Fig. 2: NXP800 induces UPR through eIF2A phosphorylation.

A RNA-seq was performed on MNNG/HOS cells treated with DMSO (CT) or 300 nM NXP800 for 6 h and 24 h (N = 3). The Principal Component Analysis (PCA) plot illustrates the variance among different samples. Each point represents an individual sample, with clustering indicating similarities in global gene expression profiles across conditions. B GSEA was performed on Gene Ontologies (GO) biological processes with the genes ordered by their expression on MNNG/HOS cells between the treatment with 300 nM NXP800 for 6 h and the corresponding control. C The volcano plot illustrates the distribution of differentially expressed genes on RNA-seq data between the NXP800 treatment for 6 h and the corresponding control. D The heatmap of differentially expressed genes associated with the UPR pathway (GO) was generated from RNA-seq data. E MNNG/HOS and U2OS cells were treated with 300 nM NXP800 for 6 and 24 h. Relative mRNA expression of UPR genes was evaluated by RT-qPCR and normalized to DMSO control condition (CT; N = 4). F MNNG/HOS and U2OS cells were treated with 300 nM NXP800 for the indicated durations. Proteins expression the UPR pathway were evaluated by western blot (representative images of three independent biological replicates). G MNNG/HOS and U2OS cells were treated with 300 nM NXP800 for 4 h treatment and immunofluorescent labeling of CHOP and nucleus (DAPI) was performed (representative images of three independent biological replicates). H MNNG/HOS and U2OS cells were treated with 10 µM CHX used as a reference translation inhibitor or 300 nM NXP800. Translation rate was determined by OPP assay and normalized to DMSO (CT). In (E, H), bars indicate means ± SD of the different values, *: p < 0.05 (Mann-Whitney test).