Journal: Cellular & Molecular Biology Letters

Article Title: FTH1 overexpression using a dCasRx translation enhancement system protects the kidney from calcium oxalate crystal-induced injury

doi: 10.1186/s11658-024-00582-w

Figure Lengend Snippet: Application of the dual-vector dCasRx translation enhancement tool in a CaOx crystal-induced cell injury model. A Volcano plot shows differential expression genes (DEGs) between CaOx crystal-induced cell injury models and negative controls. B The protein expression of FTH1, GPX4, ASCL4, and CD71 (also called TFRC) in models were determined by 4D-LFQ proteomic analysis. C After constructing the dual-vector CRISPR-dCasRx-eIF4GI tool targeting FTH1 (DV-FTH1), the mRNA expression level of FTH1 were detected using qRT–PCR analysis. D , E WB analysis results show the protein expression levels of FTH1, CD71 GPX4, and ASCL4 between CaOx + Neg. and CaOx + DV-FTH1, and the bar graph shows the relative protein levels. F The cell viability was detected using CCK8 assay kit. G Several indicators of anti-oxidative status, including T-AOC content, SOD content and CAT content, were measured. H Two indicators of ferroptosis, including MDA content and Fe 2+ content, were measured. I The level of cellular ROS was observed and images were taken under dark field (magnification ×400). Brighter green indicates higher levels of cellular ROS. J The level of cellular Fe 2+ was observed and images were taken under dark field (magnification ×400). Brighter red indicates higher levels of cellular Fe 2+ . K The degree of lipid peroxidation was observed and images were taken under dark field (magnification ×400). Higher ratio of green to red indicates higher degree of lipid peroxidation. L The mitochondrial morphology was observed using transmission electron microscopy (scale bars, 2 μm or 1 μm). Data are presented as the means ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, no significant difference, represents P > 0.05

Article Snippet: The proteins were subsequently transferred onto Immun-Blot polyvinylidene fluoride (PVDF) membranes (1620177, BIO-RAD, USA).Membrane blocking was accomplished by applying a rapid blocking buffer (PS108P, Epizyme, China) for 10 min, then proceeding with multiple washes in Tris-buffered saline containing Tween 20 (TBST).Afterward, the membranes were left to incubate overnight at 4 °C with primary antibodies that targeted FTH1 (ab65080, 1:1000, Abcam, UK), GPX4 (A1933, 1:1000, ABclonal, China), CD71 (A5865, 1:1,000, ABclonal, China), ASCL4 (22401-1-AP, 1:4000, Proteintech, USA), SIRT1 (13161-1-AP, 1:2,000, Proteintech, USA), NIX (A24803, 1:1000, ABclonal, China), Tubulin (11224-1-AP, 1:10,000, Proteintech, USA), and GAPDH (10494-1-AP, 1:20,000, Proteintech, USA).

Techniques: Plasmid Preparation, Quantitative Proteomics, Expressing, CRISPR, Quantitative RT-PCR, CCK-8 Assay, Transmission Assay, Electron Microscopy

Journal: Cellular & Molecular Biology Letters

Article Title: FTH1 overexpression using a dCasRx translation enhancement system protects the kidney from calcium oxalate crystal-induced injury

doi: 10.1186/s11658-024-00582-w

Figure Lengend Snippet: Application of the single-vector dCasRx translation enhancement tool in a mouse kidney stone model. A To package into adenoassociated viral vectors, the dCasRx-eIF4GI fusion protein and sgRNA complementary to DNA sequence of related genes was inserted into the consent skeleton containing the CMV and U6 promoters to form the plasmid dCasRx-eIF4GI-sgRNA. B Flow chart of animal model construction. C The mRNA expression level of FTH1 were detected using qRT–PCR analysis between Neg. stone model and SV-FTH1 stone model. D , E The protein expression of FTH1, ASCL4, and GPX4 were verified using western blot and the bar graph shows the relative expression levels. F Two indicators of kidney injury, including BUN content and serum CRE content, were measured. G Two indicators of ferroptosis, including Fe 2+ content and MDA content, were measured. H The images show the degree of renal expression for GPX4 and ASCL4 in two groups (magnification ×400) and bar graph shows relative protein levels. I The mitochondrial morphology alterations associated with ferroptosis were observed using transmission electron microscopy (scale bar, μm or 1 μm). J Crystal deposition was observed. Data are presented as the means ± SEM from six independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, no significant difference, represents P > 0.05

Article Snippet: The proteins were subsequently transferred onto Immun-Blot polyvinylidene fluoride (PVDF) membranes (1620177, BIO-RAD, USA).Membrane blocking was accomplished by applying a rapid blocking buffer (PS108P, Epizyme, China) for 10 min, then proceeding with multiple washes in Tris-buffered saline containing Tween 20 (TBST).Afterward, the membranes were left to incubate overnight at 4 °C with primary antibodies that targeted FTH1 (ab65080, 1:1000, Abcam, UK), GPX4 (A1933, 1:1000, ABclonal, China), CD71 (A5865, 1:1,000, ABclonal, China), ASCL4 (22401-1-AP, 1:4000, Proteintech, USA), SIRT1 (13161-1-AP, 1:2,000, Proteintech, USA), NIX (A24803, 1:1000, ABclonal, China), Tubulin (11224-1-AP, 1:10,000, Proteintech, USA), and GAPDH (10494-1-AP, 1:20,000, Proteintech, USA).

Techniques: Plasmid Preparation, Sequencing, Animal Model, Expressing, Quantitative RT-PCR, Western Blot, Transmission Assay, Electron Microscopy