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. 2018 Jun 6;37(14):e97072. doi: 10.15252/embj.201797072

Figure EV3. TRAF2 acts as a significant regulator of caspase‐2 activation across multiple scenarios, while its genetic ablation leads to compensation from TRAF3.

Figure EV3

  1. Casp2pro BiFC cells were transfected with TRAF2 siRNA for 48 h and then treated with 20 μM cisplatin, 50 μM etoposide, or 100 nM paclitaxel in the presence of 10 μM Q‐VD(OMe)‐OPh for 24 h. Caspase‐2 dimerization was assessed by flow cytometry. n = 3 independent experiments (means + s.e.m.).
  2. Caspase‐2 or TRAF2 was knocked down by siRNA in BT474 breast cancer cells for 48 h, followed by treatment with 40 μM cisplatin, 100 μM etoposide, or 100 nM paclitaxel for 72 h. Apoptosis was assessed by annexin V staining and flow cytometry. n = 4 independent experiments (means + s.e.m.).
  3. shNT or shTRAF2 #1 HeLa cells were treated with 20 μM cisplatin in the presence of 10 μM Q‐VD(OMe)‐OPh for 24 h. Cells were collected and incubated with BMH for protein crosslinking, and then, lysates were prepared and oligomerized caspase‐2 was detected by IB. Relative signal intensity of oligomerized caspase‐2 was quantified and indicated below each lane (signal was normalized to mock treatment of shNT).
  4. TRAF2 CRISPR‐KO HeLa cells were transfected with 50 ng pair of Casp2pro BiFC constructs and cultured for 24 h. Cells were treated with 20 μM cisplatin in the presence of 10 μM Q‐VD(OMe)‐OPh for 24 h, and caspase‐2 BiFC was assessed by flow cytometry. n = 4 independent experiments (means + s.e.m.).
  5. TRAF2 CRISPR‐KO HeLa cells were treated with 20 μM cisplatin for 24 h. Apoptosis was assessed by annexin V staining and flow cytometry. n = 3 independent experiments (means + s.e.m.).
  6. Lysates of TRAF2 CRISPR‐KO HeLa (left) or shTRAF2 #1 HeLa cells (right) were prepared, followed by IB. Relative band intensity of TRAF3 was quantified and indicated below each lane [signal was normalized to control (left) or shNT (right)].
  7. Lysates of several clones of TRAF2 CRISPR‐KO HeLa cells were prepared, followed by IB.
  8. Control or TRAF2 CRISPR‐KO HeLa cells were transfected with TRAF3 siRNA, followed by Casp2pro BiFC construct transfection. Cells were treated with 20 μM cisplatin in the presence of 10 μM Q‐VD(OMe)‐OPh for 24 h. Caspase‐2 BiFC was assessed by flow cytometry. n = 4 independent experiments (means + s.e.m.). *P < 0.01 (TRAF2KO #1), P < 0.05 (TRAF2KO #2) by unpaired two‐tailed t‐test. n.s.; not significant.

Source data are available online for this figure.