Fig 2: Targeting EML4 with METLL3 small-molecule inhibitor reduces metastasis in vivo. A, Representative IHC staining of EML4 in primary tumor and matched lymph node metastases from one LUAD patient (left). Protein levels of EML4 in 39 matched LUAD tumors and metastases by IHC score (right). B and C, Impact of METTL3 knockdown on lung metastasis in a tail vein metastasis model. Representative images of the NSG mouse lungs (B, top) and H&E staining of lung tissues (B, bottom) approximately 7 weeks after tail vein injection of 1 × 106METTL3-knockdown and control A549 cells. Quantification of the proportions of tumor metastases in mouse lungs based on H&E staining (C, left). Quantification of METTL3 (C, middle) and EML4 protein expression (C, right) by IHC score in METTL3-knockdown and control lung metastases. D, Relative m6A levels near the stop codon of EML4 gene in A549 cells with the indicated treatment, determined by m6A MeRIP qPCR. E, Rescue assay of STM2457 treatment. Western blot analysis of EML4 protein expression in control and EML4-overexpressing A549 cells with the indicated treatment (E, left). Cell migration abilities of control and EML4-overexpressing A549 cells with the indicated treatment (E, right). F, Rescue assay of METTL3 knockdown. Western blot analysis of METTL3 and EML4 protein expression in control and EML4-overexpressing A549 cells transfected with the indicated siRNAs (F, left). Cell migration abilities of control and EML4-overexpressing A549 cells transfected with the indicated siRNAs (F, right). Forty-eight hours after siRNA transfection, cells were harvested for Western blot and migration analysis. G, Experimental design of STM2457 treatment in the lung metastasis model (top). Representative images of the formalin-fixed NOD/SCID mouse lungs harvested 60 days after treatment with STM2457 (bottom). H, Representative images of H&E staining of mouse lung tissues (left), quantification of the numbers of metastatic nodules in mouse lungs based on H&E staining (middle), and body weight of the mice (right) at the experimental endpoint in the lung metastasis model. All histogram data are presented as mean ± SD. Compared with the indicated group, **, P < 0.01; ****, P < 0.0001; n.s., not significant.

Fig 3: EML4 promotes NSCLC cell migration and invasion in vitro and metastasis in vivo. A, Western blot analysis of EML4 protein expression in EML4-knockdown (left), EML4-overexpressing (middle), and KO (right) cells. For EML4-knockdown assay, 48 hours post siRNA transfection, A549 and H358 cells were harvested for analysis. B, Cell migration (left) and invasion (right) abilities of EML4-knockdown and control A549 and H358 cells. C, Cell migration abilities of EML4 KO and wild-type (WT) A549 and H358 cells. D, Cell migration abilities and migration rates of EML4-overexpressing and control A549 and H358 cells. E, Western blot analysis of EML4 protein expression upon shRNA-mediated EML4 knockdown in A549 and H358 cells. F, Impact of EML4 knockdown on lung metastasis in a tail vein metastasis model. Representative images of the formalin-fixed NOD/SCID mouse lungs (left) and hematoxylin and eosin (H&E) staining of lung tissues (middle) approximately 8 weeks after tail vein injection of 1 × 106EML4-knockdown and control A549 and H358 cells. Quantification of the proportions of tumor metastases in mouse lungs based on H&E staining (right). G, Survival of NOD/SCID gamma (NSG) mice bearing EML4-knockdown and control H358 lung metastases (log-rank test). H,EML4 overexpression enhanced lung metastasis in a tail vein metastasis model. Representative images of the formalin-fixed NOD/SCID mouse lungs (left) and H&E staining of lung tissues (top middle) approximately 8 weeks after tail vein injection of 1 × 106EML4-overexpressing and control H358 cells. Quantification of the proportions of tumor metastases in mouse lungs based on H&E staining (right). Representative IHC staining of EML4 (bottom middle) and quantification of EML4 protein expression (right) by IHC score in EML4-overexpressing and control lung metastases. I, F-actin staining in EML4 KO, EML4-overexpressing, and the corresponding control A549 and H358 cells. Nuclei were visualized with 4',6-diamidino-2-phenylindole (DAPI). J, IF staining of EML4 and α-tubulin in EML4-overexpressing and control cells. Nuclei were visualized with DAPI. K, Top enriched CC terms in LUAD tumors with high EML4 protein expression vs. LUAD tumors with low EML4 protein expression from the CPTAC cohort (top 50% vs. bottom 50%; left) and in EML4-overexpressing vs. control H358 cells (right). L, Co-IP assays showing the interactions of EML4 with ARPC1A in Flag-tagged EML4-overexpressing NSCLC cells. M, IF staining of Flag (Flag-tagged EML4) and ARPC1A in Flag-tagged EML4-overexpressing cells. Nuclei were visualized with DAPI. N, IF staining of Flag and ARPC1A in Flag-tagged EML4-overexpressing and control cells (left). Nuclei were visualized with DAPI. Quantification of the percentages of cells with ARPC1A enriched at the edge of lamellipodia in EML4-overexpressing and control cells (right). Protein expression levels of EML4 and ARPC1A (O), F-actin staining (P), cell migration abilities (Q, left), and the relative lamellipodia lengths (Q, right) of EML4-overexpressing and control cells transfected with the indicated siRNAs. Forty-eight hours after siRNA transfection, cells were harvested for the above analysis. All histogram data are presented as mean ± SD. Compared with the indicated group, ***, P < 0.001; ****, P < 0.0001.