Journal: Molecular Pharmacology

Article Title: Transient receptor potential ankyrin 1 promotes the expression of interferon-stimulated antiviral genes in human A549 lung epithelial cells

doi: 10.1016/j.molpha.2025.100098

Figure Lengend Snippet: TRPA1 antagonists inhibit ISRE-dependent transcription induced by IFN beta. A549 cells were transfected for 24 hours with dual luciferase plasmids with Firefly luciferase as a reporter gene under the control of an ISRE-dependent promoter or control promoter (“empty”); and in both cases, Renilla luciferase under a constitutive promoter was used as an internal control. Thereafter, cells were treated with IFN beta (10 ng/mL), with or without the TRPA1 antagonists HC-030031 (HC; 100 μ M) or A-967079 (A96; 100 μ M), or vehicle (DMSO) for 7 hours 30 minutes. After protein extraction, Firefly and Renilla luciferase activity was read. Firefly activity in each sample was normalized to the respective Renilla activity, and ISRE-transfected activity was then normalized to the mean empty-transfected activity of the respective treatment. Control was set as 1, and the other values are given in relation to that value. Individual values and mean ± SD are presented, n = 6. Statistical analysis was performed using one-way ANOVA with Holm- Šídák post-test. Asterisks on bars indicate comparison against the condition treated with IFN beta, and asterisks on brackets indicate comparison between indicated conditions. ∗∗, ∗∗∗, and ∗∗∗∗ denote P < .01, <.001, and <.0001. Ns = not significant.

Article Snippet: After 24 hours, cells were transfected with 60 ng/well of vectors containing a Firefly luciferase reporter under an ISRE-dependent promoter or a control promoter (“empty”); and Renilla luciferase under a constitutive promoter (BPS-60613; BPS Bioscience) as an internal control for 24 hours.

Techniques: Transfection, Luciferase, Control, Protein Extraction, Activity Assay, Comparison

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

Journal: Cell reports

Article Title: Neuronal hyperactivity becomes mTORC1 independent due to transcriptional changes in tuberous sclerosis complex disease models

doi: 10.1016/j.celrep.2025.116664

Figure Lengend Snippet: (A) Luciferase expression driven by the human EGR1 promoter compared to Renilla expression driven by a constitutive promoter. Experiments were performed in hiPSC-derived neurons of the indicated genotype on day 25 of differentiation. Each dot represents an independent well of neurons across three separate differentiations. Bar plot shows mean ± SEM. * p < 0.05 ( n = 16–32, ANOVA with Dunnett’s test). (B) Representative trace of luciferase expression induced by 50 mM KCl across all three genotypes of hiPSC-derived neurons and quantification of the maximum induction of luciferase due to 50 mM KCl treatment. Experiments were performed in hiPSC-derived neurons of the indicated genotype on day 25 of differentiation. Each dot represents an independent well of neurons across four separate differentiations. Bar plot shows mean ± SEM. * p < 0.05 ( n = 30, ANOVA with Dunnett’s test). (C) Immunoblots of proteasome subunits and proteasome-associated proteins in hiPSC-derived neurons of the indicated genotypes on day 30 of differentiation. Quantification of these immunoblots compared to total protein shows a significant increase in TSC2 −/− neurons of all three proteins compared to TSC2 +/+ neurons. Bar plot shows mean ± SEM. N = 3 separate differentiations, ANOVA with Dunnett’s test. (D) qPCR of proteasome subunits and associated proteins in TSC2 −/− compared to TSC2 +/+ neurons on day 30 of differentiation. * p < 0.05 ( n = 3–4 separate differentiations, t test). (E) Proteasome activity assessed by cleavage of succinate-LLVY-AMC in lysates from hiPSC-derived neurons of the indicated genotypes on day 25 of differentiation5. Each dot represents an independent well from four separate differentiations. Bar plot shows mean ± SEM. * p < 0.05 ( n = 15, ANOVA with Dunnett’s test).

Article Snippet: Renilla luciferase under a constitutive promoter was used as a control (pLX313-Renilla luciferase; Addgene, # 118016).

Techniques: Luciferase, Expressing, Derivative Assay, Western Blot, Activity Assay