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Site-directed mutagenesis Mutant forms of mito GCL refractory to the shRNA against GCL ( mito GCLmut) or catalytically inactive mito GCL ( mito GCL (E103A)) were generated using the site-directed mutagenesis QuikChange XL kit (Stratagene, La Jolla, CA, USA), followed by Dpn I digestion