Fig. 2

Paraptosis proceeds with activation of proteostatic dynamics and transcriptional regulation involved in redox homeostasis and proteostasis. (A, B) Cancer cells were pretreated with the transcription inhibitor actinomycin A (ActD, 1 µM) or the translation inhibitor cycloheximide (CHX, 20 µM) for 30 min, followed by treatment with CPYPP (10 µM), cyclosporin A (CsA, 20 µM), or curcumin (CUR, 30 µM) for 24 h. The cells were then assessed for morphological vacuolization alterations using crystal violet staining (A, MDA-MB-435 cells) or for cell viability via MTT assay (B). The results are expressed as the mean ± SD from three independent experiments. **P < 0.01, compared to the control group (CTL). (C, D) CPYPP-treated MDA-MB-435 cells underwent next-generation sequencing (NGS) to analyze differential gene expressions (DGEs) The volcano plot represents the distribution of DGEs based on the FPKM analysis with at least 2-fold changes (C). Results from the Gene Ontology (GO) analysis classify molecular function, cellular component, and biological process (D), along with the number of related significant changed genes. (E) CPYPP-treated MDA-MB-435 cells were subjected to mass spectrometric analysis for proteomic profiling with at least 1.5-fold changes. (F–J) Western blot analysis confirmed the involvement of differentially expressed genes in ER stress (F), HSP chaperones (G), redox homeostasis (H), protein ubiquitination (I), and translational activity (J) in cancer cells treated with CPYPP, CsA, or CUR for 24 h