Journal: Molecular Cancer

Article Title: DTX3L and ARTD9 inhibit IRF1 expression and mediate in cooperation with ARTD8 survival and proliferation of metastatic prostate cancer cells

doi: 10.1186/1476-4598-13-125

Figure Lengend Snippet: DTX3L is constitutively overexpressed together with ARTD8 and ARTD9 in mPCa associated with increased IFNγ/STAT1 signaling. (A) Immunoblot analyses of untreated p53-negative, mPCa cell lines PC3 and DU145, androgen-sensitive and JAK1-negative LNCaP cell line and of the immortalized normal prostate luminal epithelial cell lines HPE and RWPE1. The HR-DLBCL-SUDHL7 cell line constitutively expressing DTX3L, ARTD9 and ARTD8 was used as a positive control. Whole cell extracts were separated by SDS PAGE, blotted and subsequently probed with antibodies for DTX3L, ARTD1, ARTD8 and ARTD9 pSTAT1(Y701) and tubulin. (B) Immunoblot analyses of STAT1-signaling in p53-negative mPCa cell lines PC3 and DU145 and in the androgen-sensitive and JAK1-negative LNCaP cell line treated with or without IFNγ or IFNαβ. PC3, DU145 and LNCaP cells were treated with or without IFNγ (200 U/ml) or IFNαβ (50 U/ml each) for 2 h and then whole cell extracts separated by SDS PAGE and subsequently probed with antibodies for DTX3L, ARTD9, STAT1, pSTAT1(Y701), pSTAT1(S727) and tubulin. The immunoblots are representative of at least three independent experiments. (C) Immunofluorescence microscopy analyses and sub-cellular localization of endogenous STAT1, pSTAT1-(pY701) and pSTAT1-(pS727) in PC3 cells, in presence or absence of 1000 U/ml IFNγ. Original magnification × 400. Images are representative of at least three independent experiments. (D) Immunoblot analyses of basal expression levels of IRF1 in PC3, DU145 and LNCaP cell lines. Whole cell extracts were separated by SDS PAGE and subsequently probed with antibodies for IRF1 and tubulin. The immunoblot is representative of at least three independent experiments. (E) Immunofluorescence microscopy analyses and sub-cellular localization of endogenous DTX3L, ARTD8 and ARDT9 in PC3 cells, in presence or absence of 1000 U/ml IFNγ. Original magnification × 400. Images are representative of at least three independent experiments.

Article Snippet: : 7932–1), anti-IRF1 (RabMab, Cell Signaling Technology, Cat.No.

Techniques: Western Blot, Expressing, Positive Control, SDS Page, Immunofluorescence, Microscopy

Journal: Molecular Cancer

Article Title: DTX3L and ARTD9 inhibit IRF1 expression and mediate in cooperation with ARTD8 survival and proliferation of metastatic prostate cancer cells

doi: 10.1186/1476-4598-13-125

Figure Lengend Snippet: DTX3L and ARTD9 repress tumor suppressor IRF1 expression in mPCa cells. (A and B) Immunoblot analyses of the tumor suppressor gene product IRF1. PC3-siMock, PC3-siDTX3L (A) or PC3-siARTD9 (B) single knockdown cells were treated with or without IFNγ (200 U/ml) for 6 h and then whole cell extracts separated by SDS PAGE, blotted and subsequently probed with antibodies for ARTD9, DTX3L, IRF1, IRF7 and tubulin. The immunoblots are representative of at least three independent experiments. (C) Quantification of IRF1 levels shown in Figure A, B. IRF1 levels were normalized to tubulin. Values represent the means of three independent experiments. (D) Quantification of ARTD9 and DTX3L protein levels in PC3-siMock, PC3-siDTX3L and PC3-siARTD9 single knockdown, respectively, as represented in Figure A, B. ARTD9 and DTX3L protein levels were normalized to tubulin. Values represent the means of three independent experiments. (E) Cell proliferation analyses of PC3-CMVprom-empty-control and PC3-CMVprom-IRF1 cells were assessed in presence or absence of IFNγ (100U/ml) by the trypan blue exclusion assay. Relative cell proliferation and cell numbers are presented as means of three independent experiments performed in triplicates. (F) Cell proliferation analyses of PC3-siMock, PC3-siIRF1 and PC3-siARTD9 single knockdown cells were assessed in presence or absence of IFNγ (200 U/ml) by the trypan blue exclusion assay. Relative cell proliferation and cell numbers are presented as means of three independent experiments performed in triplicate. (G) Cell proliferation analyses of LNCaP-siMock and LNCaP-siIRF1 single knockdown cells were assessed by the trypan blue exclusion assay. Relative cell proliferation and cell numbers are presented as means of two independent experiments performed in triplicate. All error bars shown in A to G represent the SE. Statistical analysis was performed using the Student's t test. * P < 0.05, ** P < 0.001 and *** P < 0.0001.

Article Snippet: : 7932–1), anti-IRF1 (RabMab, Cell Signaling Technology, Cat.No.

Techniques: Expressing, Western Blot, SDS Page, Trypan Blue Exclusion Assay

Journal: Molecular Cancer

Article Title: DTX3L and ARTD9 inhibit IRF1 expression and mediate in cooperation with ARTD8 survival and proliferation of metastatic prostate cancer cells

doi: 10.1186/1476-4598-13-125

Figure Lengend Snippet: Proposed working models for the postulated crosstalk among DTX3L, ARTD8 and ARTD9 in chemotherapy-resistant mPCa cells. (A) Constitutively active IL6/STAT3-signaling and enhanced IFNGR-JAK1/2-STAT1- signaling in chemotherapy-resistant mPCa cells, including CRPC-like cells causes overexpression of DTX3L and ARTD9, which in turn, further stimulates their own expression through a positive feedback loop. (B) Crosstalk between DTX3L/ARTD9-STAT1 and ARTD8-STAT6-signaling pathways is required for proliferation and cell survival of chemotherapy-resistant mPCa cells, including CRPC-like cells. (C) Similar to the situation in HR-DLBCL, DTX3L and ARTD9, together with STAT1β repress the transcriptional activation of the tumor suppressor IRF1 and other anti-proliferative and pro-apoptotic genes while together with STAT1α both, DTX3L and ARTD9 might activate genes required for cell proliferation and survival of mPCa cells. (D) Overexpression of DTX3L but not ARTD8 or ARTD9 also mediates cell migration of mPCa cells, dependent on STAT1 and STAT3-signaling. DTX3L might form migration-specific, ARTD9-independent STAT1 homodimer or non-canonical STAT1/STAT3 heterodimer complexes.

Article Snippet: : 7932–1), anti-IRF1 (RabMab, Cell Signaling Technology, Cat.No.

Techniques: Over Expression, Expressing, Activation Assay, Migration