Fig 1: miR-125a regulates the expression levels of key enzymes in the mevalonate signaling pathway. (A) Protein expression levels of HMGCR, FDPS, SQS and GGTase-I were measured by western blotting. miR-125a mimic transfection reversed HG-induced dysregulation of these proteins in VSMCs. (B) miR-125a inhibitor transfection induced the activation of the mevalonate signaling pathway in VSMCs cultured under normal conditions (5.6 nM glucose). Data are presented as the mean ± SD. n=3. *P<0.05, **P<0.01. ns, not significant; miR, microRNA; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; FDPS, farnesyl diphosphate synthase; SQS, squalene synthase; GGTase-I, geranylgeranyltransferase type I; H.G, high glucose; N.G, normal glucose; VSMC, vascular smooth muscle cell; NC, negative control.
Fig 2: High glucose-induced atherosclerosis is associated with miR-125a-mediated dysregulation of the mevalonate signaling pathway. (A) Media thickness and MCSA of the thoracic aorta from Sprague Dawley rats at time 0, and 5, 10 and 20 weeks after streptozotocin injection. (B) miR-125a expression in the aortic media. (C) Protein expression levels of HMGCR, FDPS, SQS and GGTase-I in the aortic media were measured by western blotting. Data are presented as the mean ± SD. n=6. *P<0.05, **P<0.01. ns, not significant; MCSA, media cross-sectional area; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; FDPS, farnesyl diphosphate synthase; SQS, squalene synthase; GGTase-I, geranylgeranyltransferase type I; miR, microRNA.
Fig 3: Qki depletion leads to downregulation of genes in the cholesterol biosynthesis pathway in eye lens.a IPA of the genes with a significant reduction of mRNA expression in isolated Qk-iCKO mouse lenses compared to Ctrl mouse lenses at P17-19 according to RNA-seq (p < 0.05; right-tailed Fischer’s exact t-test). The top enriched downregulated canonical cellular pathways are ranked according to p-value. Exact p-value is listed in Supplementary Table 2. Cellular pathways involved in cholesterol biosynthesis are labeled in red. b Heatmap showing the relative expression value (Z score) of all 19 genes involved in cholesterol biosynthesis in Ctrl (n = 3) and Qk-iCKO (n = 3) lenses according to RNA-seq data ranked by fold change. c Cholesterol biosynthesis pathway with the genes encoding the enzymes involved in each step of the pathway. Red labeled indicate the cholesterol biosynthesis genes downregulated shown in b. d Results of RT-qPCR analysis of cholesterol biosynthesis genes in isolated Ctrl (n = 3) and Qk-iCKO (n = 3) lenses at P17-19. p = 0.0001552 (Hmgcs1); 0.001142 (Hmgcr); 0.002307 (Pmvk); 0.000008831 (Mvd); 0.0005463 (Idi1); 0.00003504 (Fdps); 0.0009219 (Fdft1); 0.00001013 (Lss); 0.00003438 (Cyp51); 0.00002009 (Tm7sf2); 0.000001402 (Msmo1); 0.000006230 (Nsdhl); 0.00003707 (Hsd17b7); 0.00003894 (Sc5d), **p < 0.01; ***p < 0.001; ****p < 0.0001 (two-tailed unpaired t-test). The results are presented as means with SD. e, f Immunoblots and quantification of enzymes involved in cholesterol biosynthesis (Hmgcs1, Hmgcr, and Fdps) and immunoblots of Qki-5 and Qki-6 in isolated Ctrl (n = 3) and Qk-iCKO (n = 3) lenses at P19. ß-actin: loading control. p = 0.009136 (Hmgcs1); 0.02481 (Hmgcr); 0.0006804 (Fdps), *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test). The results are presented as means with SD. g Schematic of the differentiation of NSCs to NLPCs (right). Bright-field images and immunostains for Pax6 (green) and aB-crystallin (red) in NSCs and NLPCs (left). DAPI (blue): nuclei. Scale bar: 50 µm. h Immunofluorescent staining of WT and Qki-depleted (Qk-/-) NSCs for Qki-5. DAPI (blue): nuclei. Scale bar: 50 µm. i The top enriched downregulated canonical cellular pathways in Qk-/- NLPCs (n = 3) compared to WT NLPCs (n = 3) according to RNA-seq (IPA; p < 0.05; right-tailed Fischer’s exact t-test) are ranked according to p-value. Exact p-value is listed in Supplementary Table 3. Cellular pathways involved in cholesterol biosynthesis are labeled in red. j Heatmap of the relative average expression value (Z score) of all 19 genes involved in cholesterol biosynthesis according to RNA-seq data in WT (n = 3) and Qk-/- (n = 3) NLPCs and WT (n = 3) and QKI KO (n = 3) HLE-B3 cells. k Results of RT-qPCR analysis of cholesterol biosynthesis genes in WT (n = 3) and Qk-/- (n = 3) NLPCs. p = 0.01012 (Hmgcs1); 0.02979 (Hmgcr); 0.003582 (Mvd); 0.0009995 (Idi1); 0.00006936 (Fdps); 0.001929 (Cyp51); 0.03582 (Msmo1); 0.001525 (Nsdhl); 0.0000009839 (Sc5d); 0.02260 (Dhcr24), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (two-tailed unpaired t-test). The results are presented as means with SD. All the experiments were replicated three times in the lab.
Supplier Page from Abcam for Anti-FDPS/FPS antibody - C-terminal