Fig 2: Expression of PML in malignant melanoma cell lines and JAK–STAT pathway analysis in IFNα treated and untreated FM82 and FM55 control and FM82 and FM55 shRNA malignant melanoma cell lines. (a) Left panel: IF with antibodies to PML (green), ABCB5 (green) and HAGE (red) on FM82 control and FM82 shRNA1 and 2 (HAGE knocked down). Right panel: IB with antibodies to HAGE, ABCB5, PML and actin (loading control) using whole lysates from FM82 and FM55 control and FM82 and FM55 shRNA1 and 2. (b) Semi-quantitative real-time PCR of PML mRNAs isolated from FM82 control and FM82 shRNA1. Mann–Whitney U-test: ***P=<0.001. (c) Schematic representation of JAK–STAT signalling pathway upon induction by IFN type I. (d and e) IB with antibodies to PML, HAGE and actin using whole extracts from IFNα treated (16 h) and untreated FM82 and FM55 control and FM82 and FM55 shRNA1 and 2. Quantification of PML protein expression in plus and minus IFNα-treated cells. (f) IB with antibodies to the phosphorylated forms of Jak1, Tyk2, STAT1 and STAT2 using whole cell extracts from IFNα treated (0.25 h) and untreated FM82 control and FM82 shRNA1 and 2. (g) Quantification of the phosphorylated forms of Jak1, Tyk2, STAT1 and STAT2 using whole cell extracts from IFNα treated (0.25 h) and untreated FM82 control and FM82 shRNA1 and 2. (h) IB with antibodies to total Jak1, Tyk2, STAT1 and STAT2 using whole extracts from FM82 control and FM82 shRNA1 and 2. (i) Values expressed in Arbitrary Units (AU) using Aida Image Analyser (v.3.52) IB with antibodies to the phosphorylated forms of Jak1, Tyk2, STAT1 and STAT2 using whole cell extracts from IFNα treated (0.25 h) and untreated FM55 control and FM55 shRNA1 and 2. (j) Quantification of the phosphorylated forms of Jak1, Tyk2, STAT1 and STAT2 using whole cell extracts from IFNα treated (0.25 h) and untreated FM55 control and FM55 shRNA1 and 2. (k) IB with antibodies to total Jak1, Tyk2, STAT1 and STAT2 using whole extracts from FM55 control and FM55 shRNA1 and 2. (l) IB with antibody to IFN α/β receptor (IFN receptor type I) and actin using whole extracts from untreated FM82 and FM55 control and FM82 and FM55 shRNA1 and 2

Fig 3: SOCS1 induces ubiquitination of p-Jak1 and p-Tyk2 in FM82 control malignant melanoma cell lines. (a) IP with antibody to SOCS1 or rabbit IgG followed by IB with antibodies to SOCS1, p-Jak1 and p-Tyk2 from FM82 control whole cell extracts. (b and c) IP with antibody to p-Jak1 or p-Tyk2 and corresponding IgG (rabbit or goat, respectively) followed by IB with antibodies to p-Jak1 or p-Tyk2 and ubiquitin from FM82 control and FM82 shRNA1 whole cell extracts. (d) IP with antibody to p-Jak1 followed by whole cell extracts IB with an antibody to ubiquitin from FM82 shRNA1 transfected with HAGE cDNA or SOCS1 cDNA. The protein expression of HAGE and SOCS1 was determined by IB with the corresponding antibodies

Fig 4: An IL-20RB–neutralizing antibody effectively suppresses bone metastasis of lung cancer.(A–C) JAK1/STAT3 phosphorylation (A), STAT3-responsive reporter activity (B), and organoid formation (C) of A549 with or without IL20RB overexpression after treatment with LTMA1G11 (5 μg/mL) and/or OC CM for 24 hours. (D–I) LTMA1G11 treatment of mice with intracardiac injection of A549 with or without IL20RB overexpression for bone metastasis analysis. Weekly whole-body BLI analysis (D, n = 7 mice per group), ex vivo hind limb BLI quantitation (E), micro-CT quantification of relative bone volumes of hind limbs (F), representative BLI and micro-CT images (G, arrows point to osteolytic areas in the legs), quantitation of EdU+ tumor cells (H) and representative H&E and immunofluorescent images of bone sections (I). Scale bars: 100 μm. P values were obtained by Mann-Whitney U test (D and E) and 2-tailed unpaired t test (B, C and HE). For multiple comparisons, the P values were corrected by the Benjamini-Hochberg procedure and were shown as q values (adjusted P values). Box plots display values of minimum, first quartile, median, third quartile, and maximum. Data are represented as mean ± SD.

Fig 5: Conventional HDAC inhibitors activate JAK1-STAT3-BCL2-mediated drug resistance in solid tumours but not in Roxyl-zhc-84-treated tumours. (A) Immunoblot of STAT3, BCL-2, BIM and STAT3 phosphorylation in cells treated with the histone deacetylase inhibitor vorinostat and JAK1 inhibitor filgotinib. (B) GSEA analysis for enrichment of JAK-STAT3 pathway in vorinostat-treated SK-OV-3 cells. (C) HDACi panobinostat-treated DLBCL cell lines. Data were acquired on GEO database (GSE53603, GSE27226). (D) Heatmap showing upregulated genes in the JAK-STAT3 pathway based on data from GSE53603. (E) Dose-response curve showing that Roxyl-zhc-84 (0.1 nM-1 µM) inhibits the activity of recombinant human JAK1 dose-dependently by in vitro kinase assay. (F) Representation of predicted binding modes of Roxyl-zhc-84 (purple) and filgotinib (green) in the ATP pocket of JAK1. Dash lines indicate H-bond interactions between compounds and JAK1. (G) Immunoblot of JAK-1 phosphorylation in MDA-MB-231 and MDA-MB-468 cell lysates. Cells were treated with Roxyl-zhc-84 at different concentrations. Vorinostat is shown as a negative control.