Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 1. Tagging of the CYP4F2 and CYP4F3 loci. Position and linkage disequilibrium (LD) of genotyped (tag-)SNPs across the CYP4F2 (left) and the CYP4F3 locus (right). The position of (tag-)SNPs on the genes is indicated at the top of the figure. Tall black boxes are coding exons. Short black boxes are untranslated exons. LD is represented by shades of grey as a function of r2 values (black diamonds for r2 ≥0.90, white diamonds for r2 = 0).

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques:

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 3. Impact of rs2108622 and rs3093105 on CYP4F2 protein and mRNA levels. CYP4F2 protein and mRNA levels were measured in ABCD1-KD cell lines (n = 4 per variant) seeded simultaneously and grown under similar conditions. CYP4F2 protein levels were markedly lower upon expression of p.433M variants (A and B). The p.W12G SNP did not affect CYP4F2 protein levels (B). The CYP4F2 mRNA levels were not affected by rs2108622 or rs3093105 (C). Data are presented as mean ± SD. ***p b 0.001 by one-way ANOVA followed by Bonferroni's multiple comparison test. NS is not significant.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Variant Assay, Expressing, Comparison

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 4. CYP4F2 p.V433M decreases the clearance of VLCFA by reducing their ω-oxidation. VLCFA homeostasis was assessed in ABCD1-KD (n = 6) (A) and PEX13-KD (n = 6) (B and C) cell lines exposed to D3-C22:0. For each cell line 3 biological replicates were included. ω-OH-C22:0 and ω-OH-C24:0 are markers for the ω-oxidation of D3-C22:0 and D3-C24:0, respectively; while D3-C24:0, D3-C26:0 reflect the elongation of D3-C22:0. (A) Assessment of the effect of CYP4F2 expression in the ABCD1-KD cell lines showed a non-significant decrease in the amount of the elongation products D3-C24:0 and D3-C26:0 from D3-C22:0. (B) Expression of CYP4F2 p.433 V resulted in a significant decrease in D3-C22:0 elongation to D3-C24:0 and D3-C26:0 in PEX13-KD cells, and (C) significant higher levels of ω-OH-C22:0 and ω-OH-C24:0. Fatty acid levels are in nmol/mg protein. Data are presented as mean ± SD. ***p b 0.001 by unpaired student's t-test.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Expressing

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 5. p.V433M decreases the ω-oxidation of C26:0. The ω-oxidation of C26:0 was measured in ABCD1-KD (n = 6) (A) and PEX13-KD (n = 6) (B and C) and cell lines exposed to D4-C26:0. For each cell line 3 biological replicates were included. (A) Increased ω-oxidation of D4-C26:0 was observed in all ABCD1-KD cell lines expressing CYP4F2 but this effect was more pronounced upon expression of p.433V variants. (B and C) Increased ω-oxidation of C26:0 was observed in all PEX13-KD cell lines expressing CYP4F2, but this effect was more pronounced upon expression of p.433V variants. PEX13-KD cell lines that expressed CYP4F2 p.433V produced significantly higher levels of both ω-OH-C26:0 (B) and C26:0- dicarboxylic acid (C26:0-DCA) (C) than PEX13-KD cell lines expressing CYP4F2 p.433M. n.d. = not detected. Fatty acid levels are in nmol/mg protein. Data are presented as mean ± SD. **p b 0.01 and ***p b 0.001 by unpaired student's t-test.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Expressing, Produced

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 6. p.V433M does not affect the catalytic activity of CYP4F2 in vitro. Microsomes isolated from clones expressing either CYP4F2 p.433V (n = 4) (●) or p.433M (n = 4) (■) were used to determine the kinetic parameters, Km and Vmax, of CYP4F2 for both C22:0 and ω-OH-C22:0. Omega oxidation activity for an increasing concentration of C22:0 (A) or ω-OH-C22:0 (B) was determined and used to calculate the kinetic parameters with the Michaelis-Menten equation. The insert shows the Lineweaver-Burk plot for the collected data. Omega oxidation activities (V) are in nmol/min/mg protein. Substrate concentrations (S) are in μM. Values represent the mean and error bars represent the SD. (C) Differences in CYP4F2 content of microsomes were expressed as a ratio between two variants, using calregulin as a loading control. ***p b 0.001 by unpaired student's t-test.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Activity Assay, In Vitro, Isolation, Clone Assay, Expressing, Concentration Assay, Control

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 7. Increased proteasomal degradation of CYP4F2 p.433M. (A) The effect of CYP4F2 p.433V and p.433M on protein stability was assessed by exposure of microsomes isolated from cells (n = 4) expressing CYP4F2 p.433V or p.433M at 50 °C for different time periods followed by CYP4F2 enzyme activity measurement using 200 μM C22:0 as substrate. (B) ABCD1-KD Flp-In 293 cells expressing CYP4F2 p.433V or p.433M were cultured for 24 h in the presence of bortezomib at different concentrations (0, 10, 30 or 100 nM) and CYP4F2 protein levels were determined by Western blotting.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Isolation, Expressing, Activity Assay, Cell Culture, Western Blot