Journal: Frontiers in Cell and Developmental Biology

Article Title: HIIT-induced lactate/GPR81 signaling with dual branches converging on ERK1/2 contributes to hippocampal synaptic remodeling and memory improvement

doi: 10.3389/fcell.2026.1699042

Figure Lengend Snippet: GPR81 mediates the regulation of HIIT on the AC-RAP1 and PLC-PKC pathways and ERK1/2 activation in the hippocampus of mice, as well as the expression of synaptic plasticity-related proteins. (A,D,H,M) Representative Western blotting images. (B,C,E,F) Quantitative analysis of AC, RAP1, PLC, and PKC protein expression levels in the hippocampus of different groups. (I–L) Quantitative analysis of protein expression levels of phosphorylated/total c-Raf and ERK1/2 in the hippocampus of different groups. (G) Quantitative analysis of gene expression levels of ERK1/2 in the hippocampus of each group. * p < 0.05, ** p < 0.01 vs . the SC group. ns, no significant difference. (N–S) Quantitative analysis of protein expression levels of NMDAR, PSD95, SYN, BDNF, c-FOS, and Zif268 in the hippocampus of mice from different groups. Protein expression levels were normalized to Tubulin. Phosphorylated proteins were further normalized to their corresponding total protein levels. Relative gene expression was quantified and normalized to Gapdh. For comparisons among multiple groups, one-way ANOVA was applied. The data are presented as mean ± SD. n = 6. * p < 0.05, ** p < 0.01 vs . the SC group. ns, no significant difference. SC, scramble control, sedentary; SCH, scramble control + HIIT; GKD, GPR81 knockdown, sedentary; GKDH, GPR81 knockdown + HIIT.

Article Snippet: GPR81, PA5-114741, Thermo; AC (55067-1-AP, Proteintech), PKA (4782S, CST), RAP1 (2399S, CST), PKC (SC-80 Santa), PLC (14247S, CST), Raf1 (9422S, CST), MSK1 (3489S, CST), P90RSK (9355S, CST), ERK (4370T, CST), CREB (9197S, CST), c-Fos (2250S, CST), Zif268 (4154S, CST), NMDAR (5704S, CST), SYN (4329S, CST), BDNF (28205-1-AP, Proteintech), ARC (16290-1-AP, Proteintech), PSD95 (3450S, CST) p-Raf1 (9421S, CST), p-ERK (4370T, CST), p-MSK (9595S, CST), p-P90RSK (11989S, CST), p-CREB (9198S, CST), Tubulin (10068-1-AP, Proteintech), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00001-2, Proteintech), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (SA00001-1, Proteintech).

Techniques: Activation Assay, Expressing, Western Blot, Gene Expression, Control, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: HIIT-induced lactate/GPR81 signaling with dual branches converging on ERK1/2 contributes to hippocampal synaptic remodeling and memory improvement

doi: 10.3389/fcell.2026.1699042

Figure Lengend Snippet: GPR81-induced modulation of ERK1/2 via AC-RAP1 and PLC-PKC pathways regulates synaptic plasticity-related protein expression in N2a cells. (A,I) Representative Western blotting images. (B–H,J–P) Quantitative analysis of NMDAR, PSD95, SYN, BDNF, Zif268, c-Fos, and ARC protein expression levels. Protein expression levels were normalized to Tubulin. Phosphorylated proteins were further normalized to their corresponding total protein levels. For comparisons among multiple groups, one-way ANOVA was applied. The data are presented as mean ± SD. * p < 0.05 vs . the DMSO group. n = 3. # p < 0.05, ## p < 0.01, ### p < 0.001 vs . the Ga group. ns, no significant difference. Ga, GPR81 agonist; Ei, ERK1/2 inhibitor; Aa, AC agonist; Pi, PLC inhibitor.

Article Snippet: GPR81, PA5-114741, Thermo; AC (55067-1-AP, Proteintech), PKA (4782S, CST), RAP1 (2399S, CST), PKC (SC-80 Santa), PLC (14247S, CST), Raf1 (9422S, CST), MSK1 (3489S, CST), P90RSK (9355S, CST), ERK (4370T, CST), CREB (9197S, CST), c-Fos (2250S, CST), Zif268 (4154S, CST), NMDAR (5704S, CST), SYN (4329S, CST), BDNF (28205-1-AP, Proteintech), ARC (16290-1-AP, Proteintech), PSD95 (3450S, CST) p-Raf1 (9421S, CST), p-ERK (4370T, CST), p-MSK (9595S, CST), p-P90RSK (11989S, CST), p-CREB (9198S, CST), Tubulin (10068-1-AP, Proteintech), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00001-2, Proteintech), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (SA00001-1, Proteintech).

Techniques: Expressing, Western Blot

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) Violin plots of expression of top-ranked transcription factors with enriched motifs in differentially expressed genes from cluster 9. This plot shows log 2 -transformed expression in cluster 9 in Tsc2 fl/fl SynCre − and Tsc2 del/fl SynCre + animals. (B) qPCR of dissected lower-layer regions of the cortex comparing Tsc2 del/fl SynCre + to Tsc2 fl/fl SynCre − animals. Bar plot shows mean ± SEM. * p < 0.05 ( n = 3 animals per genotype; t test). (C) Immunohistochemistry of EGR1 in the lower layer of the cortex from Tsc2 fl/fl SynCre − (left) and Tsc2 del/fl SynCre + (right) animals. These sections are also stained for BCL11B, a marker of layer 5 pyramidal neurons. Scale bar = 20 μm. (D) Quantification of EGR1 signal intensity within BCL11B + neurons of the cortex across genotypes. Each dot represents a different animal, which is the mean of 2–3 images per slice and 3–4 slices per animal. Bar plot shows mean ± SEM. * p < 0.05 ( n = 4; t test). (E) Immunoblotting of EGR1 across all genotypes of neurons on day 30 of differentiation and quantification of EGR1 level compared to total protein across all genotypes. * p < 0.05 ( n = 4 separate differentiations; ANOVA with Dunnett’s test).

Article Snippet: Antibodies used included: EGR1, Cell Signaling Technology, 4153S, 1:1000; TAP1, Proteintech, 11114–1-AP, 1:1000; PSMB8, Proteintech, 14859–1-AP, 1:1000; PSME1, Proteintech, 10543–1-AP, 1:1000; DNMT1, Proteintech, 24206–1-AP, 1:200; Phospho-CREB Ser133, Cell Signaling Technology, 9198S, 1:1000; CREB, EMD Millipore, 06–863, 1:1000; Phospho-S6 Ser235/236, Cell Signaling Technology, 2211, 1:1000; S6, Cell Signaling Technology, 2317, 1:1000.

Techniques: Expressing, Transformation Assay, Immunohistochemistry, Staining, Marker, Western Blot

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: Antibodies used included: EGR1, Cell Signaling Technology, 4153S, 1:1000; TAP1, Proteintech, 11114–1-AP, 1:1000; PSMB8, Proteintech, 14859–1-AP, 1:1000; PSME1, Proteintech, 10543–1-AP, 1:1000; DNMT1, Proteintech, 24206–1-AP, 1:200; Phospho-CREB Ser133, Cell Signaling Technology, 9198S, 1:1000; CREB, EMD Millipore, 06–863, 1:1000; Phospho-S6 Ser235/236, Cell Signaling Technology, 2211, 1:1000; S6, Cell Signaling Technology, 2317, 1:1000.

Techniques: Luciferase, Expressing, Derivative Assay, Western Blot, 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) Methylation of the EGR1 promoter in TSC2 +/+ (blue) and TSC2 −/− (red) neurons throughout differentiation. The y axis represents the log 10 p value of change in methylation of individual CpG sites, which is given a sign based on the direction of the change (negative corresponds to demethylation in neurons compared to stem cells; positive corresponds to hypermethylation in neurons compared to stem cells). The x axis shows the genomic coordinates of the EGR1 locus, and the features of this region are shown below. (B) Volcano plot of hypomethylated regions in TSC2 +/+ neurons, comparing methylation levels of these regions in TSC2 +/+ neurons compared to TSC2 +/+ hiPSCs. (C) Volcano plot of hypomethylated regions in TSC2 +/+ neurons, comparing methylation levels of these regions in TSC2 −/− neurons compared to TSC2 −/− hiPSCs. (D) Quantile-quantile plot of log 10 -transformed p values of demethylated genes in TSC2 +/+ vs. TSC2 −/− neurons, where the red line indicates the identity. The deviation of the quantiles demonstrates more significant demethylation of regions in the TSC2 +/+ neurons compared to the TSC2 −/− neurons. (E) Highest-ranked motif across regions that showed impaired demethylation in TSC2 −/− neurons. (F) Bar plot of observed/expected genomic features overlapping with regions that showed impaired demethylation in TSC2 −/− neurons. * p < 0.05 (chi-squared test).

Article Snippet: Antibodies used included: EGR1, Cell Signaling Technology, 4153S, 1:1000; TAP1, Proteintech, 11114–1-AP, 1:1000; PSMB8, Proteintech, 14859–1-AP, 1:1000; PSME1, Proteintech, 10543–1-AP, 1:1000; DNMT1, Proteintech, 24206–1-AP, 1:200; Phospho-CREB Ser133, Cell Signaling Technology, 9198S, 1:1000; CREB, EMD Millipore, 06–863, 1:1000; Phospho-S6 Ser235/236, Cell Signaling Technology, 2211, 1:1000; S6, Cell Signaling Technology, 2317, 1:1000.

Techniques: Methylation, Transformation 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) Heatmap of correlation of gene expression between hiPSC-derived neurons of all three genotypes that were treated with rapamycin or vehicle starting on day of differentiation ( n = 6–8 per genotype per treatment). TSC2 −/− vehicle-treated neurons form a distinct cluster, while TSC2 −/− rapamycin-treated neurons are similar to other genotypes of neurons treated with rapamycin. (B) Heatmap of correlation of gene expression between hiPSC-derived neurons of all three genotypes that were treated with rapamycin or vehicle starting on day 30 of differentiation. TSC2 −/− rapamycin-treated neurons remain distinct from other genotypes and are more closely related to TSC2 −/− vehicle-treated neurons. (C) Gene expression from the co-expression module (ME9) that is most strongly associated with persistent gene expression changes after late rapamycin treatment. (D) Gene Ontology analysis of genes from ME9 that shows persistent changes in genes related to synapses and DNA binding. (E) GSEA of genes near five or more distal enhancers that do not show demethylation during differentiation of TSC2 −/− neurons across ME9. The green line shows the weighted cumulative sum of enrichment of EGR1 target genes, denoted by black lines across the x axis. The color at the bottom of the graph denotes the correlation of all genes with ME9 (red, positive correlation; blue, negative correlation). (F) Cumulative distribution function for k ME in ME9 of EGR1 target genes (red) compared to all other genes (gray).

Article Snippet: Antibodies used included: EGR1, Cell Signaling Technology, 4153S, 1:1000; TAP1, Proteintech, 11114–1-AP, 1:1000; PSMB8, Proteintech, 14859–1-AP, 1:1000; PSME1, Proteintech, 10543–1-AP, 1:1000; DNMT1, Proteintech, 24206–1-AP, 1:200; Phospho-CREB Ser133, Cell Signaling Technology, 9198S, 1:1000; CREB, EMD Millipore, 06–863, 1:1000; Phospho-S6 Ser235/236, Cell Signaling Technology, 2211, 1:1000; S6, Cell Signaling Technology, 2317, 1:1000.

Techniques: Gene Expression, Derivative Assay, Expressing, Binding Assay

Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: EGR1 binds at the PKD1 proximal promoter. a The percentage of recovery relative to input chromatin was measured using qPCR with primers flanking the putative EGR1 binding site. The binding of EGR1 at the EGR1 consensus of the PKD1 promoter was confirmed by the recovery of immunoprecipitated fragments for all 3 conditions tested after PMA-induced differentiation, no treatment (GM + Nil), AngII (GM + AngII) and AngII/pioglitazone (GM + A + P). Undifferentiated THP-1 cells (GM only) express just detectable levels of EGR1, and therefore, this condition was used as a negative control of EGR1 recovery from the EGR1 binding locus and for comparison with the other conditions. b Non-specific background recovery was very low, as measured by ChIP using an IgG antibody. c Primers designed around a non-EGR1-specific locus within the proximal promoter of PKD1 also showed very low recovery, confirming the specificity of the EGR1 recovery. The data represent the means ± standard deviation from 3 independent experiments with p values as indicated.

Article Snippet: EGR1 short hairpin RNA (shRNA) (sc-29303-V; Santa Cruz, Insight Biotech) lentiviral particles (5,000 IFU/μl) that carry constructs encoding 3–5 target-specific shRNAs and a puromycin antibiotic resistance cassette were used at a multiplicity of infection of 5:1.

Techniques: Binding Assay, Immunoprecipitation, Negative Control, Comparison, Standard Deviation

Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: Analyses of available ChIP-seq data reveal a conserved EGR1 binding peak within the PKD1 proximal promoter of multiple cell lines. a Duplicate EGR1 binding peak coordinates for PKD1 were extracted from NCBI GEO (accession number GSE32465; part of the ENCODE project). Overlapping peaks were merged and graphically aligned to genomic loci (hg19) using R code based on Gviz and ggplot2 packages. A common conserved EGR1 binding peak within the PKD1 proximal promoter was identified in the above cell lines. The widths in the bar graphs indicate the position and heights proportional to the average ‘score’ values provided by ENCODE data. b Sequence of the conserved EGR1 binding peak within the PKD1 proximal promoter. c Four out of the above 5 cell lines also harbour EGR1 binding peaks within the PKD2 promoter.

Article Snippet: EGR1 short hairpin RNA (shRNA) (sc-29303-V; Santa Cruz, Insight Biotech) lentiviral particles (5,000 IFU/μl) that carry constructs encoding 3–5 target-specific shRNAs and a puromycin antibiotic resistance cassette were used at a multiplicity of infection of 5:1.

Techniques: ChIP-sequencing, Binding Assay, Sequencing

Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: Expression of PKD1 and EGR1 in THP-1 cells. Relative RNA expression of PKD1 (a) and EGR1 (b). a, b The THP-1 cells were induced for 4 hours with PMA (t = 0), then the PMA was washed out and AngII (GM + AngII), AngII/pioglitazone (GM + A+P) or no treatment control (GM + Nil) were applied for the time points indicated on the x-axis. No treatment control THP-1 cells after 24 h were set as reference for comparison with other treatments/time points (relative expression = 1). Data represent the means ± standard error of the mean from 3 independent experiments with p values as indicated. Rel. = Relative; arb. = arbitrary. c Representative staining after 48 h of treatment. Polycystin-1 expression was assessed by immunocytochemistry under the same conditions. Polycystin-1 was localised at the cell periphery (arrowheads). The data are representative of 3 independent experiments, with i-iii representing 100% magnifications of the boxed areas in corresponding merged images; scale bars as indicated.

Article Snippet: EGR1 short hairpin RNA (shRNA) (sc-29303-V; Santa Cruz, Insight Biotech) lentiviral particles (5,000 IFU/μl) that carry constructs encoding 3–5 target-specific shRNAs and a puromycin antibiotic resistance cassette were used at a multiplicity of infection of 5:1.

Techniques: Expressing, RNA Expression, Control, Comparison, Staining, Immunocytochemistry

Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: Expression of PKD1 and EGR1 in EGR1 shRNA lentivirally transduced THP-1 cells. Relative expression of PKD1 (a) and EGR1 (b). The THP-1 cells were induced for 12 h with PMA (t = 0), then PMA was washed out and no treatment control (Nil), AngII (AngII) or AngII/pioglitazone (A + P) were applied for the time points indicated on the x-axis. No treatment control THP-1 cells after 24 h were set as reference for comparison with other treatments/time points (relative expression = 1). Data represent the average ± standard error of the mean from 3 independent experiments with p values as indicated. Rel. = Relative; arb. = arbitrary.

Article Snippet: EGR1 short hairpin RNA (shRNA) (sc-29303-V; Santa Cruz, Insight Biotech) lentiviral particles (5,000 IFU/μl) that carry constructs encoding 3–5 target-specific shRNAs and a puromycin antibiotic resistance cassette were used at a multiplicity of infection of 5:1.

Techniques: Expressing, shRNA, Control, Comparison

Journal: Journal of Vascular Research

Article Title: Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

doi: 10.1159/000430986

Figure Lengend Snippet: Effect of Egr1 deficiency on changes in aortic diameter in CaCl 2 -induced aneurysm formation

Article Snippet: EGR1 short hairpin RNA (shRNA) (sc-29303-V; Santa Cruz, Insight Biotech) lentiviral particles (5,000 IFU/μl) that carry constructs encoding 3–5 target-specific shRNAs and a puromycin antibiotic resistance cassette were used at a multiplicity of infection of 5:1.

Techniques: