Fig 2: METTL14/PLAGL2/ß-catenin axis promoted NSCLC development in vivo. (a) Tumor images and tumor volume. (b) Tumor weight. (c) METTL14, PLAGL2, and ß-catenin mRNA expressions. (d) METTL14, PLAGL2, ß-catenin (total), and ß-catenin (nuclear) protein expressions. Comparisons among multiple groups were conducted by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. *P < 0.05 vs. sh-NC, #P < 0.05 vs. sh-METTL14 + oe-NC.

Fig 3: PLAGL2 promotes BCa metastasis in vitro and in vivo.Transwell invasion assays showed that knockdown of endogenous PLAGL2 expression in 5637 and 253J cells inhibited invasion (A, C), whereas ectopic expression of PLAGL2 in T24 and RT4 cells promoted invasion (B, C). Wound-healing assays showed that ectopic expression of PLAGL2 in T24 and RT4 cells promoted migration (D, F), whereas knockdown of endogenous PLAGL2 expression in 5637 and 253J cells inhibited the migration of cells (E, F). G EMT-related gene expression was assessed by Western blotting in the indicated cells. H Nude mice were injected with 5637-shPLAGL2/5637-shNC cells six weeks after injection, and lung fluorescence imaging was performed to detect lung metastases. I Fluorescence intensity quantification in lung metastasis. *P < 0.05, **P < 0.01 ***P < 0.001.

Fig 4: PLAGL2 activates YAP1 by inhibiting Hippo signaling.KEGG enrichment analysis (A) and GSEA (B) of the RNA-seq data in 5637-siPLAGL2 versus 5637-siNC cells. YAP1 signaling was enriched and increased in PLAGL2-overexpressing cells. C Left panel: Distribution of enrichment scores of the YAP signature between high- and low-PLAGL2 expression groups (TCGA-BLCA, transcriptome profiling). Right panel: Distribution of YAP-pS127 expression between the highest and lowest quartiles of PLAGL2 (TCGA-BLCA, proteome profiling). D Immunoblotting assays of the indicated protein expression in the indicated cells after PLAGL2 interference. GAPDH and P84 were used as loading controls for total and nuclear extracts, respectively. E–G Immunofluorescence assays indicated the expression and subcellular localization of YAP1 in the indicated cells after PLAGL2 interference. The percentage of subcellular localization of YAP1 in the indicated cells is shown. N nucleus, C cytoplasm. H HOP/HIP luciferase reporter assays showed YAP1/TAZ-TEAD transcriptional activity in the indicated cells after PLAGL2 interference. I The fold change in mRNA levels of the indicated genes was calculated by log2 transformation in qRT?PCR analysis, comparing cells overexpressing PLAGL2 (OE) versus cells with vector or siPLAGL2 (siPL) versus siNC. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.

Fig 5: PLAGL2 promotes proliferation and inhibits apoptosis of BCa in vitro and in vivo.The efficiencies of siRNA and plasmid were verified by qRT?PCR (A) and immunoblotting assays (B). CCK-8 (C), colony formation (D), and EdU assays (E) showed that knockdown of endogenous PLAGL2 expression in 5637 and 253J cells inhibited proliferation, whereas ectopic expression of PLAGL2 in T24 and RT4 cells promoted proliferation. F Flow cytometry assays showed the increased apoptosis rate by PLAGL2 overexpression and the reduced rate by PLAGL2 deficiency. G The efficiency of shRNA-constructed 5637 cell lines was verified by qRT?PCR and immunoblotting assays. H Growth of xenografts in shNC- or shPLAGL2-transfected 5637 cells was measured every 3 days. Xenografts were dissected from nude mice at 35 days after injection with shNC or shPLAGL2-transfected 5637 cells, and the average weight of excised xenografts is shown. Ki-67 staining showed that shPLAGL2-transfected 5637 cells had decreased proliferation. *P < 0.05, **P < 0.01 ***P < 0.001.