Figure 2

Crosstalk among DTX3L, ARTD8 and ARTD9 mediates proliferation in PC3 cells. (A-F) Cell proliferation analyses of PC3-siMock, PC3-siDTX3L, PC3-siARTD8, PC3-siARTD9 single knockdown cells (A-C) and PC3-siDTX3L/siARTD8, PC3 siDTX3L/siARTD9 or PC3-siARTD9/siARTD8 double knockdown cells (D-F) in presence or absence of IFNγ (200 U/ml) was assessed by the trypan blue exclusion assay. Relative cell proliferation and cell numbers are presented as mean from three independent experiments performed in triplicates. All error bars represent the SE. Statistical analysis was performed using the Student's t test. *P < 0.05, **P < 0.001 and ***P < 0.0001. (G) Co-immunoprecipitation analyses of endogenous DTX3L and ARTD family members in PC3 cells. PC3 cells were stimulated for 1 h with or without IFNγ (200 U/ml) and endogenous DTX3L complexes were then co-immunoprecipitated, separated on SDS PAGE, blotted and subsequently probed with antibodies for DTX3L, ARTD1 (positive control), ARTD2, ARTD8 and ARTD9. (H) Interaction of endogenous ARDT9 and ARTD1 or ARTD8 is mediated by (mono)-ADP-ribosylation. PC3 cells were stimulated for 1 h with IFNγ (200 U/ml) and endogenous ARTD9-ARTDx complexes subsequently co-immunoprecipitated in presence or absence of 5 mM mono-ADP-ribose using an anti-ARTD9 antibody. Complexes were then separated on SDS PAGE, blotted and subsequently probed with antibodies against endogenous ARTD1 (positive control), ARTD2, ARTD8 and ARTD9. (I, J) Interaction between endogenous DTX3L and ARTD8 or ARTD9 is independent of (mono)-ADP-ribosylation. Endogenous DTX3L-ARTD8/9 complexes were co-immunoprecipitated from extracts of PC3 cells in presence or absence of 5 mM mono-ADP-ribose using either an anti-ARTD9 (I) or an anti-DTX3L (J) antibody. Complexes were then separated on SDS PAGE, blotted and subsequently probed with antibodies against endogenous ARTD8, ARTD9 and DTX3L.