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Addgene inc plko 1 control shrna vector targeting gfp
Plko 1 Control Shrna Vector Targeting Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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DNA‐PKcs deletion suppresses ferroptosis in diabetic cardiomyopathy. In vivo, cardiomyocyte‐specific DNA‐PKcs knockout ( DNA‐PKcs Cko ) and wild‐type DNA‐PKcs f/f mice were injected intraperitoneally with streptozotocin (STZ, 50 mg kg −1 in 0.1 mol L −1 citrate buffer) for five consecutive days to induce diabetes. Age‐ and sex‐matched non‐diabetic control mice were injected with PBS. In vitro, HL‐1 cells were transduced with shRNA targeting DNA‐PKcs (sh/DNA‐PKcs) or with scramble control (sh/scramble), and then cultured in high‐glucose (HG, 30 mmol L −1 ) medium for 48 h to mimic hyperglycemic stress. Cells incubated in normal glucose (NG, 5.5 mmol L −1 ) medium served as controls. A) RNA‐seq analysis of differentially expressed genes in STZ‐treated DNA‐PKcs Cko and STZ‐injected DNA‐PKcs f/f mice. B,C) KEGG and GO analyses of differentially expressed genes. D,E) Single‐cell RNA sequencing (scRNA‐seq) data from the Genome Sequence Archive in the BIG Data Center ( http://bigd.big.ac.cn/ , accession code CRA007245) was used to assess the correlation between DNA‐PKcs expression levels and ferroptosis pathway activity scores. F,G) Representative images of TUNEL staining in heart tissues from vehicle‐ and STZ‐treated mice, and corresponding quantification data. DAPI was used for nuclear staining. H) Western blot analysis of ferritin and SLC7A11 protein expression in heart tissues. I) qPCR analysis of Ptgs2 transcription in heart tissues. J–L) ELISA‐based quantification of malondialdehyde (MDA), glutathione (GSH), and the GSH/GSSG ratio in heart tissues. M,N) ELISA quantification of MDA levels, GSH levels, and the GSH/GSSG ratio in HL‐1 cells. HL‐1 cells were treated with ferrostatin‐1 (Fer‐1) or deferoxamine (DFO) prior to HG exposure. O) Cell death was assessed by MTT assay and lactate dehydrogenase (LDH) release in HL‐1 cells treated with Fer‐1 or DFO before HG exposure. Each group consisted of 4 animals or 4 independent cell culture experiments ( n = 4). For each animal or independent cell culture experiment, measurements were repeated three times under the same experimental conditions. In each panel, dots represent individual measurements from animals or independent cell culture experiments. Bars represent group means, and error bars indicate ± standard error (SD). * p < 0.05 ** p < 0.01, *** p < 0.001, ns, not significant.

Journal: Advanced Science

Article Title: DNA‐PKcs‐Driven YAP1 Phosphorylation and Nuclear Translocation: a Key Regulator of Ferroptosis in Hyperglycemia‐Induced Cardiac Dysfunction in Type 1 Diabetes

doi: 10.1002/advs.202412698

Figure Lengend Snippet: DNA‐PKcs deletion suppresses ferroptosis in diabetic cardiomyopathy. In vivo, cardiomyocyte‐specific DNA‐PKcs knockout ( DNA‐PKcs Cko ) and wild‐type DNA‐PKcs f/f mice were injected intraperitoneally with streptozotocin (STZ, 50 mg kg −1 in 0.1 mol L −1 citrate buffer) for five consecutive days to induce diabetes. Age‐ and sex‐matched non‐diabetic control mice were injected with PBS. In vitro, HL‐1 cells were transduced with shRNA targeting DNA‐PKcs (sh/DNA‐PKcs) or with scramble control (sh/scramble), and then cultured in high‐glucose (HG, 30 mmol L −1 ) medium for 48 h to mimic hyperglycemic stress. Cells incubated in normal glucose (NG, 5.5 mmol L −1 ) medium served as controls. A) RNA‐seq analysis of differentially expressed genes in STZ‐treated DNA‐PKcs Cko and STZ‐injected DNA‐PKcs f/f mice. B,C) KEGG and GO analyses of differentially expressed genes. D,E) Single‐cell RNA sequencing (scRNA‐seq) data from the Genome Sequence Archive in the BIG Data Center ( http://bigd.big.ac.cn/ , accession code CRA007245) was used to assess the correlation between DNA‐PKcs expression levels and ferroptosis pathway activity scores. F,G) Representative images of TUNEL staining in heart tissues from vehicle‐ and STZ‐treated mice, and corresponding quantification data. DAPI was used for nuclear staining. H) Western blot analysis of ferritin and SLC7A11 protein expression in heart tissues. I) qPCR analysis of Ptgs2 transcription in heart tissues. J–L) ELISA‐based quantification of malondialdehyde (MDA), glutathione (GSH), and the GSH/GSSG ratio in heart tissues. M,N) ELISA quantification of MDA levels, GSH levels, and the GSH/GSSG ratio in HL‐1 cells. HL‐1 cells were treated with ferrostatin‐1 (Fer‐1) or deferoxamine (DFO) prior to HG exposure. O) Cell death was assessed by MTT assay and lactate dehydrogenase (LDH) release in HL‐1 cells treated with Fer‐1 or DFO before HG exposure. Each group consisted of 4 animals or 4 independent cell culture experiments ( n = 4). For each animal or independent cell culture experiment, measurements were repeated three times under the same experimental conditions. In each panel, dots represent individual measurements from animals or independent cell culture experiments. Bars represent group means, and error bars indicate ± standard error (SD). * p < 0.05 ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: Lentiviral‐based short hairpin RNAs (shRNAs) targeting mouse FANCD2 (Santa Cruz Biotechnology, Cat. No. sc‐35357), UBE2T (Santa Cruz Biotechnology, Cat. No. sc‐106661), DNA‐PKcs (Santa Cruz Biotechnology, Cat. No. sc‐35201), or scramble shRNA targeting enhanced green fluorescent protein (GFP) (Santa Cruz Biotechnology, Cat. No. sc‐45924) were used for the knockdown experiments.

Techniques: In Vivo, Knock-Out, Injection, Control, In Vitro, Transduction, shRNA, Cell Culture, Incubation, RNA Sequencing, Sequencing, Expressing, Activity Assay, TUNEL Assay, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, MTT Assay

Co‐immunoprecipitation (Co‐IP)‐based proteomics analysis of DNA‐PKcs interactions. In vivo, cardiomyocyte‐specific DNA‐PKcs knockout ( DNA‐PKcs Cko ) and wild‐type DNA‐PKcs f/f mice were injected intraperitoneally with streptozotocin (STZ, 50 mg kg −1 in 0.1 mol L −1 citrate buffer) for five consecutive days to induce diabetes. Age‐ and sex‐matched non‐diabetic control mice were injected with PBS. Diabetic DNA‐PKcs Cko and DNA‐PKcs f/f mice were subsequently injected intraperitoneally with RSL3 (10 mg kg −1 ) for 24 weeks to induce ferroptosis. A) A Co‐IP‐based proteomics assay was performed to identify DNA‐PKcs‐interacting proteins. The Venn diagram illustrates the overlap of proteins between the IgG control and DNA‐PKcs groups. B) KEGG and GO analyses of DNA‐PKcs‐interacting proteins. C) Protein‐protein interaction (PPI) network analysis of DNA‐PKcs‐specific binding proteins, focusing on a curated gene list associated with the Hippo pathway. D) CytoHubba plugin was used to score the PPI network. Higher scores, indicated by red color, represent proteins that are more central within the network. E) Immunoprecipitates of DNA‐PKcs or YAP1 from heart tissues of mice with hyperglycemia‐induced diabetic cardiomyopathy were immunoblotted as indicated. F) His‐tagged DNA‐PKcs or HA‐tagged YAP1 was transfected into HL‐1 cells, followed by a Co‐IP assay to assess protein interactions. G) HL‐1 cells were treated with shRNA targeting YAP1 (sh/YAP1) before high glucose (HG) exposure, and qPCR analysis was performed to assess Ptgs2 transcription. H) HL‐1 cells were treated with sh/YAP1 before HG exposure, and Western blotting was used to determine ferritin expression in heart tissues. I) ELISA kits were used to analyze GSH/GSSG ratios in HL‐1 cells treated with sh/YAP1 prior to HG exposure. J) Cell death was measured in HL‐1 cells treated with sh/YAP1 before HG exposure using MTT and LDH release assays. K) HL‐1 cells were treated with sh/YAP1 before AsiDNA treatment, and qPCR analysis was used to assess Ptgs2 transcription. L) Western blot analysis of ferritin expression in heart tissues from HL‐1 cells treated with sh/YAP1 prior to AsiDNA treatment. M) ELISA kits were used to determine GSH/GSSG ratios in HL‐1 cells treated with sh/YAP1 before AsiDNA treatment. N) Cell death was measured using MTT and LDH release assays in HL‐1 cells treated with sh/YAP1 before AsiDNA treatment. O) Molecular docking analysis of DNA‐PKcs and YAP1 showing potential binding sites, highlighted in different colors. P) Mapping of functional regions in DNA‐PKcs involved in binding to YAP1. Q) Immunoprecipitation and immunoblotting were applied to evaluate interactions between specific DNA‐PKcs mutants and YAP1. R) Mapping of functional regions in YAP1 involved in interaction with DNA‐PKcs. S) Immunoprecipitation and immunoblotting were used to assess interactions between region‐specific DNA‐PKcs and YAP1 mutants in HL‐1 cells. Each group consisted of 4 animals or 4 independent cell culture experiments ( n = 4). For each animal or independent cell culture experiment, measurements were repeated three times under the same experimental conditions. In each panel, dots represent individual measurements from animals or independent cell culture experiments. Bars represent group means, and error bars indicate ± standard error (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Journal: Advanced Science

Article Title: DNA‐PKcs‐Driven YAP1 Phosphorylation and Nuclear Translocation: a Key Regulator of Ferroptosis in Hyperglycemia‐Induced Cardiac Dysfunction in Type 1 Diabetes

doi: 10.1002/advs.202412698

Figure Lengend Snippet: Co‐immunoprecipitation (Co‐IP)‐based proteomics analysis of DNA‐PKcs interactions. In vivo, cardiomyocyte‐specific DNA‐PKcs knockout ( DNA‐PKcs Cko ) and wild‐type DNA‐PKcs f/f mice were injected intraperitoneally with streptozotocin (STZ, 50 mg kg −1 in 0.1 mol L −1 citrate buffer) for five consecutive days to induce diabetes. Age‐ and sex‐matched non‐diabetic control mice were injected with PBS. Diabetic DNA‐PKcs Cko and DNA‐PKcs f/f mice were subsequently injected intraperitoneally with RSL3 (10 mg kg −1 ) for 24 weeks to induce ferroptosis. A) A Co‐IP‐based proteomics assay was performed to identify DNA‐PKcs‐interacting proteins. The Venn diagram illustrates the overlap of proteins between the IgG control and DNA‐PKcs groups. B) KEGG and GO analyses of DNA‐PKcs‐interacting proteins. C) Protein‐protein interaction (PPI) network analysis of DNA‐PKcs‐specific binding proteins, focusing on a curated gene list associated with the Hippo pathway. D) CytoHubba plugin was used to score the PPI network. Higher scores, indicated by red color, represent proteins that are more central within the network. E) Immunoprecipitates of DNA‐PKcs or YAP1 from heart tissues of mice with hyperglycemia‐induced diabetic cardiomyopathy were immunoblotted as indicated. F) His‐tagged DNA‐PKcs or HA‐tagged YAP1 was transfected into HL‐1 cells, followed by a Co‐IP assay to assess protein interactions. G) HL‐1 cells were treated with shRNA targeting YAP1 (sh/YAP1) before high glucose (HG) exposure, and qPCR analysis was performed to assess Ptgs2 transcription. H) HL‐1 cells were treated with sh/YAP1 before HG exposure, and Western blotting was used to determine ferritin expression in heart tissues. I) ELISA kits were used to analyze GSH/GSSG ratios in HL‐1 cells treated with sh/YAP1 prior to HG exposure. J) Cell death was measured in HL‐1 cells treated with sh/YAP1 before HG exposure using MTT and LDH release assays. K) HL‐1 cells were treated with sh/YAP1 before AsiDNA treatment, and qPCR analysis was used to assess Ptgs2 transcription. L) Western blot analysis of ferritin expression in heart tissues from HL‐1 cells treated with sh/YAP1 prior to AsiDNA treatment. M) ELISA kits were used to determine GSH/GSSG ratios in HL‐1 cells treated with sh/YAP1 before AsiDNA treatment. N) Cell death was measured using MTT and LDH release assays in HL‐1 cells treated with sh/YAP1 before AsiDNA treatment. O) Molecular docking analysis of DNA‐PKcs and YAP1 showing potential binding sites, highlighted in different colors. P) Mapping of functional regions in DNA‐PKcs involved in binding to YAP1. Q) Immunoprecipitation and immunoblotting were applied to evaluate interactions between specific DNA‐PKcs mutants and YAP1. R) Mapping of functional regions in YAP1 involved in interaction with DNA‐PKcs. S) Immunoprecipitation and immunoblotting were used to assess interactions between region‐specific DNA‐PKcs and YAP1 mutants in HL‐1 cells. Each group consisted of 4 animals or 4 independent cell culture experiments ( n = 4). For each animal or independent cell culture experiment, measurements were repeated three times under the same experimental conditions. In each panel, dots represent individual measurements from animals or independent cell culture experiments. Bars represent group means, and error bars indicate ± standard error (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, In Vivo, Knock-Out, Injection, Control, Binding Assay, Transfection, shRNA, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Functional Assay, Cell Culture

DNA‐PKcs phosphorylates YAP1 at Thr226 and promotes its nuclear localization. A) Amino acid sequence alignment of YAP1 across various species. B) HL‐1 cells were transfected with different HA‐tagged YAP1 constructs, including full‐length YAP1 (HA‐YAP1), YAP1 lacking Thr226 (HA‐YAP1ΔT226), Gln227 (HA‐YAP1ΔQ227), or both Thr226 and Gln227 (HA‐YAP1ΔT226Q227). After high glucose exposure, HA immunoprecipitates were collected and immunoblotted to assess the interaction between DNA‐PKcs and HA‐YAP1. C) YAP1 phosphorylation was assessed in heart tissues from mice subjected to STZ‐induced diabetic cardiomyopathy. D) YAP1 phosphorylation was measured in HL‐1 cells treated with AsiDNA. E,F) HL‐1 cells were treated with shRNA targeting YAP1 (sh/YAP1) prior to AsiDNA treatment, and YAP1 phosphorylation was assessed. G) In vitro kinase assay: recombinant mouse DNA‐PKcs and recombinant mouse YAP1 were incubated together in kinase assay buffer with ATP in the presence or absence of NU7441. YAP1 phosphorylation and DNA‐PKcs levels were assessed by Western blotting. H) HL‐1 cells were treated with NU7441 under high glucose conditions, and YAP1 phosphorylation levels were determined by Western blotting. I) HL‐1 cells were transfected with HA‐tagged YAP1 constructs, including phosphorylation‐defective HA‐YAP1 T226A and phosphorylation‐mimetic HA‐YAP1 T226D . After high glucose exposure, YAP1 protein expression in the cytoplasm and nucleus was analyzed by Western blotting. Histone H3 and β‐actin were used as loading controls. J) Volcano plot of RNA‐seq data from the GSE110268 database, which includes isogenic YAP knockout human embryonic stem cells (hESCs) generated via CRISPR/Cas9‐mediated gene editing. K) WIKI pathway and Gene Ontology (GO) analyses of altered genes from the GSE110268 database. L) Gene Set Enrichment Analysis (GSEA) plots of altered genes from the GSE110268 database. M) Single‐cell RNA sequencing (scRNA‐seq) data from the Genome Sequence Archive ( http://bigd.big.ac.cn/ , accession code CRA007245) was analyzed to assess the correlation between YAP1 expression levels and ferroptosis‐related gene expression. N) HL‐1 cells were transfected with phosphorylation‐defective HA‐YAP1 T226A and phosphorylation‐mimetic HA‐YAP1 T226D constructs. After high glucose exposure, ferritin expression was determined by Western blotting. O) HL‐1 cells transfected with YAP1 constructs were exposed to high glucose, and GSH/GSSG ratios were analyzed using ELISA kits. P,Q) HL‐1 cells transfected with YAP1 constructs were exposed to high glucose, and cell death was measured by MTT assay and lactate dehydrogenase (LDH) release assay. Each group consisted of 4 animals or 4 independent cell culture experiments ( n = 4). For each animal or independent cell culture experiment, measurements were repeated three times under the same experimental conditions. In each panel, dots represent individual measurements from animals or independent cell culture experiments. Bars represent group means, and error bars indicate ± standard error (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Journal: Advanced Science

Article Title: DNA‐PKcs‐Driven YAP1 Phosphorylation and Nuclear Translocation: a Key Regulator of Ferroptosis in Hyperglycemia‐Induced Cardiac Dysfunction in Type 1 Diabetes

doi: 10.1002/advs.202412698

Figure Lengend Snippet: DNA‐PKcs phosphorylates YAP1 at Thr226 and promotes its nuclear localization. A) Amino acid sequence alignment of YAP1 across various species. B) HL‐1 cells were transfected with different HA‐tagged YAP1 constructs, including full‐length YAP1 (HA‐YAP1), YAP1 lacking Thr226 (HA‐YAP1ΔT226), Gln227 (HA‐YAP1ΔQ227), or both Thr226 and Gln227 (HA‐YAP1ΔT226Q227). After high glucose exposure, HA immunoprecipitates were collected and immunoblotted to assess the interaction between DNA‐PKcs and HA‐YAP1. C) YAP1 phosphorylation was assessed in heart tissues from mice subjected to STZ‐induced diabetic cardiomyopathy. D) YAP1 phosphorylation was measured in HL‐1 cells treated with AsiDNA. E,F) HL‐1 cells were treated with shRNA targeting YAP1 (sh/YAP1) prior to AsiDNA treatment, and YAP1 phosphorylation was assessed. G) In vitro kinase assay: recombinant mouse DNA‐PKcs and recombinant mouse YAP1 were incubated together in kinase assay buffer with ATP in the presence or absence of NU7441. YAP1 phosphorylation and DNA‐PKcs levels were assessed by Western blotting. H) HL‐1 cells were treated with NU7441 under high glucose conditions, and YAP1 phosphorylation levels were determined by Western blotting. I) HL‐1 cells were transfected with HA‐tagged YAP1 constructs, including phosphorylation‐defective HA‐YAP1 T226A and phosphorylation‐mimetic HA‐YAP1 T226D . After high glucose exposure, YAP1 protein expression in the cytoplasm and nucleus was analyzed by Western blotting. Histone H3 and β‐actin were used as loading controls. J) Volcano plot of RNA‐seq data from the GSE110268 database, which includes isogenic YAP knockout human embryonic stem cells (hESCs) generated via CRISPR/Cas9‐mediated gene editing. K) WIKI pathway and Gene Ontology (GO) analyses of altered genes from the GSE110268 database. L) Gene Set Enrichment Analysis (GSEA) plots of altered genes from the GSE110268 database. M) Single‐cell RNA sequencing (scRNA‐seq) data from the Genome Sequence Archive ( http://bigd.big.ac.cn/ , accession code CRA007245) was analyzed to assess the correlation between YAP1 expression levels and ferroptosis‐related gene expression. N) HL‐1 cells were transfected with phosphorylation‐defective HA‐YAP1 T226A and phosphorylation‐mimetic HA‐YAP1 T226D constructs. After high glucose exposure, ferritin expression was determined by Western blotting. O) HL‐1 cells transfected with YAP1 constructs were exposed to high glucose, and GSH/GSSG ratios were analyzed using ELISA kits. P,Q) HL‐1 cells transfected with YAP1 constructs were exposed to high glucose, and cell death was measured by MTT assay and lactate dehydrogenase (LDH) release assay. Each group consisted of 4 animals or 4 independent cell culture experiments ( n = 4). For each animal or independent cell culture experiment, measurements were repeated three times under the same experimental conditions. In each panel, dots represent individual measurements from animals or independent cell culture experiments. Bars represent group means, and error bars indicate ± standard error (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Techniques: Sequencing, Transfection, Construct, Phospho-proteomics, shRNA, In Vitro, Kinase Assay, Recombinant, Incubation, Western Blot, Expressing, RNA Sequencing, Knock-Out, Generated, CRISPR, Gene Expression, Enzyme-linked Immunosorbent Assay, MTT Assay, Lactate Dehydrogenase Assay, Cell Culture

Initial validation of AAV-shRNA injections. ( A ) Schematic of CMT-specific knock-down (KD) animal generation by injecting Cacna1g shRNA (or scrambled shRNA as control). ( B ) Image shows GFP-transfected neurons only, counter-stained with DAPI. ( C ) T-current traces from GFP-positive neurons from animals injected with scrambled shRNA (control, black trace) or Cacna1g shRNA (green trace). ( D ) Average T-current amplitudes in GFP-positive neurons after control and Cacna1g shRNA injections (unpaired two-tailed t -test: t (16) = 4.023, p < 0.001; Cohen’s d = −2.02; effect size r = -0.71). n = 11 control and 7 Cacna1g shRNA-transfected GFP-positive neurons; *** p < 0.001. ( E ) q-PCR data pooled from the tissues of 3 Cacna1g shRNA and 4 control animals showing 76.72% decrease in relative Ca V 3.1 expression in Cacna1g shRNA group in comparison to control (unpaired two-tailed t -test: t (2) = 6.893, p = 0.02; Cohen’s d = −6.89; effect size r = −0.96). n = 2, technical replicates; * p < 0.05.

Journal: International Journal of Molecular Sciences

Article Title: Role of Thalamic Ca V 3.1 T-Channels in Fear Conditioning

doi: 10.3390/ijms26083543

Figure Lengend Snippet: Initial validation of AAV-shRNA injections. ( A ) Schematic of CMT-specific knock-down (KD) animal generation by injecting Cacna1g shRNA (or scrambled shRNA as control). ( B ) Image shows GFP-transfected neurons only, counter-stained with DAPI. ( C ) T-current traces from GFP-positive neurons from animals injected with scrambled shRNA (control, black trace) or Cacna1g shRNA (green trace). ( D ) Average T-current amplitudes in GFP-positive neurons after control and Cacna1g shRNA injections (unpaired two-tailed t -test: t (16) = 4.023, p < 0.001; Cohen’s d = −2.02; effect size r = -0.71). n = 11 control and 7 Cacna1g shRNA-transfected GFP-positive neurons; *** p < 0.001. ( E ) q-PCR data pooled from the tissues of 3 Cacna1g shRNA and 4 control animals showing 76.72% decrease in relative Ca V 3.1 expression in Cacna1g shRNA group in comparison to control (unpaired two-tailed t -test: t (2) = 6.893, p = 0.02; Cohen’s d = −6.89; effect size r = −0.96). n = 2, technical replicates; * p < 0.05.

Article Snippet: The mice were randomized into two treatment groups: scrambled control (AAV2-GFP-U6-scrmb-shRNA; titer: 1.1 × 10 13 GC/mL; Vector Biolabs, Malvern, PA) or Cacna1g shRNA targeting Ca V 3.1 (AAV2-GFP-U6-mCACNA1G-shRNA; titer: 6.8 × 10 12 GC/mL; Vector Biolabs) groups.

Techniques: Biomarker Discovery, shRNA, Knockdown, Control, Transfection, Staining, Injection, Two Tailed Test, Expressing, Comparison

Different firing properties of GFP-positive thalamic neurons from control (scrambled shRNA) and Cacna1g shRNA-injected animals. ( A ) Original traces from representative thalamic neurons from GFP-positive control (black) and Cacna1g shRNA-transfected (green) neurons showing active membrane responses to depolarizing (275 pA) and hyperpolarizing (−225 pA) current injections. Note that GFP-positive neurons from Cacna1g shRNA cohort did not show action potentials (APs) with low threshold spikes (LTSs) after membrane hyperpolarization. ( B ) There was no difference in average tonic AP firing frequency (50–275 pA current injections) between GFP-positive thalamic neurons from control and Cacna1g shRNA mice. ( C ) Number of APs in rebound burst was statistically significantly smaller in GFP-positive neurons from Cacna1g shRNA animals (two-way RM ANOVA: interaction F (8,88) = 0.963, p = 0.470; injected current F (8,88) = 4.92, p < 0.001; GFP F (1,11) = 5.133, p = 0.045). ( D ) LTSs were not observed in thalamic GFP-positive Cacna1g shRNA-transfected neurons (two-way RM ANOVA: interaction F (8,88) = 3.50, p = 0.001; injected current F (8,88) = 3.748, p < 0.001; GFP F (1,11) = 15.02, p = 0.003; Sidak’s post hoc test). ( E ) Threshold for rebound burst firing was higher in thalamic neurons from Cacna1g shRNA-injected animals (unpaired two-tailed t -test: t (7) = 6.314, p < 0.001). Note that this rebound firing had APs without expected T-channel-dependent LTSs. n = 6 control and 7 Cacna1g shRNA-transfected GFP-positive neurons; * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: International Journal of Molecular Sciences

Article Title: Role of Thalamic Ca V 3.1 T-Channels in Fear Conditioning

doi: 10.3390/ijms26083543

Figure Lengend Snippet: Different firing properties of GFP-positive thalamic neurons from control (scrambled shRNA) and Cacna1g shRNA-injected animals. ( A ) Original traces from representative thalamic neurons from GFP-positive control (black) and Cacna1g shRNA-transfected (green) neurons showing active membrane responses to depolarizing (275 pA) and hyperpolarizing (−225 pA) current injections. Note that GFP-positive neurons from Cacna1g shRNA cohort did not show action potentials (APs) with low threshold spikes (LTSs) after membrane hyperpolarization. ( B ) There was no difference in average tonic AP firing frequency (50–275 pA current injections) between GFP-positive thalamic neurons from control and Cacna1g shRNA mice. ( C ) Number of APs in rebound burst was statistically significantly smaller in GFP-positive neurons from Cacna1g shRNA animals (two-way RM ANOVA: interaction F (8,88) = 0.963, p = 0.470; injected current F (8,88) = 4.92, p < 0.001; GFP F (1,11) = 5.133, p = 0.045). ( D ) LTSs were not observed in thalamic GFP-positive Cacna1g shRNA-transfected neurons (two-way RM ANOVA: interaction F (8,88) = 3.50, p = 0.001; injected current F (8,88) = 3.748, p < 0.001; GFP F (1,11) = 15.02, p = 0.003; Sidak’s post hoc test). ( E ) Threshold for rebound burst firing was higher in thalamic neurons from Cacna1g shRNA-injected animals (unpaired two-tailed t -test: t (7) = 6.314, p < 0.001). Note that this rebound firing had APs without expected T-channel-dependent LTSs. n = 6 control and 7 Cacna1g shRNA-transfected GFP-positive neurons; * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Control, shRNA, Injection, Positive Control, Transfection, Membrane, Two Tailed Test

Targeted reduction in thalamic Ca V 3.1 T-channels increases fear responses during expression but not during acquisition of contextual fear conditioning. ( A ) Schematic diagram of contextual fear conditioning: Day 1—training (acquisition); Day 2—different context; Day 3—testing. ( B ) Percentage of time spent freezing in control (scrambled shRNA, gray) and Cacna1g shRNA-injected (blue) mice during training phase of contextual fear conditioning paradigm, analyzed every 30 s (left) and over whole training phase (right). ( C ) Increased freezing after each tone/shock pairing in control and Ca V 3.1 KD mice without any differences in freezing behavior between groups (left; two-way RM ANOVA: interaction F (2,36) = 0.182, p = 0.834; tone F (2,36) = 23.56, p < 0.001; shRNA F (1,18) = 0.090, p = 0.768). Control and Cacna1g shRNA-injected animals had similar baseline freezing percentages (right). ( D ) Compared to control mice, percentage of time freezing in Cacna1g shRNA-injected mice during testing significantly increased over time (left; two-way RM ANOVA: interaction F (13,234) = 0.551, p = 0.891; time F (13,234) = 0.991, p = 0.461; shRNA F (1,18) = 6.642, p = 0.019) and over whole 7 min testing period (right; unpaired two-tailed t -test: t (18) = 2.577, p = 0.019; Cohen’s d = 1.17; effect size r = 0.50). n = 9 control and 11 Cacna1g shRNA-injected mice; * p < 0.05, *** p < 0.001.

Journal: International Journal of Molecular Sciences

Article Title: Role of Thalamic Ca V 3.1 T-Channels in Fear Conditioning

doi: 10.3390/ijms26083543

Figure Lengend Snippet: Targeted reduction in thalamic Ca V 3.1 T-channels increases fear responses during expression but not during acquisition of contextual fear conditioning. ( A ) Schematic diagram of contextual fear conditioning: Day 1—training (acquisition); Day 2—different context; Day 3—testing. ( B ) Percentage of time spent freezing in control (scrambled shRNA, gray) and Cacna1g shRNA-injected (blue) mice during training phase of contextual fear conditioning paradigm, analyzed every 30 s (left) and over whole training phase (right). ( C ) Increased freezing after each tone/shock pairing in control and Ca V 3.1 KD mice without any differences in freezing behavior between groups (left; two-way RM ANOVA: interaction F (2,36) = 0.182, p = 0.834; tone F (2,36) = 23.56, p < 0.001; shRNA F (1,18) = 0.090, p = 0.768). Control and Cacna1g shRNA-injected animals had similar baseline freezing percentages (right). ( D ) Compared to control mice, percentage of time freezing in Cacna1g shRNA-injected mice during testing significantly increased over time (left; two-way RM ANOVA: interaction F (13,234) = 0.551, p = 0.891; time F (13,234) = 0.991, p = 0.461; shRNA F (1,18) = 6.642, p = 0.019) and over whole 7 min testing period (right; unpaired two-tailed t -test: t (18) = 2.577, p = 0.019; Cohen’s d = 1.17; effect size r = 0.50). n = 9 control and 11 Cacna1g shRNA-injected mice; * p < 0.05, *** p < 0.001.

Techniques: Expressing, Control, shRNA, Injection, Two Tailed Test

Global deletion and targeted reduction in thalamic Ca V 3.1 T-channels did not change fear responses during cued fear conditioning. ( A ) Schematic of cued fear conditioning: Day 1—training (acquisition); Day 2—different context; Day 3—testing. ( B ) Percentage of time spent freezing in control (WT, gray) and Ca V 3.1 KO (blue) mice during cued fear conditioning paradigm, analyzed every 30 s (left; two-way RM ANOVA: interaction F (13,182) = 1.249, p = 0.248; time F (13,182) = 2.791, p = 0.001; shRNA F (1,14) = 0.156, p = 0.698), and average freezing responses at baseline and after each tone (right; two-way RM ANOVA: interaction F (2,28) = 2.86, p = 0.074; tone F (2,28) = 6.39, p = 0.005; shRNA F (1,14) = 0.039, p = 0.846). ( C ) Percentage of time freezing in control (gray) and Cacna1g shRNA-injected mice (green) during cued fear conditioning, analyzed over time (left; two-way RM ANOVA: interaction F (13,234) = 0.919, p = 0.534; time F (13,234) = 6.164, p < 0.001; shRNA F (1,18) = 0.124, p = 0.729) and averaged (right; two-way RM ANOVA: interaction F (2,36) = 0.182, p = 0.834; tone F (2,36) = 23.56, p < 0.001; shRNA F (1,18) = 0.090, p = 0.768). n = 8 WT, 8 Ca V 3.1 KO, 9 control, and 11 Cacna1g shRNA-injected mice; ** p < 0.01, *** p < 0.001.

Journal: International Journal of Molecular Sciences

Article Title: Role of Thalamic Ca V 3.1 T-Channels in Fear Conditioning

doi: 10.3390/ijms26083543

Figure Lengend Snippet: Global deletion and targeted reduction in thalamic Ca V 3.1 T-channels did not change fear responses during cued fear conditioning. ( A ) Schematic of cued fear conditioning: Day 1—training (acquisition); Day 2—different context; Day 3—testing. ( B ) Percentage of time spent freezing in control (WT, gray) and Ca V 3.1 KO (blue) mice during cued fear conditioning paradigm, analyzed every 30 s (left; two-way RM ANOVA: interaction F (13,182) = 1.249, p = 0.248; time F (13,182) = 2.791, p = 0.001; shRNA F (1,14) = 0.156, p = 0.698), and average freezing responses at baseline and after each tone (right; two-way RM ANOVA: interaction F (2,28) = 2.86, p = 0.074; tone F (2,28) = 6.39, p = 0.005; shRNA F (1,14) = 0.039, p = 0.846). ( C ) Percentage of time freezing in control (gray) and Cacna1g shRNA-injected mice (green) during cued fear conditioning, analyzed over time (left; two-way RM ANOVA: interaction F (13,234) = 0.919, p = 0.534; time F (13,234) = 6.164, p < 0.001; shRNA F (1,18) = 0.124, p = 0.729) and averaged (right; two-way RM ANOVA: interaction F (2,36) = 0.182, p = 0.834; tone F (2,36) = 23.56, p < 0.001; shRNA F (1,18) = 0.090, p = 0.768). n = 8 WT, 8 Ca V 3.1 KO, 9 control, and 11 Cacna1g shRNA-injected mice; ** p < 0.01, *** p < 0.001.

Techniques: Control, shRNA, Injection

General activity and anxiety-related behaviors in Cacna1g shRNA-injected mice. ( A ) Graphic presentation of open-field test. ( B ) Time spent in the central zone of open-field arena. ( C ) Number of entries to central zone of open-field arena. ( D ) Distance traveled in open-field arena in control and Cacna1g shRNA mice. ( E ) Graphic presentation of zero-maze test. ( F ) Time spent in open quadrants. ( G ) Number of entries into open quadrants. ( H ) Distance traveled during zero-maze test in control and Cacna1g shRNA mice.

Journal: International Journal of Molecular Sciences

Article Title: Role of Thalamic Ca V 3.1 T-Channels in Fear Conditioning

doi: 10.3390/ijms26083543

Figure Lengend Snippet: General activity and anxiety-related behaviors in Cacna1g shRNA-injected mice. ( A ) Graphic presentation of open-field test. ( B ) Time spent in the central zone of open-field arena. ( C ) Number of entries to central zone of open-field arena. ( D ) Distance traveled in open-field arena in control and Cacna1g shRNA mice. ( E ) Graphic presentation of zero-maze test. ( F ) Time spent in open quadrants. ( G ) Number of entries into open quadrants. ( H ) Distance traveled during zero-maze test in control and Cacna1g shRNA mice.

Techniques: Activity Assay, shRNA, Injection, Control

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