Fig 1: eIF5A hypusination contributes to TCF4 protein translation. (A) TCF4 contains 7 conserved Pro-Pro motifs. (B) Expression of TCF4, AMD1, eIF5A and eIF5A hypusination was examined by Western blotting in BT549 cells with or without eIF5A knockout (eIF5A-KO) as well as eIF5A-KO-expressing BT549 cells with eIF5A-WT or eIF5A-K50R expression. (C) Expression of TCF4, AMD1, eIF5A and eIF5A hypusination was examined by Western blotting in BT549 cells with or without eIF5A knockout (eIF5A-KO) as well as eIF5A-KO-expressing BT549 cells with eIF5A-WT or eIF5A-K50R expression following treatment with or without spermidine (30ng/ml) for a period of 24 h. (D) Expression of AMD1, TCF4 and cyclinD mRNA was analyzed by quantitative real-time PCR in BT549 cells with stable empty vector or ADM1 expression as well as SUM159 cells with stable empty vector or knockdown of AMD1 expression. Data are shown as mean ± SD based on three independent experiments
Fig 2: An eIF5A1 SUMOylation-deficient mutant cannot completely substitute for yeast HYP2. A Histidine-tagged proteins were purified from yeast cells transformed with an empty vector or with Histidine- and Flag-tagged human eIF5A1–WT under denaturing conditions. Then, purified proteins were analyzed by western blot using anti-Smt3 antibody. Arrow indicates Smt3-conjugated eIF5A1 protein. B Histidine-tagged proteins were purified from yeast cells transformed with an empty vector, Histidine- and Flag-tagged human eIF5A1–WT or Histidine- and Flag-tagged human eIF5A1–5KR under denaturing conditions. Purified proteins were then analyzed by western blot using anti-Smt3 antibody. C WT and hyp2-1 (upper row) or hyp2-3 (lower row) yeast strains were transformed with different alleles of Histidine- and Flag-tagged human eIF5A1 in a pAG425GPD vector or with the empty vector. The resulting strains were streaked on SC–Leu plates, incubated at 25 °C or 37 °C for 3 days and imaged in a GelDoc documentation system. D Expression levels of the different versions of eIF5A in the indicated strains, growing at 25 °C or after 4 h at 37 °C, were analyzed by western blot. Ponceau staining of the membrane is shown as a loading control
Fig 3: OGFRP1 exerts its role through regulating miR-4640-5p/eIF5A axis. A The sequences of miR-4640-5p, wide type of eIF5A (WT) and mutated eIF5A (Mut). B The protein expression of eIF5A in 25 pairs of LUAD and normal lung tissues. Overall (C) and disease-free (D) survival curves of LUAD patients with different eIF5A expression. The result of C and D derived from GEPIA (http://gepia.cancer-pku.cn/), which was based on the database of TCGA and GTEx. E The correlation between miR-4640-5p with eIF5A levels in 25 pairs of clinically collected LUAD and normal lung tissue samples. F The correlation between OGFRP1 with eIF5A levels in 25 pairs of clinically collected LUAD and normal lung tissue samples. G The expression levels of luciferase of A549 cells transfected with wild-type (WT) or mutated (Mut) eIF5A reporters plus miR-4640-5p mimic or miR-NC were determined. H The protein expression of eIF5A. * represented P < 0.05
Fig 4: In vitro validation of radiation resistance by EIF5A. (A, B) The transfection efficiency of siEIF5A in HCT116 cell line, which was examined by western blot and qRT-PCR. (C) Representative wound healing images of migration after knocking down EIF5A. (D) Representative transwell images of migration and invasion after knocking down EIF5A. (E) CCK-8 assay to analyze the effect of radiation on cell proliferation of the irradiated and non-irradiated groups after knocking down EIF5A. (F) Representative colony formation images of the irradiated and non-irradiated groups after knocking down EIF5A. (G) Representative ROS production images of the irradiated and non-irradiated groups after knocking down EIF5A. (H) Flow cytometry assay to analyze the cell apoptosis changes in irradiated and non-irradiated groups after knocking down EIF5A. (I) Calcein-AM/PI staining assay and EdU incorporation assay to detected the cell death and proliferation in irradiated and non-irradiated groups after knocking down EIF5A. qRT-PCR, Quantitative reverse transcription polymerase chain reaction; CCK-8, Cell counting kit-8; ROS, Reactive oxygen species; EdU, 5-Ethynyl-2’-deoxyuridine; NC, Negative control; KD, Knock down; IR, Irradiation; ns, Non-statistics significance;*: P < 0.05; **: P < 0.01; ***: P < 0.001.
Fig 5: Landscape and potential mechanism of radiation resistance via scRNA-seq. (A) The t-SNE plot of 26 cell clusters from the multicellular ecosystem of three CRC scRNA-seq cohorts. (B) Dotplot showing the percentage of expressed cells and average expression levels of canonical marker genes of major cell types in 26 cell clusters. (C) t-SNE plot and bar plots indicating the landscape and proportion of major cell lineages between radioresistant and radiosensitive cells. (D) Pseudotime analysis of epithelial cell and cancer stem cell in high- and low-EIF5A expression groups. scRNA-seq, Single-cell RNA sequencing; t-SNE, Stochastic neighbor embedding; CRC, Colorectal cancer.
Supplier Page from Abcam for Anti-eIF5A antibody [EP526Y]