Fig 2: Pharmacological inhibition of STUB1 with expressed biologic phenocopies the genetic knockout. (a, b) Validation of the binding of synthetic peptides to STUB1 (aa25–aa153) by isothermal titration calorimetry (a) and thermal shift assay (b). Representative of three independent experiments (a). Data are mean ± s.d. of six replicates derived from three independent experiments (b). (c) Competitive fluorescence polarization assay. Synthetic peptides were assessed for their ability to compete with 15 nM of tracer peptide (5-FAM-SSGPTIEEVD) for binding to 1 µM STUB1 (aa25–aa153). Data are mean ± s.d. of six replicates derived from two independent experiments. (d) Design of the inhibitory biologic by grafting the peptide (SIWWPD) to the C-terminus of an mCherry2 (red) scaffold. The fused peptide blocks the function of the tetratricopeptide repeat domain (blue) of STUB1 (PDB code 2C2L) and inhibits its substrate binding. U-box domain (orange) which recruits the E2 ubiquitin-conjugating enzyme is not affected. (e) Generation of tumour cell lines stably expressing the biologic or its control. Plasmid encoding the biologic was electroporated into tumour cells, followed by antibiotics selection of the stable clones. The mCherry2-positive cells (red dotted box) were further gated for mCherry2hi population (top 50th percentile, red box). Gating example represents IFN?-treated B16-F10 stable cell lines. (f, g) Flow cytometry analysis of the relative cell surface level of IFNGR1 (f) and MHC-I (g) expressed by the mCherry2hi population in B16-F10, A375 or A549 cells. The cells were either untreated or treated with mouse IFN? (0.03 ng ml-1) or human IFN? (0.01 ng ml-1) for 24 h. The expression levels were normalized to the average value of the control (mCherry2-SIWWHR). n = 5 biological replicates from two independent experiments (f–g). Bars are mean with all data points (f–g). P values were determined by ordinary two-way ANOVA in each cell type with Sidak’s multiple comparisons test, ****P = 0.0001 (f–g). (h) Co-immunoprecipitation (co-IP) of FLAG-mCherry2-peptide and STUB1 from the cellular lysate of B16-F10 using anti-FLAG antibody. Synthetic peptide (SIWWPD) was added into the co-IP mixture to assess specificity of the interaction. Blot is representative of three independent experiments.

Fig 3: Stub1 dampens IFN? sensing by downregulating IFNGR1. (a–c) Flow cytometry analysis of cell surface IFNGR1 on parental, control or independent Stub1-null B16-F10 cells which were either untreated (Nil) or treated with IFN? for 24 h. See Supplementary Fig. 3a for additional plots. (d) Flow cytometry analysis of the surface level of other cytokine receptors on the tumour cells. See Supplementary Fig. 3b–c for the plots. (e) Heatmap showing genes (Supplementary Table S3) being upregulated by > twofold in both gStub1 #1 and #2 cells relative to untreated gControl cells. The cells were treated with 0.03 ng ml-1 IFN? for 6 or 24 h. See Supplementary Fig. 4a for the full heatmap. FC, fold change. (f) Volcano plot showing differential protein expression in gStub1 #2 versus gControl cells, following stimulation with 0.03 ng ml-1 IFN? for 24 h. Red or blue circles highlight proteins significantly enriched in gStub1 #2 or gControl cells respectively (twofold cutoff, adjusted P = 0.05; n = 6 replicates per cell group, 3 biological replicates × 2 MS replicates). See Supplementary Fig. 4e for data of gStub1 #1 cell. (g) MS proteomics uncovered 13 proteins commonly enriched in both gStub1 #1 and #2 cells. The overlapping proteins are explicitly labeled in panel (f). (h) Proposed model whereby Stub1 is an intracellular checkpoint that curbs the tumour cells’ ability to sense and respond to IFN? by downregulating IFNGR1. Representative of three independent experiments (a). Data are mean ± s.d. (b) or mean with all data points (c) from three independent experiments. Data are mean with all data points from four independent experiments (d). P values were determined by ordinary two-way ANOVA (c) or one-way ANOVA (d) on Log2-transformed data with Dunnett’s multiple comparisons test, ****P = 0.0001.

Fig 4: Inactivation of STUB1 or PTPN2 sensitized human tumour cells to growth inhibition induced by cytokines. (a) Western blot analysis of the expression level of PTPN2 and STUB1 in tumour cells. (b) Flow cytometry plot showing the surface expression level of IFNGR1 in tumour cells. In parallel, parental cells were stained with PE-conjugated isotype control antibody to demonstrate low level of non-specific binding. (c) Level of phosphorylated Tyr701-STAT1 after 30-min response to varying doses of IFN? as measured by Lumit immunoassay. (d) Fold change (FC) in ATP level relative to the parental cells as a quantification of viable cells. Measurements were performed using CellTiter-Glo 2.0 assay after 6-day treatment with the cytokines (10 ng ml-1 each). Data are mean ± s.d. from two biological replicates (c) or mean ± s.e.m. from three biological replicates (d). P values were determined by ordinary two-way ANOVA on Log2-transformed data with Dunnett’s multiple comparisons test versus parental cells, **P = 0.01, ****P = 0.0001, ns P > 0.90 (d). Representative of two independent experiments (a–d).