Fig 2: The DTX3L-LIPG-ISG15 signaling axis is essential for malignancies of LIPG-expressing TNBC cells in vitro and in vivo.(A) DTX3L knockdown inhibits growth, migration, invasion and CSC sphere formation of MCF10DCIS cells. The scale bars indicate 100 μm. (B) DTX3L knockdown increases EpCAM+ cells in MCF10DCIS. (C) DTX3L inactivation downregulates the expression of multiple stem cell genes in MCF10DCIS cells. (D) DTX3L depletion induces the decreased expression of multiple basal/EMT genes in MCF10DCIS cells. (E) DTX3L knockdown leads to E-cadherin upregulation and vimentin downregulation in MCF10DCIS cells. The scale bar indicates 25 μm. (F) DTX3L knockdown results in vimentin downregulation in MDA-MB-468 cells. The scale bar indicates 25 μm. (G) The LIPG-ISG15 axis mediates DTX3L overexpression-enhanced invasion of MDA-MB-468 cells. Transwell-based invasion analysis was performed on DTX3L-overexpressing MDA-MB-468 cells with or without siRNA-mediated knockdown of a gene component in the DTX3L-LIPG-ISG15 axis and parental control cells. (H) The LIPG-ISG15 axis mediates DTX3L overexpression-promoted secondary CSC sphere formation of MDA-MB-468 cells. Secondary CSC sphere formation analysis was performed on cells as described in (G). (I) The LIPG-ISG15 axis mediates DTX3L overexpression-induced increases in expression of stem cell and EMT genes in MDA-MB-468 cells. qRT-PCR analysis of three stem cell and three EMT genes was performed on cells as describe in (G). ¥ p<0.01 versus the control dataset (Vector + siControl); # p<0.05 versus the dataset of MDA-MB-468-DTX3L OE cells transfected with siControl (DTX3L OE +siControl). (J) The DTX3L-LIPG-ISG15 axis is required for in vivo tumor formation of MDA-MB-468 cells in nude mice. 1 × 106 of DTX3L-overexpressing MDA-MB-468 cells with or without shRNA-mediated knockdown of a gene component in the DTX3L-LIPG-ISG15 axis or parental control cells were transplanted into the mammary fat pads of nude mice for xenograft tumor formation. The picture of isolated tumors and plotted tumor growth curves are shown on the left and right panels, respectively. Error, SD (n = 3); *p<0.05; **p<0.01; ***p<0.001.10.7554/eLife.31334.039Figure 8—source data 1.Source data for Figure 8. The source data file contains numerical data that were used to generate data graphs presented in sub-figures (8B, C, D, G, H, I, J) of Figure 8.

Fig 3: Interruption of the PLpro-ISG15 binding interface by binding of chloroxine near Arg65 in a two-state manner. a) The representative conformations of State 1 and State 2 with the same coloring scheme as in Fig. 3a. Arg65 is individually colored magenta. b) The heavy-atom RMSD of chloroxine with State 1 as the reference and the distance between the center of mass of chloroxine and the CZ (the terminal carbon) atom of Arg65 side chain. c) Apo-PLpro, the structures of State 1 and State 2 aligned to the holo-PLpro (PLpro with ISG15 bound) structure. The region of the helix where Arg65 resides is zoomed and rotated 180°for a clearer view. ISG15 in the holo-PLpro structure is colored marine and shown as surface. The structures of apo-PLpro and holo-PLpro were taken from PDB 6XAA[12] and 6YVA [35], respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 4: Cas9-mediated BL3SSO tagging of the endogenous human ISG15 locus. (A) Schematic GFP BL3SSO template for N-terminus of the ISG15 locus. (B) FACS analysis of GFP-positive HEK293FT cells after IFN-α treated at 48 hours after transfection of Cas9-sgRNA RNP and BL3SSO. (C) Genomic PCR and Sanger sequencing of single FACS sorted GFP positive clones reveal the variable outcomes of HDR tagging categorized as bona fide HDR and improper HDR. The small pink block represents various mutations that likely terminate translation downstream. (D) GFP fluorescence of endogenously tagged ISG15 locus in selected HDR positive clones at 1 and 10 nM IFN-α treatment for 24 and 48 hours. (E) Western blot of endogenously tagged ISG15 protein in HDR positive HEK293FT cell clones. Arrowheads point to the bands that are specifically recognized by the antibody. The offset of the GFP-ISG15 fusion arrowheads is because only clones #1D4 and #1D6 express the fusion.

Fig 5: Two assays established for screening inhibitors targeting SARS-CoV-2 PLpro. a) Size exclusion chromatography curve of PLpro purification. In the inset (i) the PLpro protein band is present at ~36.8kD on 12% SDS-PAGE, (ii) the PLpro-C111S protein band is present at ~36.8 kD on 12% SDS-PAGE and the ISG15 protein band at ~9.5 kD. b) The schematic diagram for the PLpro protease activity-based assay for inhibitor screening. Cleavage of a fluorogenic peptide by PLpro will release a free AMC fluorophore with its fluorescence signal correlated to the protease cleavage kinetics. The cleavage kinetics will be changed upon inhibition of the protease activity by an inhibitor (i.e., a drug candidate). c) Dependence of reaction kinetics (shown as fluorescence changes) on PLpro concentrations at a constant concentration of ALKGG-AMC (125 μM). d) The schematic diagram for the competitive fluorescence polarization-based assay for inhibitor screening. The binding between inactive C111S mutant of SARS-CoV-2 PLpro with FITC labeled ISG15 will restrict the rotation of ISG15-FITC. This restriction will be indicated with higher fluorescence polarization signals. The competition between an inhibitor and PLpro for binding with ISG15-FITC will be reflected from the concentration dependence of the fluorescence polarization signal. e) Florescence polarization changes in the reaction between PLpro and ISG15-FITC. f) Florescence polarization changes in competition with an unlabeled ISG15.