Fig. 3: Impact of AVJ16 treatment on the steady state of RNAs in H1299 cells.

RNA-seq was performed on RNA from cells treated with 1.5 µM AVJ16 or DMSO for 48 h. A Heat map of the most significantly affected genes after AVJ16 treatment, with downregulated genes shown in blue and upregulated genes shown in red. B KEGG annotation of gene sets downregulated by AVJ16 treatment, showing significant enrichment in IGF2BP1-related pathways, such as axon guidance, ECM-receptor interactions, WNT signaling, and PI3K-Akt signaling. C Enrichment map of the AVJ16-downregulated genes analyzed by Gene Set Enrichment Analysis (GSEA) for all human gene sets, showing significant enrichment of pathways associated with transformed phenotypes and signaling pathways activated during cancer progression, with WNT and ECM being the most clustered pathways. D Enrichment map of the AVJ16-upregulated genes analyzed by GSEA for all human gene sets. E RNA from cells treated with 1.5 µM AVJ16 or DMSO for 48 h was analysed for expression of WNT-related genes by qPCR. The seven WNT-related genes downregulated in RNAseq were significantly downregulated in H1299 cells upon AVJ16 treatment, compared to the DMSO-treated control group (RPLPO was added as a housekeeping gene control). The WNT inhibitor, DKK3, was upregulated in the RNAseq analysis, and its expression was significantly upregulated in the AVJ16-treated H1299 cells. F Western blot analysis of beta catenin protein levels in H1299 cells treated with 1.5 µM AVJ16 or DMSO for 48 h. Beta catenin protein levels were downregulated, consistent with the reduction in its RNA level observed in the RNAseq data. G Correlation between the normalized expression of WNT pathway genes (listed on the X-axis) and IGF2BP1 expression was analyzed in data collected from human databases. Gene expression data was combined from TCGA, TARGET and GTEx lung cohorts and segregated into those expressing the highest levels of IGF2BP1 (top 25%, High IGF2BP1) and those expressing the lowest levels of IGF2BP1 (low 25%, Low IGF2BP1). H Table comparing changes in RNA levels (RNAseq) and in protein levels. (Mass spec) in H1299 cells treated with 1.5 µM AVJ16 or DMSO for either 48 h (RNAseq) or 96 h (Mass spectrometry). I Heat map of all statistically significant upregulated and downregulated proteins. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns not significant.