Fig 1: DTX3L is the upstream activator of the LIPG-ISG15 signaling axis in basal-like TNBC cells.(A) DTX3L inactivation leads to a dramatic reduction in LIPG protein expression in MCF10DCIS and MDA-MB-468. Western blot analysis of DTX3L, LIPG and tubulin was performed on MCF10DCIS and MDA-MB-468 cells with or without DTX3L knockdown by two different siRNAs. (B) DTX3L knockdown results in a modest decrease in LIPG mRNA levels in MCF10DCIS and MDA-MB-468 cells. qRT-PCR analysis of LIPG mRNA expression was performed on siRNA-transfected cells as described in (A). (C) DTX3L deficiency shortens the half-life of LIPG protein in MDA-MB-468 cells. After treatment with the protein synthesis inhibitor cycloheximide (200 μg/ml), MDA-MB-468 cells with or without DTX3L knockdown were harvested at different time points as indicated for western blot analysis of LIPG and tubulin. Kinetic changes in LIPG protein levels during the time course are shown in the quantitative plot on the bottom panel. (D) DTX3L depletion facilitates proteasome-mediated LIPG protein degradation. Western blot analysis of DTX3L, LIPG and tubulin was performed on control and DTX3L-knockdown MDA-MB-468 cells with or without MG-132 treatment (10 μM) for 10 hr. The quantitative data are shown on the bottom panel. (E) The DTX3L-LIPG signaling axis is required for ISG15 expression in MCF10DCIS and MDA-MB-468 cells. Western blot analysis of DTX3L, LIPG, ISG15 and tubulin was performed on MCF10DCIS and MDA-MB-468 cells with or without DTX3L knockdown, and on their respective DTX3L-overexpressing cells with or without knockdown of either DTX3L or LIPG. (F) DTX3L is aberrantly overexpressed in breast cancer. DTX3L mRNA expression in normal breast and IDC was analyzed in silico using the TCGA and Curtis datasets. TCGA: breast (n = 61), IDC (n = 389); Curtis: breast (n = 144), IDC (n = 1556). The 25th and 75th percentiles are indicated as a vertical box and the minimal and maximal data values are indicated as outliers. (G) DTX3L protein expression positively correlates with LIPG expression in TNBC. IHC analysis of DTX3L and LIPG was performed on nine cases of TNBC. The linear regression correlation was plotted based on H-scores of DTX3L and LIPG IHC-stained TNBC cases. (H) DTX3L mRNA expression is positively associated with ISG15 expression in human basal-like breast cancers. The linear regression correlation was plotted based on in silico analysis of DTX3L and ISG15 expression in 45 cases of BLBCs derived from the Gluck dataset. Error, SD (n = 3); *p<0.05; **p<0.01; ***p<0.001.10.7554/eLife.31334.034Figure 7—source data 1.Source data for Figure 7.The source data file contains numerical data that were used to generate data graphs presented in sub-figures (7B, C, D) of Figure 7. 10.7554/eLife.31334.035Figure 7—source data 2.DTX3L expression in breast cancer.The source data file contains numerical data that were used to generate the data graph presented in Figure 7F. 10.7554/eLife.31334.036Figure 7—source data 3.DTX3L expression is positively associated with LIPG and ISG15.The source data file contains numerical data that were used to generate data graphs presented in sub-figures (7G, H) of Figure 7. 10.7554/eLife.31334.037Figure 7—source data 4.DTX3L expression in different molecular subtypes of breast cancer.The source data file contains numerical data that were used to generate the data graph presented in Figure 7—figure supplement 2.