constitutive promoter  (Vector Biolabs)

Journal: Glia

Article Title: Lactate Dehydrogenase Inhibition Reverts the Fatty Acid‐Induced Neurotoxic Phenotype of Astrocytes

doi: 10.1002/glia.70136

Figure Lengend Snippet: Fatty acid treatment induces a proinflammatory phenotype in astrocytes. Relative luminescence produced by firefly luciferase expressed under the control of an NF‐κB‐driven promoter after 24 h of treatment with (A) oleic acid (OA, 50 and 100 μM) and (B) linoleic acid (LA, 50 and 100 μM), in nontransgenic neonatal spinal cord astrocytes cultures. Control cultures were treated with BSA (used as a carrier for FAs). Relative firefly luciferase luminescence was corrected by the amount of Renilla luciferase activity controlled by a constitutive promoter and expressed as a percentage of vehicle (BSA)‐treated cultures ( n = 3, mean ± SD). (C–F) Representative images depicting immunostaining against NF‐κB‐p65 (red) and GFAP (green) in neonatal spinal cord astrocytes 4 h after treatment with (C) OA (50 μM) or (E) LA (50 μM). Nuclei were counterstained with DAPI (blue). Scale bar: 25 μm. Quantification of NF‐κB‐p65‐positive nuclei in neonatal spinal cord astrocytes 4 h after treatment with (D) OA (50 μM) or (F) LA (50 μM). Data are expressed as NF‐κB‐p65 positive nuclei over the total number of nuclei analyzed, percentage of vehicle (BSA)‐treated cultures ( n = 5, mean ± SD). (G and H) ELISA quantification of CXCL10 (G) and TNFα (H) levels in conditioned media from astrocytes treated with OA (50 μM) and LA (50 μM). Data are expressed as percentage of vehicle (BSA)‐treated control cultures ( n = 2, 5 treatment replicates per experiment, mean ± SD). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Adenovirus expressing a firefly luciferase gene under the control of a synthetic promoter that contains direct repeats of the NF‐κB binding site (Ad‐NFkb‐Luc) or a Renilla luciferase under a constitutive promoter (Ad‐pRL‐Luc) were obtained from Vector Biolabs.

Techniques: Produced, Luciferase, Control, Activity Assay, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Glia

Article Title: Lactate Dehydrogenase Inhibition Reverts the Fatty Acid‐Induced Neurotoxic Phenotype of Astrocytes

doi: 10.1002/glia.70136

Figure Lengend Snippet: Lactate dehydrogenase inhibition reverts the effect of fatty acid treatment in neonatal spinal cord astrocytes. Neonatal spinal cord astrocytes were treated with vehicle (BSA) or fatty acids for 24 h, followed by treatment with vehicle (DMSO) or GSK2837808A (2.5 μM, LDHi) for 24 h. Representative images of LD staining (LipidGreen2) in spinal cord astrocyte cultures treated with (A) oleic acid (OA, 50 μM) in the presence or absence of LDHi (2.5 μM) and (B) linoleic acid (LA, 50 μM) in the presence or absence of LDHi (2.5 μM). Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (C) Quantification of LD in spinal cord astrocytes treated as indicated above. LD numbers were normalized by the number of nuclei analyzed ( n = 4, mean ± SD). (D) Relative luminescence produced by firefly luciferase expressed under an NF‐κB‐driven promoter in cultures treated as indicated above. Relative firefly luciferase luminescence was corrected by the amount of Renilla luciferase activity controlled by a constitutive promoter and expressed as percentage of cultures treated with vehicle (BSA and DMSO) ( n = 3, mean ± SD). (E and F) Motor neuron survival determined 72 h after being plated on top of astrocytes treated as indicated above ( n = 4, mean ± SD). (G) Representative images of LD staining (LipidGreen2) in spinal cord astrocyte cultures treated with OA (50 μM) in the presence or absence of LDHi (2.5 μM), Etomoxir (10 μM, Eto), or both combined (Eto + LDHi). Neonatal spinal cord astrocytes were treated with vehicle (BSA) or fatty acids for 24 h, followed by treatment with vehicle (DMSO) or GSK2837808A (2.5 μM, LDHi) and/or Etomoxir (10 μM) for 24 h. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (H) Quantification of LD in spinal cord astrocytes treated as indicated above. LD numbers were normalized by the number of nuclei analyzed ( n = 3, mean ± SD). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Adenovirus expressing a firefly luciferase gene under the control of a synthetic promoter that contains direct repeats of the NF‐κB binding site (Ad‐NFkb‐Luc) or a Renilla luciferase under a constitutive promoter (Ad‐pRL‐Luc) were obtained from Vector Biolabs.

Techniques: Inhibition, Staining, Produced, Luciferase, Activity Assay

Journal: Glia

Article Title: Lactate Dehydrogenase Inhibition Reverts the Fatty Acid‐Induced Neurotoxic Phenotype of Astrocytes

doi: 10.1002/glia.70136

Figure Lengend Snippet: Lactate dehydrogenase gene ablation reverts the effect of fatty acid treatment in neonatal spinal cord astrocytes. (A) Primary confluent spinal cord astrocyte cultures obtained from neonatal Ldha flox/flox mice were transduced with an adenovirus expressing Cre recombinase (CRE) or an empty ORF (NULL). Seventy‐two hours later lactate dehydrogenase A (LDHA) expression was analyzed by Western blot. (B) LDHA expression was quantified, normalized by actin levels, and expressed as a percentage of NULL‐treated cultures ( n = 3, mean ± SD). (C) Ldha flox/flox spinal cord astrocyte cultures treated as above were subsequently exposed to oleic acid (OA, 50 μM) for 24 h. Representative images of LD staining (LipidGreen2). Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (D) Quantification of LD in spinal cord astrocytes treated as indicated in (C). LD numbers were normalized by the number of nuclei analyzed ( n = 3, mean ± SD). (E) Relative luminescence produced by firefly luciferase expressed under an NF‐κB‐driven promoter in cultures treated as indicated in (C). Relative firefly luciferase luminescence was corrected by the amount of Renilla luciferase activity controlled by a constitutive promoter and expressed as percentage of control cultures (NULL/BSA) ( n = 4, mean ± SD). (F) Motor neuron survival determined 72 h after being plated on top of Ldha flox/flox spinal astrocytes treated as indicated in (C) ( n = 4, mean ± SD). **** p < 0.0001, ** p < 0.01, *p < 0.05.

Article Snippet: Adenovirus expressing a firefly luciferase gene under the control of a synthetic promoter that contains direct repeats of the NF‐κB binding site (Ad‐NFkb‐Luc) or a Renilla luciferase under a constitutive promoter (Ad‐pRL‐Luc) were obtained from Vector Biolabs.

Techniques: Transduction, Expressing, Western Blot, Staining, Produced, Luciferase, Activity Assay, Control

Journal: Glia

Article Title: Lactate Dehydrogenase Inhibition Reverts the Fatty Acid‐Induced Neurotoxic Phenotype of Astrocytes

doi: 10.1002/glia.70136

Figure Lengend Snippet: Lactate dehydrogenase inhibition ameliorates the neurotoxic phenotype of spinal cord astrocytes isolated from symptomatic hSOD1 G93A mice. (A) Representative LD staining (LipidGreen2) in spinal cord astrocyte cultures from symptomatic hSOD1 G93A (G93A) mice treated with GSK2837808A (2.5 μM, LDHi) or vehicle (DMSO) for 24 h. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (B) Quantification of LD normalized by the number of nuclei analyzed ( n = 3, mean ± SD). (C) Relative luminescence produced by firefly luciferase expressed under an NF‐κB‐driven promoter in cultures treated as indicated above. Relative firefly luciferase luminescence was corrected by the amount of Renilla luciferase activity controlled by a constitutive promoter and expressed as percentage of cultures treated with vehicle (DMSO) ( n = 3, mean ± SD). (D) Motor neuron survival in co‐cultures with spinal cord astrocytes from symptomatic hSOD1 G93A mice treated as in (A) ( n = 4, mean ± SD). * p < 0.05.

Article Snippet: Adenovirus expressing a firefly luciferase gene under the control of a synthetic promoter that contains direct repeats of the NF‐κB binding site (Ad‐NFkb‐Luc) or a Renilla luciferase under a constitutive promoter (Ad‐pRL‐Luc) were obtained from Vector Biolabs.

Techniques: Inhibition, Isolation, Staining, Produced, Luciferase, Activity Assay