Journal: PLoS ONE

Article Title: Opposing Effects of Sirtuins on Neuronal Survival: SIRT1-Mediated Neuroprotection Is Independent of Its Deacetylase Activity

doi: 10.1371/journal.pone.0004090

Figure Lengend Snippet: (A) Sirtuin family expression and cerebellar granule neuron survival. Expression plasmids encoding Flag-tagged forms of SIRT 1–7 were transfected into CGNs and the cultures were switched to HK or LK medium. The proportion of transfected neurons that were apoptotic was quantified 24 hrs after the switch and compared with control cultures which were transfected with CMV-GFP. Viability for transfected cells was determined using DAPI staining of chromatin (*indicates p<.05 compared with GFP HK (control); # indicates p<.05 compared with GFP LK). (B) Expression of other SIRT1 constructs in neurons confirms neuroprotection. Neurons were transfected with Flag-SIRT1 (tag on the N-terminus), HA-SIRT1, or Trunc-SIRT1-Flag and switched to HK or LK medium. The proportion of transfected neurons that were apoptotic was quantified 24 hrs after the switch and compared with control cultures which were transfected with CMV-GFP. Apoptosis was determined for transfected cells using DAPI staining (* indicates p<.05 compared with GFP LK; control = GFP HK). (C) SIRT5 localization and CGNs survival. SIRT5 localization in CGNs was determined by immunocytochemistry following SIRT5-Flag transfection and viability assessed using DAPI staining 24 after HK and LK treatment. The localization of viable and apoptotic SIRT5-Flag expressing cells was scored as nuclear and cytoplasmic or mitochondrial (* indicates p<.05 compared with nuclear-cytoplasmic).

Article Snippet: Expression plasmids for Flag-tagged SIRT1–SIRT7, HA-tagged SIRT1, Flag-tagged SIRT1-H363Y and Flag-tagged SIRT-G261A were purchased from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Transfection, Staining, Construct, Immunocytochemistry

Journal: PLoS ONE

Article Title: Opposing Effects of Sirtuins on Neuronal Survival: SIRT1-Mediated Neuroprotection Is Independent of Its Deacetylase Activity

doi: 10.1371/journal.pone.0004090

Figure Lengend Snippet: Expression plasmids encoding Flag-tagged forms of SIRT 1–7 were transfected into CGNs and the cultures were switched to HK or LK medium. The localization of each sirtuin was visualized by immunocytochemistry using a Flag antibody. DAPI staining was used to label the nucleus. In some cases, Cox4 antibody was used to visualize mitochondria. Images were obtained with a Nikon Eclipse 80i using a 60× objective. (A) Subcellular localization of SIRT1, SIRT2, SIRT6, and SIRT7 in HK and LK-treated neuronal cultures. Non-apoptotic cells were used for SIRT2 and SIRT6 in HK to show localization. (B) Mitochondrial localization of SIRT3 and SIRT4. The punctate pattern of SIRT3 and SIRT4 correspond to mitochondrial localization as evidenced by the overlap with Cox4 immunostaining. (C) Subcellular localization of SIRT5 in CGNs. In HK treatment SIRT5 predominately localizes to the cytoplasm, mitochondria, and nucleus (HK panel) while in LK SIRT5 can localize to the cytoplasm, mitochondria, and nucleus (upper LK panel) or to mitochondria (lower LK panel). Mitochondrial localization of SIRT5 is associated with apoptosis.

Article Snippet: Expression plasmids for Flag-tagged SIRT1–SIRT7, HA-tagged SIRT1, Flag-tagged SIRT1-H363Y and Flag-tagged SIRT-G261A were purchased from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Transfection, Immunocytochemistry, Staining, Immunostaining

Journal: PLoS ONE

Article Title: Opposing Effects of Sirtuins on Neuronal Survival: SIRT1-Mediated Neuroprotection Is Independent of Its Deacetylase Activity

doi: 10.1371/journal.pone.0004090

Figure Lengend Snippet: (A) Neurons treated with nicotinamide. CGNs were transfected with Flag-tagged SIRT1 or GFP control vector and the cultures switched to LK medium or LK-medium supplemented with 5 mM nicotinamide. Viability of transfected neurons (detected by Flag immunocytochemistry) was quantified 24 hours later (* indicates p<.05 compared with GFP LK; # indicates p<.05 compared with GFP HK). (B) Sirtinol does not affect SIRT1 protection. Flag-tagged SIRT1 and GFP vectors were transfected into CGNs. The neurons were then treated with LK medium or LK-medium supplemented with 100 µM sirtinol. Survival for SIRT1-transfected neurons was assessed using immunocytochemistry and DAPI staining 24 hours following treatment (* indicates p<.05 compared with GFP LK; # indicates p<.05 compared with GFP HK). (C) Resveratrol cannot rescue neurons in LK. Cultured CGNs were switched to HK medium, LK medium, or LK medium containing 10 or 30 µM resveratrol. Neuronal viability was quantified 24 hours later by DAPI staining.

Article Snippet: Expression plasmids for Flag-tagged SIRT1–SIRT7, HA-tagged SIRT1, Flag-tagged SIRT1-H363Y and Flag-tagged SIRT-G261A were purchased from Addgene (Addgene, Cambridge, MA).

Techniques: Transfection, Plasmid Preparation, Immunocytochemistry, Staining, Cell Culture

Journal: PLoS ONE

Article Title: Opposing Effects of Sirtuins on Neuronal Survival: SIRT1-Mediated Neuroprotection Is Independent of Its Deacetylase Activity

doi: 10.1371/journal.pone.0004090

Figure Lengend Snippet: CGNs were transfected with vectors encoding GFP, Flag-tagged wild-type form of SIRT1, or the deacetylase-dead mutation forms of SIRT1, H363Y or H355A. The cultures were then switched to HK or LK medium for 24 hours and viability of transfected neurons was quantified by DAPI staining (* indicates p<.05 compared with GFP LK).

Article Snippet: Expression plasmids for Flag-tagged SIRT1–SIRT7, HA-tagged SIRT1, Flag-tagged SIRT1-H363Y and Flag-tagged SIRT-G261A were purchased from Addgene (Addgene, Cambridge, MA).

Techniques: Transfection, Histone Deacetylase Assay, Mutagenesis, Staining

Journal: PLoS ONE

Article Title: Opposing Effects of Sirtuins on Neuronal Survival: SIRT1-Mediated Neuroprotection Is Independent of Its Deacetylase Activity

doi: 10.1371/journal.pone.0004090

Figure Lengend Snippet: (A) Effect of sirtuin expression on HT-22 cell survival. Sirtuin expression levels were enhanced in HT-22 neuroblastoma cells via transfection with mammalian expression plasmids. HT-22 cells were treated 24 hours following transfection with normal growth medium (untreated), 1 mM HCA or Serum-free (SF) medium for 24 hours. The viability of transfected cells was determined using a TUNEL assay. The mean of 5 experiments were taken (* indicates p<.05 compared with Flag untreated sample; # indicates p<.05 compared with Flag HCA sample; ˆ indicates p<.05 compared with Flag SF sample). (B) Subcellular localization of exogenous SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6 in HT-22 cells. HT-22 cells were transfected with SIRT-Flag constructs and immunocytochemistry performed using a Flag antibody. DAPI staining was used to visualize nuclei/chromatin. SIRT5 shows colocalization with the mitochondrial protein Cox4.

Article Snippet: Expression plasmids for Flag-tagged SIRT1–SIRT7, HA-tagged SIRT1, Flag-tagged SIRT1-H363Y and Flag-tagged SIRT-G261A were purchased from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Transfection, TUNEL Assay, Construct, Immunocytochemistry, Staining

Journal: Frontiers in Chemistry

Article Title: Macrocycle-Antibiotic Hybrids: A Path to Clinical Candidates

doi: 10.3389/fchem.2021.659845

Figure Lengend Snippet: Chemical structures of VAN hybrids, cefilavancin ( 3 , TD-1792), and TD-1607 ( 4 ).

Article Snippet: TD-1792 , 1,984.28 , −4.68 , 673.39 , 0.03 , MRSA, hVISA , 0.19 , 1.7 , Theravance biopharma/R-pharm , IV , Phase 2 completed in 2007 ; phase 3 registered in Russia .

Techniques: