Siva1 is a XIAP-interacting protein that balances NFB and JNK signalling to promote apoptosis

JCS049940 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) HEK293 cells were transfected with increasing amounts of FLAG-Siva1 and stimulated with TNFα (20 ng/ml) for the indicated times. Siva was precipitated from whole cell extracts with anti-FLAG−matrix, and the presence of endogenous XIAP was assessed by western blotting (IP, upper panel). Higher molecular mass species of Siva1 in the precipitate were detected after 4 and 8 hours stimulation (panel below, arrows) with α-Siva antibody. Expression levels of endogenous XIAP and FLAG-Siva1 (short and long exposure), and β-actin, used as loading control, are shown in the lower panels. *An unspecific band detected by the FLAG antibody. Note that higher molecular mass forms of Siva1 are also detected in the input after long exposure. Numbers on the right of the panels are molecular masses in kilodaltons. (B) Immunoprecipitation (IP) of the endogenous proteins in SW480 cells. Precipitations were done with control IgG (c) or anti-Siva (α-Siva) followed by detection using anti-XIAP antibody. Numbers on the right of the panels are molecular masses in kilodaltons. (C) Siva1 interacts with XIAP in Jurkat T cells. Whole cell extracts from unstimulated (0) or PMA/Ionomycin (PMA/Iono) stimulated (120 or 240 minutes) cells were prepared and incubated with either control goat IgG or Siva antibodies. Precipitates were immunoblotted and membranes probed with XIAP antibodies (IP, upper panel). Expression levels of XIAP and Siva are shown in the Input (lower two panels). Note that Siva1 levels increase upon stimulation. (D) Siva1 does not interact with a RING-deletion construct of XIAP. HEK293 cells were transfected with empty vector (EV), XIAP or a RING-deleted XIAP construct (ΔRING) in the absence (-) or presence of FLAG-Siva1 (+). Siva1 was immunoprecipitated using anti-FLAG beads, and the presence of XIAP or ΔRING was assessed by western analysis using XIAP antibodies (IP, upper panel). Expression levels of XIAP, the ΔRING construct and FLAG-Siva1 in the cell extracts (Input) is shown in the lower two panels.

• Supplemental Figure S2 -

  Fig. S2. (A) Siva1 and the deletion construct SivaΔC containing the SAH domain and the death domain homology region (DDHR) similarly reduce XIAP and TAK1-TAB1-induced NF-κB activity. HEK293 cells were transfected with a 5xNF-κB-luc reporter and increasing amounts of FLAG-Siva1 or FLAG- SivaΔC along with empty vector (EV), XIAP or TAK1-TAB1 (each 0.25 mg), and luciferase activity normalized to co-transfected UBT-β-Gal was measured 48 hours after transfection. Fold induction was calculated in relation to EV transfection without Siva1 or SivaΔC. (B) Siva1 reduces TNFα-induced NF-κB activity. HEK293 cells were transfected with a 5xNF-κB-luc reporter along with increasing amounts of FLAG-Siva1 and stimulated with TNFα (20ng/ml) for 16 hours. Fold induction was calculated in relation to unstimulated, EV-transfected cells. (C) Inhibition of TNFα-induced NF-κB activity by Siva1 is independent of caspase activation. HEK293 cells were transfected with a 5xNF-κB-luc reporter along with increasing amounts of FLAG-Siva1. 12 hours post-transfection cells were treated with the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone (zVAD-fmk, 20 µM in DMSO) or vehicle and 30 minutes later stimulated with TNFα (20 ng/ml). At 36 hours post-transfection cells were lysed and luciferase activity was measured.

• Supplemental Figure S3 -

  Fig. S3. (A) Siva1 enhances AP1 activity independent of the E3 ubiquitin ligase of XIAP. HEK293 cells were transfected with an AP1-luc reporter (1 µg), in the absence (EV) or presence of XIAP or the E3 ubiquitin ligase mutant XIAP_H467A with or without (mock) Siva1. Fold induction was calculated in relation to empty vector (EV)-transfected cells without Svia1. (B) Knock down of Siva1 reduces AP1 activity induced by XIAP overexpression. Jurkat T cells stably expressing lentiviral-delivered siSiva or control siRNA were transfected with an AP1-luc reporter, UBT-β-Gal along with XIAP or empty vector (EV). Luciferase activity was normalized to β-Gal levels.

• Supplemental Figure S4 -

  Fig. S4. Siva1 is a target for XIAP-mediated, lysine-48 linked ubiquitiniation. HEK293 cells were transfected with wild-type (WT) or mutated versions of HA-tagged ubiquitin (K48R, K63R) along with FLAG-Siva1 or in combination with XIAP or XIAP_H467A. Cells were lysed after 24 hours, Siva1 was immunoprecipitated with anti-FLAG−matrix and subjected to western analysis. Polyubiquitinated Siva1 was detected with α-HA antibody (upper panel) and expression levels of Siva1 and XIAP (inputs) are shown in the lower two panels. *An unspecific band; numbers on the right of the panels are molecular masses in kilodaltons.