Fig 1: Application of the single‐vector CRISPR‐dRfxCas13d‐eIF4G translational regulatory tool in the mouse kidney stone model. A) The dRfxCas13d‐eIF4GI fusion protein and sgRNA complementary to the DNA sequence of related genes were inserted into the consent skeleton containing the CMV and U6 promoters to form the plasmid dRfxCas13d‐eIF4GI‐sgRNA for packaging into adeno‐associated viral vectors. Then the adeno‐associated viral plasmids were injected orthotopically into mice, and 80 mg kg−1 glyoxylic acid was injected i.p. into the mice every day for 14 days. B) The mRNA expression of GPX4 in HK‐2 cells was determined using qRT‐PCR analysis for the two groups. C) Western blotting results show the protein expression levels of GPX4, ASCL4, and CD71 for the two groups and the bar graph shows the relative protein levels (n = 3). D) Several indicators of ferroptosis, including MDA content and Fe2+content, were measured. E) Several indicators of kidney injury, including serum CRE content and BUN content, were measured (n = 6). F) The mitochondrial morphology alterations associated with ferroptosis were observed using transmission electron microscopy (scale bar = 2 or 1 µm). Red arrows represent solid mitochondria and red star marks represent broken mitochondria. The bar graph shows the ratio of the number of abnormal mitochondria to the total number of mitochondria in the current field. G) Crystal deposition was observed by calcium salt staining and H&E staining and the black arrows indicate crystals (not limited to these arrows) (magnification, ×400). The bar graph shows the area of crystal deposition in the current field. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the vector stone model group.
Fig 2: eIF4G1 is required for oxidative stress tolerance. (A) Schematic of protein sequence alignment of eIF4G1 and eIF4G2 where yellow sections (scored 3) are conserved residues and purple sections (scored 0) are not similar. Alignment and similarity scores from ClustalOmega (76). (B) The wild-type (BY4741) and isogenic eIF4G deletion strains (4G1Δ, 4G2Δ) or wild-type (BY4742) and isogenic homogenic strains (4G1h, 4G2h) were grown to exponential phase and the A600 adjusted to 1, 0.1, 0.01 or 0.001 before spotting onto the indicated plates. This included glucose (SCD), glycerol/ethanol, H2O2 or sulpholmeturon methyl (SMM) plates. (C) Western blot analysis of the same strains probed with antibodies that recognise eIF4G1 or eIF4G2 normalized to Zwf1 (glucose 6-phophate dehydrogenase) as a loading control. Data shown are the means of 3 independent biological repeat experiments ± SD. Significance is shown using a one-way ANOVA test. (D) Doubling times for the same strains grown on SCD media. Data shown are the means of three independent biological repeat experiments ± SD. Significance is shown using a one-way ANOVA test. **P < 0.01, *** P < 0.001, **** P < 0.0001.
Fig 3: The translational response to oxidative stress is different in strains containing eIF4G1 or eIF4G2 alone. (A) Representative polyribosome traces are shown for the wild-type and homogenic strains before or after treatment with 0.4 mM H2O2 for 15 min. The peaks that contain the small ribosomal subunit (40S), the large ribosomal subunit (60S), and both subunits (80S) are indicated along with the polysome peaks generated by 2, 3, 4, 5, etc., 80S ribosomes on a single mRNA. (B) Quantification of polysome:monosome ratios is shown from triplicate traces determined by the ratio between the area under the monosome to the polysome peaks. Error bars denote standard deviation and significance is shown comparing unstressed or stressed ratios using a one way ANOVA test. (C) Western blot analysis of eIF2α (Sui2) and eIF2α-P using the same strains and conditions as for panels A and B. Quantification is shown from triplicate experiments comparing phosphorylation in the presence or absence of hydrogen peroxide for each strain using one-way ANOVA. *P < 0.05, **P < 0.01.
Fig 4: The proportion of mRNA-bound versus free 80S ribosomes increases in eIF4G1 containing strains but not in eIF4G2-containing strains strain following oxidative stress conditions. (A) Schematic showing the effects of high magnesium concentrations in polysome buffers on either free or mRNA bound 80S ribosomes. Free 80S subunits dissociate to their 40S and 60S constituent parts whereas mRNA bound 80S subunits are stabilised by their mRNA interaction and remain as 80S monosomes. (B) Representative polyribosome traces are shown in the presence or absence of high magnesium for the wild-type and homogenic strains before or after treatment with 0.4 mM H2O2 for 15 minutes. Green diamonds (40S), red ovals (60S) and blue triangles (80S) denote ribosomes and subunits. (C) Quantification of the area of the 40S and 60S peaks (free) and the 80S and polysomes (bound) as a proportion of the total trace, where the free fraction represents ribosomal subunits separated by magnesium and the bound fraction represents ribosomes stabilised by mRNA. Significance is shown using a one-way ANOVA test for the bound fraction; *P < 0.05, ***P < 0.001.
Fig 5: Application of the dual‐vector CRISPR‐dRfxCas13d‐eIF4G translational regulatory tool in a CaOx crystal‐induced cell injury model. A) The lentiviral plasmids with the dual‐vector CRISPR‐dRfxCas13d‐eIF4G translational regulatory tool (dET‐GPX4) or vector were transfected into HK2 cells, and HK‐2 cells were treated with CaOx intervention solution (2 mm) for 24 h. B) The mRNA expression of GPX4 in HK‐2 cells was determined using qRT‐PCR analysis for the two groups. C) Western blotting results show the protein expression levels of GPX4, ASCL4, and CD71 for the two groups and the bar graph shows the relative protein levels. D) The data from flow cytometric analysis shows the degree of lipid peroxidation in the two groups and the bar graph E) shows the mean fluorescence intensity. Several indicators of ferroptosis, including MDA content (F) and Fe2+ content (G), were measured. H) CCK‐8 assays were used to detect cell viability. Several indicators of oxidative and anti‐oxidative status, including T‐AOC content (I) and SOD content (J) were measured. K) Images were taken under a dark field (magnification, x200). Brighter green indicates higher levels of cellular ROS and the bar graph shows the mean fluorescence intensity. L) The mitochondrial morphology alterations associated with ferroptosis were observed using transmission electron microscopy (scale bars = 2 µm or 1 µm). Red arrows represent abnormal mitochondria (solid and broken mitochondria) and the bar graph shows the ratio of the number of abnormal mitochondria to the total number of mitochondria in the current field. Data are presented as the means ± SEM from three independent experiments (n = 3). One set of representative images from three independent experiments is shown. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the Vector+CaOx group.
Supplier Page from Abcam for Anti-eIF4G1 antibody