Fig 1: The hypermethylated m6A-circRNAs possess potential for translation. A The cumulative fraction of coding probability scores predicted by CPAT for BSJ-distal m6A-circRNAs, BSJ-near m6A-circRNAs and non-m6A-circRNAs. The P value was calculated with the Wilcoxon rank sum test. B Significant higher ratio of binding sites of EIFs and YTHDFs in BSJ-distal m6A-circRNAs than BSJ-near m6A-circRNAs and non-m6A-circRNAs. For each clustered bar, from left to right were binding sites ratios of BSJ-distal m6A-circRNAs, BSJ-near m6A-circRNAs, and non-m6A-circRNAs. P values were calculated by chi-squared test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. C Venn diagram showing the number of m6A-circRNAs with coding potential (circRNAs with ORFs and EIFs binding sites) for non-DE hypermethylated-m6A-circRNA and all hypermethylated-m6A-circRNA. The outer layer indicated total number of corresponding m6A-circRNAs; the inner layer indicated the number of corresponding m6A-circRNAs with coding potential. Fisher’s exact test was used to derive the P value. D GO molecular function (MF) terms enrichment analysis for host genes of hypermethylated m6A-circRNAs with coding potential. E Relative fractions of unbound (free) RNAs, monosome- and polysome-bound RNAs for circZFHX3, circTSHZ1, and circLMTK2 in PDAC cell lines with METTL3 siRNA and control. The HPRT mRNA was used as control. F RIP-qPCR analysis showed EIF3A-, EIF3B-, and EIF3H-bound RNA abundance for circZFHX3, circTSHZ1, and circLMTK2 in PDAC cells with or without METTL3 knockdown. Data in E and F were means ± S.E.M. (n = 3). *, P < 0.05; **, P < 0.01; ***, and P < 0.001 of Student’s t test comparing with each control
Fig 2: The eEF1G defect in clones 1–24 and 3–9 does not inhibit cell proliferation or endogenous protein expression. (A) Cell proliferation of clones 1–24 and 3–9. The data are shown as means ± SD (n = 3). ∗∗P < 0.01 according to a two-way ANOVA followed by Dunnett’s test. (B) Proteins expressed in clones 1–24 and 3–9. Total cell lysates were subjected to SDS–PAGE and visualized with CBB staining. (C) Expression of endogenous proteins in clones 1–24 and 3–9. Total cell lysates were analyzed by western blotting using antibodies against eIF3B, SNRPA, α-tubulin, and β-actin.
Fig 3: EIF3B promoted GC cell growth in vitro and in vivo.Notes: (A) qPCR and Western blotting analysis revealed the EIF3B expression in one gastric mucosa epithelial cell line (GES1) and six GC cell lines. (B) EIF3B knockdown was evaluated through Western blot in GC cell lines. (C) EIF3B knockdown GC cells showed significantly low proliferative ability, as evaluated using an RTCA real-time analysis instrument. (D) Knockdown of EIF3B significantly inhibited cell colony formation. (E) IHC staining indicated that high EIF3B protein expression was significantly associated with PCNA expression (upper panel), and GPEIA online tools revealed that EIF3B mRNA expression was correlated with PCNA mRNA expression (lower panel). (F) EIF3B knockdown inhibited subcutaneous tumorigenicity, as indicated by tumor size and weight. ***P<0.0001.
Fig 4: EIF3B transcription in GC patients and correlation with poor prognosis.Notes: (A) EIF3B mRNA expression was significantly upregulated in GC tissues compared with adjacent normal mucosa in GSE63089 and 13,861 from GEO datasheets. (B) Western blotting analysis for EIF3B expression in five paired primary GC tissues. GAPDH was used as an internal control (left panel). Ratio (T/N) of EIF3B mRNA expression in ten paired primary GC patients, which was determined through qPCR (right panel). The expression levels were normalized using an internal control (GAPDH). (C) Kaplan–Meier survival analysis of overall survival obtained from public gene expression datasets. (D) Kaplan–Meier survival analysis of 5-year survival with low vs high EIF3B mRNA expression status. ***P<0.0001.
Fig 5: EIF3B promotes GC cell migration, invasion and metastasis in vitro and in vivo.Notes: (A) Knockdown of EIF3B inhibited cell migration, as assessed using a wound healing assay. Scale bar: 100 μm. (B) Knockdown of EIF3B reduced cell invasion, as assessed using a Matrigel invasion Boyden chamber assay. Scale bar: 50 μm. (C) Morphological changes were detected in EIF3B knockdown cells. Scale bar: 50 μm. (D) The correlation between EMT related markers and EIF3B was detected through Western blot. (E) The effect of EIF3B on lung metastatic colonization. *P<0.05.
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