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