Yamagata-1
Materials and Methods for Supplemental Data
Antibodies and chemicals
Anti-mitochondrial heat shock protein 70 (1:1000) monoclonal antibody was purchased from Affinity BioReagents. Anti-Flag M2 monoclonal antibody was obtained from Sigma. The anti-mouse Bcl-2 polyclonal antibody was raised by immunization of rabbit with recombinant GST-mouse Bcl-2 fusion protein. The IgG was purified with protein-A column, followed by purification with the affinity column on which the antigen was cross-linked. Alexa Fluor 488 conjugated anti-rabbit IgG and Alexa Fluor 568 conjugated anti-mouse IgG were purchased from Molecular Probes Inc. (Eugene, OR). CellTiter-Blue was purchased from Promega (Madison, WI).
Cell culture and transfection
Neuro2A were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum. Cells were transfected with siRNA using the lipofectamine 2000 according to the supplier’s protocol. At 24 hours after transfection with siRNA, cells were used for the experiments. Flag-tagged murine VDAC2 was cloned by PCR using Flag sequence-extended primer into a retroviral expression vector (pBabe-IRES-GFP).
Cell viability assay
Cells (1 105 per well) were seeded into 12-well dishes. After 24 hours, the cells were treated with 2 µg/ml of tunicamycin, or 5 µM thapsigargin. The CellTiter-Blue cell viability assay was performed according to the supplier’s protocols.
Immunofluorescence microscopy
For analysis of localization of Bcl-2, and Bcl-xL, cells were fixed in phosphate buffer with 4% paraformaldehyde for 20 min at room temperature. After washing with PBS, the cells were permeabilized with 0.1 % Triton X for 5 min at room temperature. After washing three times with PBS, permeabilized cells were incubated with primary antibodies, which were an anti-mitochondrial heat shock protein 70 mouse monoclonal antibody and an anti-Bcl-2 or an anti- Bcl-xL rabbit polyclonal antibodyovernight at 4OC. Next, the cells were washed three times more and were incubated with secondary antibodies, Alexa Fluor 568-labeled anti-mouse IgG and Alexa Fluor 488-labeled anti-rabbit IgG, for 1 hour at room temperature. After washing another three times, the cells were analyzed with an LSM510 META (Carl Zeiss).
Immunoprecipitation
Forimmunoprecipitation of Flag-VDAC2, the cell lysateswere incubated with an anti-Flag monoclonal antibodyor normal mouse IgG overnight at 4OC, followed by incubation with Dynabeads protein G (invitrogen). After washing, the beads were boiled in sample buffer for Western blotting.
Supplemental Figure Legends
Supple. Fig. 1. Requirement of VDAC2 for apoptosis in Bak-silenced cells.
WT and VDAC2 cells were silenced with siRNAs for the non-targeting control, Bax, or Bak, and then were incubated with 2 µg/ml of tunicamycin (a-c), 40 µM etoposide (d-f), 5 µM thapsigargin (g-i), and 3 µM paclitaxel (j-l). Data are shown as the mean ± SD (n=3, * P < 0.05 versus WT cells).
Supple. Fig. 2. Requirement of VDAC2 for cell death of Bak-silenced cells.
WT and VDAC2 cells were silenced with siRNAs for the non-targeting control, Bax, or Bak, and then were treated with 2 µg/ml of tunicamycin (TM) (a), or 5 µM thapsigargin (TG) (b)for the indicated periods. Cell viability assessed by CTB assay.
Supple. Fig. 3. Lack of a nonspecific effect on cell viability after silencing of Bax and Bak.
Bax and Bak cells were silenced with siRNAs for the non-targeting control, Bax, or Bak, and then were incubated with 2 µg/ml of tunicamycin for the indicated periods. Data are shown as the mean ± SD (n=3).
Supple.Fig. 4. Efficient reduction of Bax and Bak expression by siRNA treatment. WT, V2, and mV2 cells were silenced with the indicated siRNAs. Then cell lysates were subjected to immunoblot analysis with antibodies for Bak, Bax, and GAPDH (the loading control).
Supple. Fig. 5. No change in Bcl-2 localization by silencing of VDAC2.
WT and Bak cells were silenced with siRNAs for the non-targeting control or VDAC2, and then were immunostained with an anti-Bcl-2 polyclonal antibody (Green) and an anti-mitochondrial heat shock protein 70 monoclonal antibody (Red).
Supple. Fig. 6. No change in Bcl-xL localization by silencing of VDAC2.
WT and Bak cells were silenced with siRNAs for the non-targeting control or VDAC2, and then were immunostained with an anti- Bcl-xL polyclonal antibody (Green) and an anti-mitochondrial heat shock protein 70 monoclonal antibody (Red).
Supple. Fig. 7. Decreased apoptosis sensitivity in Neuro2A by silencing of both VDAC2 and Bak
Neuro2A cells were silenced with siRNAs for VDAC2, Bak, Bax, or control (a), and then were incubated with 100 µM etoposide (Eto) for 36 hours(b). Data are shown as the mean ± SD (n=5, * P < 0.05 versuscontrol/siBak).
Supple. Fig. 8. Interaction of VDAC2 and Bax in tunicamycin-treated cells.
(a, b) WT (a) or Bak(b) cells were treated with 2 µg/ml tunicamycin for the indicated periods. The cells were lysed, and subjected to immunoprecipitation with an anti-Bax polyclonal antibody and control antibody (NRI). Then the VDAC2 was detected by Western blotting with an anti-VDAC2 polyclonal antibody. (c) WT cells were transfected with Flag-VDAC2 DNA and treated with 2 µg/ml tunicamycin for 24 hours. The cell lysates were immunoprecipitated with anti-Flag antibody and Bax wasdetected by Western blotting. Note that Bax did not interact with Flag-VDAC2innon-treated (NT) cells. Normal mouse IgG (NMI) was negative control.