Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial CEPT1 Promotes Angiogenesis Through PPARα and VEGF-A Signaling

doi: 10.1161/ATVBAHA.125.323302

Figure Lengend Snippet: A ) Experimental design of aortic cell isolation for scRNA-seq analysis. B ) t-distributed stochastic neighbor embedding of EC and stromal cells separated by genotypes. C ) Pie-chart representing the relative abundance of each cluster. D ) Violin plot of Cept1 gene expression in EC sub-clusters (EC 1–3). E ) Pathway analysis of Differential Expressed Genes (DEG) between Cept1 overexpression and control in EC1 sub-cluster (FDR adjusted p value of <0.05, log (FC) > 0.1 and pct.1 >0.6). F-H ) Module scores based on significantly upregulated pathways.

Article Snippet: HUVECs were transduced to overexpress human Cept1 with lentiviral ORF technology, and control lentiviral ORF particles were used to generate a control cell line for the overexpression system (Pan02 OE-CTL ; Origene NM_001007794 and PS100093V; Origene Technology).

Techniques: Over Expression, Cell Isolation, Gene Expression, Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial CEPT1 Promotes Angiogenesis Through PPARα and VEGF-A Signaling

doi: 10.1161/ATVBAHA.125.323302

Figure Lengend Snippet: A ) Graphical representation of the experiment design. B ) Representative Doppler perfusion of HLI of Cept1 fl/fl Cre − and Cept1 fl/fl Cre + mice (n=4–5 mice) treated with STZ. C-D ) Quantitative graphical representation of Doppler perfusion of the hindpaw and gastrocnemius muscle. E-F ) Limb and ischemic damage severity were evaluated on days 3, 7, 14, and 21. G ) Representative images showing HE, Isolectin B4 (IB4), and CD31 staining in the gastrocnemius muscle at 21 days after artery ligation (n=4–5 mice per group). Scale bar =50 μm. H-I ) Quantification of relative microvessel density, relative muscle fiber area CD31 staining (n=4–5 mice per group). Data are presented as mean ± SEM. Statistical analysis in panel C-F was performed using multiple t -test with FDR by the 2-stage step-up method of Benjamini, Krieger, and Yekutieli multiplicity adjustment. Statistical analysis in panel H-J was performed using an unpaired Student’s test.

Article Snippet: HUVECs were transduced to overexpress human Cept1 with lentiviral ORF technology, and control lentiviral ORF particles were used to generate a control cell line for the overexpression system (Pan02 OE-CTL ; Origene NM_001007794 and PS100093V; Origene Technology).

Techniques: Over Expression, Staining, Ligation

Journal: Cell reports. Medicine

Article Title: Genetic variants affecting NQO1 protein levels impact the efficacy of idebenone treatment in Leber hereditary optic neuropathy.

doi: 10.1016/j.xcrm.2023.101383

Figure Lengend Snippet: Figure 1. IDB effectiveness in cybrids depends on NQO1 expression (A) Western blot analysis of NQO1 expression level in cellular lysates from control (Ctr) and m.3460G>A/MT-ND1 (3460) cybrids carrying an empty plasmid (mock) or human NQO1 (NQO1). Actin was used as a loading Ctr. A representative blot of two similar ones is shown. (B) OCR measurements of Ctr (Ctrmock) and m.3460G>A/MT-ND1 LHON cybrids (3460mock). OCR values were expressed as pmolO2 consumed/min normalized for cellular protein content under resting conditions and after oligomycin (O), FCCP (U), rotenone (R), and antimycin A (A) addition as detailed under STAR Methods. Empty circles correspond to cells treated with the vehicle DMSO (10 and seven independent experiments for Ctrmock and 3460mock, respectively) and red squares to cells treated with 10 mM idebenone (IDB; six and five independent experiments for Ctrmock and 3460mock, respectively). Data are reported as mean ± SD, and statistical analysis was performed using a t test as detailed under STAR Methods. **p < 0.01, ***p < 0.001. (C) OCR measurements of Ctr (CtrNQO1) and m.3460G>A/MT-ND1 LHON cybrids (3460NQO1) overexpressing NQO1. Open circles correspond to cells treated with the vehicle DMSO (eight and five experiments for CtrNQO1 and 3460NQO1, respectively), red squares to cells treated with 10 mM IDB (six and five experiments for CtrNQO1 and 3460NQO1, respectively), and black triangles to cells treated with 10 mM IDB and 10 mM dicoumarol (IDB+DIC). Statistical analysis was performed for cells treated with vehicle and IDB as described in B). (D) Rate of ATP synthesis measured in digitonin-permeabilized cells driven by malate and pyruvate (CI substrates) or exogenous NAD(P)H. Where indicated, IDB was added at the final concentration of 10 mM as detailed under STAR Methods. Data are means ± SD of at least three to five independent experiments. Statistical analysis was performed using unpaired t test. *p < 0.05, **p < 0.01. (E) Rate of H2O2 production in mock or NOQ1-overexpressing cells grown in the presence of 10 mM IDB as described under STAR Methods. Data are reported as mean ± SEM of four to six independent experiments. Statistical analysis was performed using a paired t test. *p < 0.05, **p < 0.01, ***p < 0.001. (F) Rate of H2O2 production in mock cell lines in the presence or absence of 10 mM IDB. The measurement and data analysis were performed as described in (E). (G) Cell viability measurements were performed in the presence or absence of 10 mM IDB as detailed under STAR Methods. Values are expressed as percentage of the ratio between IDB treated and non-treated cells. Data are reported as mean ± SD of four independent experiments. Statistical analysis was performed using unpaired t test. *p < 0.05, **p < 0.01.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Neural precursors cells (control, carrying the 3460/ND1 or the 11778/ND4 mutation) This paper and Peron et al.45 N/A Oligonucleotides Primers for qPCR This paper See supplemental information Primers for NQO1 sequencing This paper See supplemental information Primers for NQO1 SNaPshot assay This paper See supplemental information Recombinant DNA pLenti-C-Myc-DDK-P2A-Puro Origene Plasmid #PS100092 human NQO1 isoform 1 in pLenti-CMyc-DDK-P2A-Puro Origene Plasmid #RC200620L3 Software and algorithms ANNOVAR Wang et al.67 https://annovar.openbioinformatics. org/en/latest/; RRID:SCR_012821 bcl2fastq v2.20 Illumina https://emea.support.illumina.com/ sequencing/sequencing_software/ bcl2fastq-conversion-software.html; RRID:SCR_015058 Burrows-Wheeler Aligner v0.7.17 Li and Durbin68 http://bio-bwa.sourceforge.net/; RRID:SCR_010910 GeneMapper Software v4.1 Applied Biosystems RRID:SCR_014290 Genome Analysis Toolkit v3.8 Van der Auwera GA, O’Connor BD.

Techniques: Expressing, Western Blot, Control, Plasmid Preparation, Concentration Assay

Journal: Cell reports. Medicine

Article Title: Genetic variants affecting NQO1 protein levels impact the efficacy of idebenone treatment in Leber hereditary optic neuropathy.

doi: 10.1016/j.xcrm.2023.101383

Figure Lengend Snippet: Figure 5. Homozygous NQO1 polymorphisms result in reduced NQO1 protein levels (A and B) Violin plots of normalized RNA expression (A) and protein expression (B), stratified by genotypes of NQO1*2. (C and D) Violin plots of normalized RNA expression (C) and protein expression (D), stratified by genotypes of NQO1*3. Each dot represents an individual observation. The black box indicates mean and 95% confidence intervals. Blue color corresponds to RNA-seq analysis and red-spectrum colors to proteomics. The black line was fitted using linear regression, with the equation and significance specified in the bottom.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Neural precursors cells (control, carrying the 3460/ND1 or the 11778/ND4 mutation) This paper and Peron et al.45 N/A Oligonucleotides Primers for qPCR This paper See supplemental information Primers for NQO1 sequencing This paper See supplemental information Primers for NQO1 SNaPshot assay This paper See supplemental information Recombinant DNA pLenti-C-Myc-DDK-P2A-Puro Origene Plasmid #PS100092 human NQO1 isoform 1 in pLenti-CMyc-DDK-P2A-Puro Origene Plasmid #RC200620L3 Software and algorithms ANNOVAR Wang et al.67 https://annovar.openbioinformatics. org/en/latest/; RRID:SCR_012821 bcl2fastq v2.20 Illumina https://emea.support.illumina.com/ sequencing/sequencing_software/ bcl2fastq-conversion-software.html; RRID:SCR_015058 Burrows-Wheeler Aligner v0.7.17 Li and Durbin68 http://bio-bwa.sourceforge.net/; RRID:SCR_010910 GeneMapper Software v4.1 Applied Biosystems RRID:SCR_014290 Genome Analysis Toolkit v3.8 Van der Auwera GA, O’Connor BD.

Techniques: RNA Expression, Expressing, RNA Sequencing

Journal: Cell reports. Medicine

Article Title: Genetic variants affecting NQO1 protein levels impact the efficacy of idebenone treatment in Leber hereditary optic neuropathy.

doi: 10.1016/j.xcrm.2023.101383

Figure Lengend Snippet: Figure 6. NQO1 variants leading to very low protein levels match the IDB responder/non-responder analysis (A, E, and I) Forest plots of odds ratios (ORs) for the binary visual outcome (responder/non-responder to IDB therapy). (A) ORs calculated by multivariable GEE modeling on the 118 LHON patients treated with IDB therapy. (E) ORs calculated by univariable GEE modeling on the LHON patients treated with IDB therapy carrying the m.11778G>A/MT-ND4 mutation. (I) ORs calculated by univariable GEE modeling on the LHON patients treated with IDB therapy carrying the m.3460G>A/MT-ND1 mutation. (B, F, and J) Boxplots of VA at last visit, with solid lines representing median values for NQO1 mut/mut, NQO1 mut/WT, and NQO1 WT/WT genotypes of all 118 LHON patients (B), LHON patients carrying the m.11778G>A/MT-ND4 (F), and LHON patients carrying m.3460G>A/MT-ND1 (J). (C, G, and K) All Italian LHON patients (C), Italian LHON patients with m.11778G>A/MT-ND4 (G), and Italian LHON patients with m.3460G>A/MT-ND1 (K). Shown are boxplots of average RNFL thickness at last visit, with solid lines representing median values for NQO1 mut/mut, NQO1 mut/WT, and NQO1 WT/WT genotypes. All of these patients were evaluated with swept-source OCT (DRI Triton OCT, Topcon, Tokyo, Japan). *p < 0.05. (D, H, and L) All German LHON patients (D), German LHON patients with m.11778G>A/MT-ND4 (H), and German LHON patients with m.3460G>A/MT-ND1 (L). Shown are boxplots of average RNFL thickness at last visit, with solid lines representing median values for NQO1 mut/mut, NQO1 mut/WT, and NQO1 WT/WT genotypes. All of these patients were evaluated with spectral-domain OCT (Heidelberg Spectralis OCT, Heidelberg Engineering, Heidelberg, Germany).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Neural precursors cells (control, carrying the 3460/ND1 or the 11778/ND4 mutation) This paper and Peron et al.45 N/A Oligonucleotides Primers for qPCR This paper See supplemental information Primers for NQO1 sequencing This paper See supplemental information Primers for NQO1 SNaPshot assay This paper See supplemental information Recombinant DNA pLenti-C-Myc-DDK-P2A-Puro Origene Plasmid #PS100092 human NQO1 isoform 1 in pLenti-CMyc-DDK-P2A-Puro Origene Plasmid #RC200620L3 Software and algorithms ANNOVAR Wang et al.67 https://annovar.openbioinformatics. org/en/latest/; RRID:SCR_012821 bcl2fastq v2.20 Illumina https://emea.support.illumina.com/ sequencing/sequencing_software/ bcl2fastq-conversion-software.html; RRID:SCR_015058 Burrows-Wheeler Aligner v0.7.17 Li and Durbin68 http://bio-bwa.sourceforge.net/; RRID:SCR_010910 GeneMapper Software v4.1 Applied Biosystems RRID:SCR_014290 Genome Analysis Toolkit v3.8 Van der Auwera GA, O’Connor BD.

Techniques: Mutagenesis