Fig. 3: NCP26 exposure induces the integrated stress response via GCN2 and leads to subsequent apoptosis in MM cells.

A Table of EC₅₀ values for NCP26 determined for wild-type and drug-resistant MM cell lines. B Serum-starved AMO1 and RPMI 8226 cells were pre-incubated with NCP26 (0.25, 0.5 and 1 µM) for 1 h and then treated with IL-6 (10 ng/ml) or IGF-1 (50 ng/ml). C AMO1 and RPMI 8226 cells were cultured with NCP26 (0.25, 0.5 and 1 µM) for 48 h in the presence or absence of BMSC. D BMMCs from MM patients were cultured with or without NCP26 (1 µmol/L) for 48 h and analysed using multi-channel flow cytometry. Viability of CD138-positive MM cells and CD138-negative normal BM stromal cells was determined by Annexin V and PI staining. The percentage of viable MM cells from five different patients is shown. E Bone marrow CD138⁺ tumour cells from three MM patients and PBMCs and B cells isolated from three healthy volunteers were cultured with NCP26 (0.01–10 µM) for 48 h. F PCA plot of transcriptomic (RNAseq) changes in AMO1 cells after a 6- or 24 h exposure with HFG or NCP26 (both 1 µM) or carfilzomib (10 nM). G Pathway analysis shows enrichment for stress responses in the RNAseq dataset. H Heatmap of selected differentially expressed genes upon NCP26 treatment. I Western blot demonstrating dose-dependent responses to NCP26 for canonical ISR activation with concomitant GCN2 and eIF2α phosphorylation. J, K AMO1 and RPMI 8226 cells were transduced with shLuc (control), shGCN2 or sheIF2α. After puromycin selection, cells were treated with or without NCP26 (0.5 µM) for 6 h (J) or 24 h at indicated doses (K). Whole-cell lysates from MM cells were subjected to immunoblotting using indicated antibodies (J). L AMO1 and RPMI 8226 cells were cultured with NCP26 at 0.5 µM for 6 or 24 h. G₀/G₁, S and G₂/M phase in cell cycle profiling was analysed by flow cytometry. Means ± SD from two independent experiments. M AMO1 and RPMI 8226 cells were cultured with NCP26 for the indicated times at the indicated dose. Whole-cell lysates were subjected to immunoblotting using indicated antibodies. Cell growth was assessed by MTT assay (A, B, E, K) or BrdU uptake (C). Data represent mean ± SD of triplicate cultures. **P < 0.01, ***P < 0.001.