Wild type mouse embryonic fibroblasts (MEF) vs Bax/BAK double knock out MEF treated with and without Nocodazole
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Gautier Stoll
PROVIDER: E-MTAB-10997 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKORM-BM- MEF): nuclei fragmented; mitochondrial membrane potential (M-bM-^HM-^FM-OM-^Hmito) depolarized; Ca2+M-BM- was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca2+] (Cacyto); caspase 3/7 was activated. DKORM-BM- MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONRM-BM- MEF). RNAseq analysis, qPCR and western blots showed that WT and DKORM-BM- MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), while DKONRM-BM- MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONRM-BM- MEF rendered them responsive to C12: M-bM-^HM-^FM-OM-^HmitoM-BM- depolarized, CacytoM-BM- increased and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control M-bM-^HM-^FM-OM-^Hmito, Ca2+M-BM- release from the ER and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin, staurosporine but activates Bax- and Bak-independent apoptosis in response to C12. Gene expression profiling of mouse embryo fibroblasts from WT and Bax/Bak double knock-out mice (C12 responsive and non-reponsive cell lines).
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Project description:Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKOR MEF): nuclei fragmented; mitochondrial membrane potential (∆ψmito) depolarized; Ca2+ was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca2+] (Cacyto); caspase 3/7 was activated. DKOR MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONR MEF). RNAseq analysis, qPCR and western blots showed that WT and DKOR MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), while DKONR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONR MEF rendered them responsive to C12: ∆ψmito depolarized, Cacyto increased and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control ∆ψmito, Ca2+ release from the ER and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin, staurosporine but activates Bax- and Bak-independent apoptosis in response to C12.
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