Deegan S, Saveljeva S, Logue SE, Pakos-Zebrucka K, Gupta S, Vandenabeele P, Bertrand MJ,Samali A. (2014) Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions. Autophagy. 2014;10(11):1921-36. doi: 10.4161/15548627.2014.981790. Figure S1. Analysis of the role of mitochondria in cells compromised in the intrinsic cell death pathway. (A) Casp9 +/+ and casp9 -/- MEFs were treated with 0.5 μm of Tg and 1 μg/ml of Tm for 24 h. CCCP was added for 6 h. Mitochondrial potential was analyzed by evaluating the amount of TMRE positive cells by flow cytometry. (B) Bax +/+ Bak1 +/+ and bax -/- bak1 -/- MEFs were treated with 0.5 μm of Tg for 48 and 72 h and harvested lysates were analyzed for the expression of cleaved CASP8. Actin was used as a loading control. (C) Casp9 +/+ were treated for with 0.5 μg/ml of Tm alone or in combination with Boc-D-FMK. casp9 -/- MEFs were treated for 72 h with 0.5 μg/ml of Tm. After indicated timepoints treatment was removed and colonies were left to form for 10 d. Boc-D-FMK was replenished for the first 3 d after the removal of the treatment. Colonies were stained with crystal violet. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD. 1
Figure S2. Validation of the blocking antibodies for the death receptor-induced cell death. (A) casp9 -/- MEFs were pretreated with 1 μg/ml of cycloheximide and 50 ng/ml of TNF blocking antibody, 100 ng/ml of FAS:Fc or 500 ng/ml of TNFSF10 blocking antibody in case of samples cotreated with blocking antibodies. One h later treatment with 100 ng/ml of TNF, 50 ng/ml of FAS or 50 ng/ml of SuperKillerTRAIL (TNFSF10) was performed. Cell viability was analyzed by propidium iodide exclusion measured by flow cytometry. (B) Casp9 +/+ MEFs were pretreated with 1 μg/ml of cycloheximide and 500 ng/ml of TNFSF10 blocking antibody for 1 h. 50 ng/ml of SuperKillerTRAIL (TNFSF10) was added for 24 h and cell viability was measured by propidium iodide (PI) staining. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD. 2
Figure S3. Autophagic flux in casp9 -/- MEFs deficient for autophagy genes. (A and B) casp9 -/- MEFs stably expressing pgipz or Atg5 shrna were treated with 20 μm chloroquine alone, 0.5 μg/ml Tm alone, or a combination of 20 μm chloroquine and 0.5 μg/ml of Tm for the indicated times and cell lysates immunoblotted for LC3 and actin. (C and D) casp9 -/- MEFs stably expressing plko or Atg7 shrna were treated with 20 μm chloroquine alone, 0.5 μg/ml Tm alone, or a combination of 20 μm chloroquine and 0.5 μg/ml of Tm for the indicated times and cell lysates immunoblotted for LC3 and actin. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD. 3
Figure S4. Knockdown of autophagy genes in Casp9 +/+ and casp9 -/- MEF cells has different outcomes on ER stress-induced cell death. Casp9 +/+ MEFs stably expressing pgipz or Atg5 shrna were generated and (A) treated with 0.5 μm Tg for 24 h after which RNA was extracted, Atg5 and Gapdh expression analyzed by RT-PCR. (B) Casp9 +/+ pgipz and Atg5 shrna MEFs were treated with 0.5 μg/ml of Tm for indicated times and cell viability analyzed by propidium iodide (PI) uptake. (C) casp9 -/- MEFs stably expressing plko or Atg7 shrna were generated via lentiviral transduction. RNA from plko and Atg7 shrna casp9 -/- MEFs was isolated and cdna expression of Atg7 and Gapdh analyzed by RT-PCR. (D) plko and Atg7 shrna casp9 -/- MEFs were treated for the indicated times with 0.5 μm Tg, cell lysates prepared and immunoblotted for cleaved CASP8, cleaved CASP3, and actin. (E) plko and Atg7 shrna casp9 -/- MEFs were treated for the indicated times with 0.5 μm Tg and cell viability analyzed by propidium iodide (PI) uptake. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD. 4
Figure S5. Inhibition of RIP1 kinase activity has no effect on ER stress-induced cell death in casp9 -/- MEFs. casp9 -/- MEFs were treated for indicated times with 0.5 μm Tg (A) and 0.5 μg/ml of Tm (B) alone or in combination with 20 μm of Necrostatin 1. Cell viability was evaluated by propidium iodide (PI) uptake. Results are representative of 3 independent experiments. Error bars represent the mean ± SD. 5
Figure S6. Apoptosome-compromised cells undergo cell death in response to genotoxic stress. Casp9 +/+ and casp9 -/- MEFs were treated for indicated times with 50 μm etoposide. (A) lysates immunoblotted for CASP9, CASP3, PARP and actin. (B) Cell viability was analyzed by PI uptake. (C) casp9 -/- MEFs were treated for indicated times with 50 μm etoposide after which CASP8 processing was assessed by immunoblotting. (D) plko and Casp8 shrna casp9 -/- MEFs were treated with 50 μm etoposide for the indicated times and cell viability analyzed by propidium iodide (PI) uptake. 6