Th NAC (100 mM, Sigma) overnight prior to antimycin A (250 nM, Sigma) treatment in neurobasal medium and images acquired at 0 h andCNS SIRT3 in AD Mitochondrial Stress12 h post-treatment. Fluorescence intensity was quantitated in 50 neurons using ImageJ.Lenti-mSIRT3iGFP GenerationLong-form mouse SIRT3 cDNA was cloned into pIRES2EGFP (Clontech) upstream of the IRES-GFP sequences. The mSIRT3-IRES-GFP construct was then inserted into a lentisynapsin plasmid (lenti-mSIRT3iGFP) and lentivirus generated and concentrated as described previously [25].Neuronal Death AnalysisPrimary 374913-63-0 custom synthesis hippocampal cultures expressing lenti-mSIRT3iGFP or lenti-GFP were treated with AA (250 nM). Time-lapse microscopy was used to acquire images of the cultures at 5 min intervals and fluorescent neuronal death recorded by morphological changes including cell rounding.Figure 17460038 S2 Expression of Sirt3 splice variants in response to AA treatment in mouse primary hippocampal neurons. TaqMan qPCR probes were designed to specifically measure expression of long-form or short-form Sirt3. PC12 cells were transfected with plasmids expressing either long-form or short-form SIRT3 to test the specificity of the probes. Sirt3 expression was measured using probes designed to bind to all Sirt3 splice forms (A), only long-form Sirt3 (B), or only short-form Sirt3 (C) n = 3. D/E/F Mouse primary hippocampal neurons were treated with AA (250 nM) for 12 h. Sirt3 mRNA expression was measured using the probe to measure either total Sirt3 (D), longform Sirt3 (E) or short-form Sirt3 (F). G Ratio of long-form Sirt3/ short-form Sirt3. n = 6. Student’s 11138725 t-test: **p,0.01, *p,0.05. (TIF) Figure S3 Lentiviral mouse Sirt3 over-expression. Neuronal-specific mouse Sirt3 over-expression increases neuronal longevity in the face of ROS augmentation. A Phase (left panel) and fluorescent (right panel) images of primary hippocampal neurons expressing the lenti-mSIRT3iGFP construct. B and C Sirt3 over-expression was measured in rat primary hippocampal cultures (n = 4) that had been transduced with either lenti-GFP, lenti-mSIRT3iGFP or untransduced (control). mRNA expression was measured by qPCR relative to 18S rRNA using a TaqMan probe specific for mouse Sirt3 (exogenous, B) or rat Sirt3 (endogenous, C). D Representative survival curve of neurons expressing either GFP or mSIRT3iGFP lentivirus and treated with AA (250 nM). E Mean time of death of neurons from D. (n = 34?48, ***P.0.0001) (TIF) Figure S4 Transcript stability and PM delay. No correlation between 18S rRNA or Sirt3 and Eno2 mRNA expression with respect to PM delay in human control and AD brain samples. (TIF) Table S1 Details of human cases studied.MiceMice were maintained on a 12-hour light/dark cycle with free access to water and mouse chow (2016 Teklad Global 16 Protein Rodent Diet, Harlan, UK). Studies were performed in accordance with the UK Animals (Scientific Procedures) Act and with approval of the University of FCCP Bristol Ethical Review Group. PDAPP mice have been described previously [20] and wild-type littermates were used as controls.Human Brain TissueBrain tissue was obtained from the Human Tissue Authoritylicensed South West Dementia Brain Bank, University of Bristol, with North Somerset and South Bristol Research Ethics Committee approval. Frozen tissue was dissected from the midfrontal and temporal neocortex (Brodmann areas 6 and 22).ImmunoblottingWestern Blot analysis was performed as previously described [25].(DOCX)Text S1 Extended materi.Th NAC (100 mM, Sigma) overnight prior to antimycin A (250 nM, Sigma) treatment in neurobasal medium and images acquired at 0 h andCNS SIRT3 in AD Mitochondrial Stress12 h post-treatment. Fluorescence intensity was quantitated in 50 neurons using ImageJ.Lenti-mSIRT3iGFP GenerationLong-form mouse SIRT3 cDNA was cloned into pIRES2EGFP (Clontech) upstream of the IRES-GFP sequences. The mSIRT3-IRES-GFP construct was then inserted into a lentisynapsin plasmid (lenti-mSIRT3iGFP) and lentivirus generated and concentrated as described previously [25].Neuronal Death AnalysisPrimary hippocampal cultures expressing lenti-mSIRT3iGFP or lenti-GFP were treated with AA (250 nM). Time-lapse microscopy was used to acquire images of the cultures at 5 min intervals and fluorescent neuronal death recorded by morphological changes including cell rounding.Figure 17460038 S2 Expression of Sirt3 splice variants in response to AA treatment in mouse primary hippocampal neurons. TaqMan qPCR probes were designed to specifically measure expression of long-form or short-form Sirt3. PC12 cells were transfected with plasmids expressing either long-form or short-form SIRT3 to test the specificity of the probes. Sirt3 expression was measured using probes designed to bind to all Sirt3 splice forms (A), only long-form Sirt3 (B), or only short-form Sirt3 (C) n = 3. D/E/F Mouse primary hippocampal neurons were treated with AA (250 nM) for 12 h. Sirt3 mRNA expression was measured using the probe to measure either total Sirt3 (D), longform Sirt3 (E) or short-form Sirt3 (F). G Ratio of long-form Sirt3/ short-form Sirt3. n = 6. Student’s 11138725 t-test: **p,0.01, *p,0.05. (TIF) Figure S3 Lentiviral mouse Sirt3 over-expression. Neuronal-specific mouse Sirt3 over-expression increases neuronal longevity in the face of ROS augmentation. A Phase (left panel) and fluorescent (right panel) images of primary hippocampal neurons expressing the lenti-mSIRT3iGFP construct. B and C Sirt3 over-expression was measured in rat primary hippocampal cultures (n = 4) that had been transduced with either lenti-GFP, lenti-mSIRT3iGFP or untransduced (control). mRNA expression was measured by qPCR relative to 18S rRNA using a TaqMan probe specific for mouse Sirt3 (exogenous, B) or rat Sirt3 (endogenous, C). D Representative survival curve of neurons expressing either GFP or mSIRT3iGFP lentivirus and treated with AA (250 nM). E Mean time of death of neurons from D. (n = 34?48, ***P.0.0001) (TIF) Figure S4 Transcript stability and PM delay. No correlation between 18S rRNA or Sirt3 and Eno2 mRNA expression with respect to PM delay in human control and AD brain samples. (TIF) Table S1 Details of human cases studied.MiceMice were maintained on a 12-hour light/dark cycle with free access to water and mouse chow (2016 Teklad Global 16 Protein Rodent Diet, Harlan, UK). Studies were performed in accordance with the UK Animals (Scientific Procedures) Act and with approval of the University of Bristol Ethical Review Group. PDAPP mice have been described previously [20] and wild-type littermates were used as controls.Human Brain TissueBrain tissue was obtained from the Human Tissue Authoritylicensed South West Dementia Brain Bank, University of Bristol, with North Somerset and South Bristol Research Ethics Committee approval. Frozen tissue was dissected from the midfrontal and temporal neocortex (Brodmann areas 6 and 22).ImmunoblottingWestern Blot analysis was performed as previously described [25].(DOCX)Text S1 Extended materi.
