Journal: International Journal of Molecular Sciences

Article Title: Type IV Collagen and SOX9 Are Molecular Targets of BET Inhibition in Experimental Glomerulosclerosis

doi: 10.3390/ijms24010486

Figure Lengend Snippet: JQ1 reduced renal type IV collagen overexpression at the gene and protein levels in nephrotoxic nephritis. Mice were treated daily with JQ1 (100 mg/kg/day) or vehicle, starting before the first NTS injection. ( A ) RNA was isolated from frozen whole kidney samples, and α3 chain type IV collagen ( Col4a3 ); type I collagen ( ColI ); fibronectin , and Ccn2 gene expression levels were evaluated by real-time qPCR. ( B ) Protein levels of COLIV were quantified by Western blot in total kidney extracts. The figure shows representative blots where GAPDH was used as a loading control. Data are expressed as the mean ± SEM of 5–7 animals per group. * p < 0.05 vs. control; # p < 0.05 vs. NTS-injected mice. ( C ) Immunohistochemistry with an anti-type IV collagen antibody in paraffin-embedded kidney sections. A representative animal from each group was shown (200× magnification). A detailed image of glomeruli was included (600× magnification). ( D ) Type IV collagen staining quantification. Scale bar represents 100 μm (200×) and 50 μm (600×).

Article Snippet: Mouse assays IDs were: TGF-β1, Mm01178820_m1; serpine-1 (Pai-1) Mm00435858_m1, Fibronectin Mm01256744_m1, Type I Collagen Mm00483888_m1; SOX9 Mm00448840_m1 and Collagen 4a3 Mm00483669_m1.

Techniques: Over Expression, Injection, Isolation, Gene Expression, Western Blot, Control, Immunohistochemistry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Type IV Collagen and SOX9 Are Molecular Targets of BET Inhibition in Experimental Glomerulosclerosis

doi: 10.3390/ijms24010486

Figure Lengend Snippet: Type IV Collagen is a direct target of iBETs. ChIP assays were performed in renal samples using an antibody specific for BRD4 or normal rabbit IgG as a negative control. Enrichment of BRD4-binding regions in the promoter of mouse Col4a3 was quantified by qPCR using specific primers. In each group, samples were pooled (4–7 mice per group). Data are from two independent experiments, and each qPCR was run in triplicate. Results are expressed as the n-fold enrichment of anti-BRD4 antibody relative to the negative control antibody (considered to be 1).

Article Snippet: Mouse assays IDs were: TGF-β1, Mm01178820_m1; serpine-1 (Pai-1) Mm00435858_m1, Fibronectin Mm01256744_m1, Type I Collagen Mm00483888_m1; SOX9 Mm00448840_m1 and Collagen 4a3 Mm00483669_m1.

Techniques: Negative Control, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Type IV Collagen and SOX9 Are Molecular Targets of BET Inhibition in Experimental Glomerulosclerosis

doi: 10.3390/ijms24010486

Figure Lengend Snippet: BET inhibition by JQ1 reduced ECM gene and protein production in human mesangial cells and murine kidney fibroblasts. Cells were pretreated with 5 uM JQ1 for 1 h and stimulated with 10 ng/mL TGF-β1 for 24 h (gene expression) or 48 h (protein expression). ( A ) Fibronectin , Col4a3 and ColIa2 gene levels were evaluated by real-time qPCR in mesangial cells. ( B ) Fibronectin and Type IV collagen (COLIV) protein levels were evaluated by Western blot in mesangial cells. ( C ) Protein levels of Fibronectin and Type I collagen (COLI) were evaluated by Western blot in kidney fibroblasts. * p < 0.05 vs. control; ** p < 0.001 vs. control; # p < 0.05 vs. treated cells with TGF-β1; ### p = 0.0001 vs. TGF-β1; #### p < 0.0001 vs. TGF-β1.

Article Snippet: Mouse assays IDs were: TGF-β1, Mm01178820_m1; serpine-1 (Pai-1) Mm00435858_m1, Fibronectin Mm01256744_m1, Type I Collagen Mm00483888_m1; SOX9 Mm00448840_m1 and Collagen 4a3 Mm00483669_m1.

Techniques: Inhibition, Gene Expression, Expressing, Western Blot, Control

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: List of Oligo used for cloning COL4A3 / COL4A5 plasmids

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: Cloning

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: sgRNA identification and design and dual plasmid cloning strategy to achieved CRISPR/Cas9 correction. In the upper lane of panel a , the sgRNA is represented in red and blue: PAM in blue and in red the mutated nucleotide for the COL4A5 CRISPR/Cas9 design. Below, the same design is reported for the COL4A3 variant. Plasmid functionality is based on the cloning of two plasmids for CRISPR/Cas9 components delivery. In the first plasmid, the sgRNA is under the control of Human U6 Promoter ( b ), while the mCherry Red/Green Reporter System is under the control of a CMV Promoter. The Cas9 plasmid is cloned with spCas9 endonuclease gene under control of a CMV promoter flanked by two Self-Cleaving Cas9 for auto-inactivation ( c ). Representative FACS analysis on HEK293T cells after 48 h of transfection with reporter harboring the wild type sequence and mutated reporter, for COL4A3 and COL4A5 variants, respectively. The population of cells mCherry + /GFP + is gated in the UR quadrant. The double fluorescence is present only in the cells co-transfected with Donor/Reporter plasmid with specific variant and spCas9 (41.9% and 27.6% for COL4A3 and COL4A5 , respectively) demonstrating the specificy of sgRNA ( d )

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: Plasmid Preparation, Cloning, CRISPR, Variant Assay, Control, Clone Assay, Transfection, Sequencing, Fluorescence

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: CasAnalyzer results on the edited bases after CRISPR/Cas9 correction

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: CRISPR, Mutagenesis

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: CasAnalyzer results on editing efficiency on COL4A3 podocyte-lineage cells. The CasAnalzyer Tool performed on the CRISPR/Cas9 treated sample and control has reported a HDR of 44.2% in patients’ mutated podocyte-lineage cells. IGV Visualization Software for this variant also exhibited the loss of heterozygosity with the restoration of the wt base in COL4A3 gene

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: CRISPR, Control, Software, Variant Assay

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: (p.(Pro72Arg)) genotype of TP53 in COL4A3 and COL4A5 mutated patients

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: Mutagenesis

Journal: Journal of Neuroinflammation

Article Title: Lipopolysaccharide-induced changes in the neurovascular unit in the preterm fetal sheep brain

doi: 10.1186/s12974-020-01852-y

Figure Lengend Snippet: Schematic illustration of the study design and quantification of the microvasculature in randomly selected areas of preterm fetal sheep cerebral cortex and white matter from saline control and LPS-treated subjects. a After recovery from surgery at 103–104 days of gestation, the preterm fetal sheep were given either normal saline infusions/boluses (saline controls, n = 8) or LPS infusion/boluses ( n = 7). LPS was infused at 100 ng/kg (50 ng/mL at 83 μL/h) for the first 24 h followed by 250 ng/kg/24 h (50 ng/mL at 207.5 μL/h) for the next 96 h. Boluses were administered as 1 μg LPS at 48, 72, and 96 h from the start of infusion. Normal saline controls received equivalent volumes of saline for both infusions and boluses. Ten days after the start of the infusions at 113–114 days of gestation, the fetal brains were collected for further immunohistochemical analysis. b Representative images of collagen type IV staining (green) in the cerebral cortex and white matter of saline control and LPS-exposed animals. × 10 (top row) and × 20 (bottom row) magnifications are shown, scale bar = 100 μm. White arrows indicate collagen IV-positive microvessels. DAPI (blue) is utilized as a counterstain. c Graphs representing the microvessel density expressed as the percent area of the fields (total area) segmented on anti-collagen type IV-stained sections in the cerebral cortex and white matter of the saline and LPS-exposed fetal sheep. An average of 15–20 fields on two slides per animal and n = 8 in the saline-exposed group and n = 7 in the LPS-exposed group were analyzed. Statistical analysis by Kruskal-Wallis test followed by Dunn’s post hoc test, * P < 0.05; ** P < 0.01. d A schematically represented coronal cross-section of the sheep brain. The Roman numeral numbered boxes indicate the areas from which the images were taken in b and quantified in c

Article Snippet: On the second day, the slides were rinsed twice in TBS for 5 min and then the sections were incubated with collagen type IV antibody (1:100 dilution, Origen, Rockville, MD, USA) at 4 °C overnight to visualize the brain microvessels.

Techniques: Saline, Control, Immunohistochemical staining, Staining

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: List of Oligo used for cloning COL4A3 / COL4A5 plasmids

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: Cloning

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: sgRNA identification and design and dual plasmid cloning strategy to achieved CRISPR/Cas9 correction. In the upper lane of panel a , the sgRNA is represented in red and blue: PAM in blue and in red the mutated nucleotide for the COL4A5 CRISPR/Cas9 design. Below, the same design is reported for the COL4A3 variant. Plasmid functionality is based on the cloning of two plasmids for CRISPR/Cas9 components delivery. In the first plasmid, the sgRNA is under the control of Human U6 Promoter ( b ), while the mCherry Red/Green Reporter System is under the control of a CMV Promoter. The Cas9 plasmid is cloned with spCas9 endonuclease gene under control of a CMV promoter flanked by two Self-Cleaving Cas9 for auto-inactivation ( c ). Representative FACS analysis on HEK293T cells after 48 h of transfection with reporter harboring the wild type sequence and mutated reporter, for COL4A3 and COL4A5 variants, respectively. The population of cells mCherry + /GFP + is gated in the UR quadrant. The double fluorescence is present only in the cells co-transfected with Donor/Reporter plasmid with specific variant and spCas9 (41.9% and 27.6% for COL4A3 and COL4A5 , respectively) demonstrating the specificy of sgRNA ( d )

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: Plasmid Preparation, Cloning, CRISPR, Variant Assay, Control, Clone Assay, Transfection, Sequencing, Fluorescence

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: CasAnalyzer results on the edited bases after CRISPR/Cas9 correction

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: CRISPR, Mutagenesis

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: CasAnalyzer results on editing efficiency on COL4A3 podocyte-lineage cells. The CasAnalzyer Tool performed on the CRISPR/Cas9 treated sample and control has reported a HDR of 44.2% in patients’ mutated podocyte-lineage cells. IGV Visualization Software for this variant also exhibited the loss of heterozygosity with the restoration of the wt base in COL4A3 gene

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: CRISPR, Control, Software, Variant Assay

Journal: European Journal of Human Genetics

Article Title: New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells

doi: 10.1038/s41431-019-0537-8

Figure Lengend Snippet: (p.(Pro72Arg)) genotype of TP53 in COL4A3 and COL4A5 mutated patients

Article Snippet: Plasmids sequences have been submitted to Addgene Vector Database ( https://www.addgene.org ) with these accession numbers: for COL4A3 Donor/Reporter (130279) and COL4A3 self-cleavingCas9 (130281) for COL4A5 Donor/Reporter (130280) and COL4A5 self-cleavingCas9 (130282), respectively.

Techniques: Mutagenesis

Journal: ASN NEURO

Article Title: Blood Flow Deficits and Cerebrovascular Changes in a Dietary Model of Hyperhomocysteinemia

doi: 10.1177/1759091419865788

Figure Lengend Snippet: TaqMan Custom Gene Array.

Article Snippet: Col4a3 , Mm00483656_m1 , Smo , Mm01162710_m1.

Techniques: