Journal: iScience

Article Title: Restored glyoxylate metabolism after AGXT gene correction and direct reprogramming of primary hyperoxaluria type 1 fibroblasts

doi: 10.1016/j.isci.2024.109530

Figure Lengend Snippet: Characterization of iHeps generated from PH1 patient-derived fibroblasts and AGXT gene corrected clones (A) Expression of hepatocyte specific genes in iHeps generated by direct cell reprogramming from PH1 patient-derived fibroblasts and AGXT gene corrected fibroblasts. The expression of genes involved in different liver functions was analyzed by RT-qPCR. Data from twenty-six independent experiments, conducted with two different PH1-donor fibroblasts (PH1-1 (n = 8) and PH1-2 (n = 4)), three AGXT point mutation corrected clones from the two PH1 donors (GC-1A (n = 4) and GC-1C (n = 1) derived from PH1-1. GC-2A (n = 2) derived from PH1-2) and four AGXT targeted knockin clones (pA-1A (n = 2), pB-1C (n = 2), and pD-1A (n = 2)), derived from PH1-1. pD-2A (n = 1) derived from PH1-2). Violin plot of data show median (bold dashed line) and interquartile range (light dashed line). Normal distribution of the data was assessed by a Kolmogorov-Smirnov test. Nonparametric Kruskal-Wallis with Dunn’s multiple comparisons test was used to compare, for each analyzed gene, iHeps with hepatocytes ($) or with fibroblasts (#). Significant differences between cell populations are marked as follows: $/#: p < 0.05; $$/##: p < 0.01; $$$/###: p < 0.001; $$$$/####: p < 0.0001. ABCC2: ATP binding cassette subfamily C member 2, CP: ceruloplasmin, FGL1: fibrinogen-like protein 1 and HP: haptoglobin. (B) Glycogen storage in the generated iHeps. Microscope images of PAS staining on starting human fibroblasts and generated iHeps, from healthy donor, PH1 patients and AGXT -corrected clones (GC-1A from PH1-1 and GC-2A from PH1-2). HepG2 cell line was used as positive control. All images were taken with 100× magnification. Scale bar: 75 μm.

Article Snippet: Three hundred micrograms of iHeps lysates and 5μg of HepG2 cell lysates were pre-incubated first with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG 1:5000 (Amersham, Cat#NA934), and second with the Protein A/G PLUS reagent (Santa Cruz Technologies, Cat#sc-2003).

Techniques: Generated, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Mutagenesis, Knock-In, Binding Assay, Microscopy, Staining, Positive Control

Journal: iScience

Article Title: Restored glyoxylate metabolism after AGXT gene correction and direct reprogramming of primary hyperoxaluria type 1 fibroblasts

doi: 10.1016/j.isci.2024.109530

Figure Lengend Snippet:

Article Snippet: Three hundred micrograms of iHeps lysates and 5μg of HepG2 cell lysates were pre-incubated first with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG 1:5000 (Amersham, Cat#NA934), and second with the Protein A/G PLUS reagent (Santa Cruz Technologies, Cat#sc-2003).

Techniques: Recombinant, Virus, Derivative Assay, Cell Culture, PCR Cloning, Reverse Transcription, SYBR Green Assay, Lactate Dehydrogenase Assay, Sequencing, Knock-In, Plasmid Preparation, Amplification, Gene Expression, Software, Extraction