Fig 2: WX8 triggered the EIF2AK3/PERK‐dependent ER‐stress response. The EIF2AK3/PERK‐dependent ER‐stress response in WX8‐treated melanoma A375 cells consisted of 17 genes that were detected by upregulation of their RNA and/or protein levels (Fig. S2B). (A) RNA levels were quantified by RNA‐seq analysis in both A375 cells and HFF1 cells. Gray bars are 0.05 μm WX8. Black bars are 1 μm WX8. Genes with RNA levels ≥ 1.5 fold change above vehicle (V)‐treated cells were considered as significant upregulation. (B) Upregulation of interleukin‐24 (IL24) RNA was detected by both RT‐PCR and RNA‐seq. (C) IL24 protein was detected by immunoblotting. Extracts from WX8‐treated cells were treated with PNGase F to remove N‐linked oligosaccharides from IL24. (D) Cells treated for 24 h with vehicle (V), 1 μm WX8, 1 μm thapsigargin (THAP) or 1 μm tunicamycin (TUN) were immunoblotted for IL24 protein. (E) IL24 RNA in samples from panel D were quantified by RT‐PCR (V indicates ‘vehicle’). (F) Cells were treated with vehicle (V), 1 μm WX8 or 10 μm WX8 for 24 h. IL24 secreted into the culture medium by was quantified by ELISA assays. (G) Melanoma A375 cells were cultured with either vehicle or 1 μm thapsigargin (THAP) for the indicated times. Whole cell extracts were then immuno‐blotted for the indicated proteins, which were identified by their molecular weight and their reaction with a specific antibody. Gene symbols are from the HUGO gene nomenclature committee. V indicates ‘vehicle’. Error bars indicate ± SEM for three samples. Statistical significance was P < 0.05 (*), P < 0.001 (***), P < 0.0001 (****). P > 0.05 was ‘not significant’ (ns). P‐values in RNA‐seq were determined by Wald Chi‐square test using the DEseq2 platform. P‐values of two data sets from the other experiments were calculated by Student's paired two‐tailed t‐test using graphpad prism.

Fig 3: Il24 gene expression is essential for WX8‐dependent cell death. Exon‐2 in the IL24 gene was ablated in melanoma A375 cells. (A) Surviving clones were isolated and screened for deletion within exon‐2 of IL24 gene and comparison with DNA size standards (std) using PCR technology. (B) Immunoblot of total cell extracts confirmed the absence of IL24 protein in A375(IL24 −/− ) clones. (C) WX8 upregulated expression of IL24 protein in wild‐type A375 cells but not in A375(IL24 −/− ) clones exemplified by c10 and c17. (D) Phase contrast images of A375 and A375(IL24 −/− ) cells. Bar indicates 20 μm. (E) ELISA assays confirmed the absence of IL24 protein in the cell culture medium of A375(IL24 −/− ) clones cultured for 24 h in the presence of either vehicle or 1 μm WX8. (F) The fractions of live (impermeable to trypan blue) and dead (permeable to trypan blue) A375 and A375(IL24 −/− ) cells after culturing A375 wild‐type and A375(IL24 −/− ) clone 10 cells for 72 h with the indicated concentrations of WX8. (G) The fraction of cells with < 2N DNA content (less DNA than G1 phase cells) were considered dead. To allow a logarithmic axis, vehicle was plotted as 0.001 μm WX8. Panels E–G include ±SD, n = 2. P‐values in RNA‐seq were determined by Wald Chi‐square test using the DEseq2 platform. P‐values of two data sets from the other experiments were calculated by Student's paired two‐tailed t‐test using graphpad prism. Images in panels A through D are representative of three samples.

Fig 4: RNA‐seq analysis of genes upregulated by WX8 in melanoma A375 cells and HFF1 foreskin fibroblasts. (A) The total number of IL24 transcripts per 106 total transcripts [±SD for A375 (n = 4) and HFF1 (n = 3)] in cells treated for 24 h with either vehicle, 0.05 μm or 1 μm WX8. (B, C) RNA sequence analysis identified 37 936 different RNAs in A375 cells. The 100 most highly expressed genes were plotted against their RNA abundance. The top 5% of RNAs upregulated with 0.05 μm WX8 were also present in the top 26% of RNAs upregulated with 1 μm WX8. (D, E) RNA sequence analysis identified 31 249 different RNAs in HFF1 cells. Of these, 19 were upregulated by 0.05 μm WX8, but 968 were upregulated by 1 μm WX8. IL24 was upregulated in A375 cells, but not in HFF1 cells. Only PLA2G3, LIN00520, and INSIG1 were upregulated in both cell lines. Ratios < 1.5 were considered not significant. In panels B and D, the number of genes upregulated ≥ 1.5 [fold change] greater than vehicle treated cells is marked by horizontal broken lines. Panels B and C are the average of four samples. Panels D and E are the average of three samples.

Fig 5: IL24 upregulation was specific for PIKFYVE inhibition. (A) Melanoma A375 cells were cultured with the indicated PIKFYVE inhibitor (WX8, Apilimod/Apl, Vacuolin‐1/Vac, YM201636/YM) or autophagy inhibitor (Chloroquine/CQ) for 24 h. Expression of IL24 RNA relative to vehicle was quantified using RT‐PCR. (B) Example of an IL24 immunoblot from panel A in which IL24 protein is visible for all five inhibitors. (C) A375 cells were cultured for 24 h with the indicated inhibitors [WX8, Apl, CQ, Bafilomycin‐A1 (BafA1), Adriamycin (Adr), etoposide (Etop)]. The ratio of IL24 RNA in cells treated with inhibitor to RNA in cells treated with vehicle was quantified by RT‐PCR. (D) A375 cells were cultured for 24 h with the indicated concentrations of WX8, CQ and BafA1. The relative amounts of LC3‐II protein were detected by immunoblotting of whole cell extracts, thereby confirming that each inhibitor blocked autophagic flux to an equivalent extent. (E) A375 cells were cultured for 24 h with either vehicle (V), or the indicated concentration of WX8, Adr, or Etop. Equal amounts of total cell extract were subjected to immunoblotting for PARP, cleaved PARP (c‐PARP), γH2AX, and the cytoplasmic protein ACTB. Only Adr and Etop induced DNA damage, as indicated by the appearance of cPARP and γH2AX. (F) Phase contrast images of A375 cells treated with vehicle, apilimod, or NCT‐504 for 48 h at the indicated concentration. Bar indicates 20 μm. (G) Percentage of live (black) and dead A375 cells (gray) after treatment of vehicle, WX8, apilimod, and NCT‐504 for 72 h. (H) A375 cells are treated with indicated concentrations of WX8, apilimod, and NCT‐504 and IL24 protein was detected through immunoblotting. (I) A375 cells are treated with either nt‐siRNA or PIP4K2C siRNA for 24–72 h and cell lysates were immunoblotted for IL24. Error bars in panels A, C and G are ±SD, n = 2. Panels B, D–F, H, and I are representative of three samples.