serum-derived exosomes ( n = 50). (h) Flowchart of co-culture between human SVF and hepatocytes. (i) Relative Pparγ and miR-206-3p expression in human mature adipocytes ( n = 4). (j) Relative mRNA expression of PPP genes ( G6pd, Pgd, and Tkt ) in hepatocytes incubated with conditioned medium from mature adipocytes ( n = 4). Human-derived SVF was induced to differentiate into mature adipocytes, and the culture medium was harvested on day 8 after induction for further study (h−j).(k) Representative H&E, G6PD, PGD, and TKT staining in liver sections of MAFLD patients and Non-MAFLD controls. Scale bar, 100 μm. (l) Quantification of positive area for immunohistochemistry staining in the liver ( n = 6). Values are means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by Student’s t test or ANOVA test.

Article Snippet: Primary antibodies against the following proteins were used: CD9 (Santa Cruz, sc-13118, 1:200), TSG101 (ProteinTech Group, 28283-1-AP, 1:200), ALIX (ProteinTech Group, 12422-1-AP, 1:1000), peroxisome proliferator-activated receptor gamma (PPARγ, CST, #2443, 1:1000), G6PD (Abcam, ab210702, 1:1000), PGD (Abcam, ab12199, 1:1000), TKT (Santa Cruz, sc-390179, 1:500), and HSP90 (Santa Cruz, sc-13119, 1:1000).

Techniques: Derivative Assay, Expressing, Co-Culture Assay, Incubation, Staining, Immunohistochemistry