Journal: Molecular Medicine Reports

Article Title: Asprosin activates multiple placental pathways in vitro : Evidence for potential involvement in angiogenesis, fatty acid metabolism and the mTOR, NOTCH and WNT signalling pathways

doi: 10.3892/mmr.2025.13674

Figure Lengend Snippet: DEGs in BeWo and JEG-3 cells treated with Asp. (A) A Venn diagram displaying the DEGs for Asp-treated BeWo and JEG-3 cells when compared with their respective UNT controls. (B and C) Geneset signature UMAP displaying up- and down-regulated genes in different samples. UMAPs display genes clustered by relative log-expression which were up- (red) or down- (blue) regulated in (B) BeWo and (C) JEG-3 cells, UNT control and 10 nM Asp-treated samples. (D) Volcano plots for BeWo and JEG-3 cells, indicating the most up- and down-regulated DEGs. RNA sequencing validation for (E) BeWo and (F) JEG-3 cells, using reverse transcription-quantitative PCR. *P<0.05, **P<0.001. DEGs, differentially expressed genes; UMAP, uniform manifold approximation and projection; UNT, untreated; Asp, asprosin; ZNF395, Zinc Finger Protein 395; DDIT4, DNA Damage Inducible Transcript 4; SLC2A1, solute carrier family 2 member 1; Non-ST, non-syncytialised; ns, not significant.

Article Snippet: DDIT4 , DNA damage inducible transcript 4 , 68 , Hs01111686_g1.

Techniques: Expressing, Control, RNA Sequencing, Biomarker Discovery, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: ATF4 activates a transcriptional program that chronically suppresses mTOR activity promoting neurodegeneration in Parkinson’s disease models

doi: 10.1101/2025.06.09.658667

Figure Lengend Snippet: A. Cortical neurons were transduced with Ad-ATF4 or Ad-GFP (50-MOI) for 24 hours and mRNA levels of Sesn2, Trib3 and Ddit4 were determined by qRT-PCR. Expression is reported as fold increase over untransduced neurons from the same culture (n=5; t-test, *p<0.05). B. ATF4+/+ and ATF4-/- cortical neurons were treated with MPP+ (50µM) or 6-OHDA (10µM) for 16 hours and Sesn2, Trib3 and Ddit4 mRNA levels was determined by qRT-PCR. Expression is reported as fold increase over vehicle treated neurons from the same culture (n=3-4; t-test, *p<0.05). C. ATF4+/+ and ATF4-/- cortical neurons were treated with MPP+ (50μM) or 6-OHDA (10μM) for the indicated times and SESN2 and DDIT4 protein levels were assessed by western blot and quantified by densitometry and normalized to the loading control Actin or Cyclophilin B (n=3; 2-way-ANOVA *p<0.05). D. Mesencephalic neurons derived from ATF4+/+ and ATF4-/- mice were treated with MPP+ (25μM) or 6-OHDA (5μM) for 16 hours and Sesn2, Trib3 and Ddit4 mRNA levels were assessed in dopaminergic neurons using multiplex RNAScope fluorescence in situ hybridization combined with tyrosine hydroxylase immunostaining. Representative images showing PD neurotoxin induced expression of Sesn2, Trib3 and Ddit4 mRNA fluorescence puncta in wildtype and ATF4-deficient dopaminergic (TH+) neurons (left scale bar=20uM, right scale bar=5uM). Data represent the mean ± SEM number of transcripts per DA neuron (n=3, 2-way-ANOVA, *p<0.05). E. Wildtype and ATF4-deficient mesencephalic neurons were seeded with α-Syn PFFs (5μg/ml) for 9 days and Sesn2, Trib3 and Ddit4 mRNA levels were assessed in dopaminergic neurons using multiplex RNAScope fluorescence in situ hybridization combined with tyrosine hydroxylase immunostaining. Data represent the mean ± SEM number of transcripts per DA neuron (n=3, t-test, *p<0.05). F-H. RNA was isolated from N2 animals or atfs-1 LoF animals treated with MPP+ [100uM] or 6-OHDA [100uM] and a-syn or a- syn::atfs-1LoF animals at indicated time points. Heat maps represent fold change in gene expression over time as calculated by RT-qPCR. Values were normalized to act-1 housekeeping mRNA and fold change was calculated based on normalized mRNA levels in untreated or wildtype animals at respective time points (n=3, 2way-ANOVA, *p<0.05).

Article Snippet: Lentiviruses expressing shRNAs specifically targeting Trib3, SESN2 and DDIT4 were purchased from Vectorbuilder (shCTRL VB# VB010000-009mxc, shTrib3 VB# VB230127- 1198jed, shSESN2 VB# VB230127-1195msp, shDDIT4 VB# VB230127-1197gke).

Techniques: Transduction, Quantitative RT-PCR, Expressing, Western Blot, Control, Derivative Assay, Multiplex Assay, RNAscope, Fluorescence, In Situ Hybridization, Immunostaining, Isolation, Gene Expression

Journal: bioRxiv

Article Title: ATF4 activates a transcriptional program that chronically suppresses mTOR activity promoting neurodegeneration in Parkinson’s disease models

doi: 10.1101/2025.06.09.658667

Figure Lengend Snippet: A. Cortical neurons were transduced with lenti-shRNA vectors targeting either Sesn2, Trib3, or Ddit4. After 5 days, neurons were treated with MPP+ (50μM) or 6-OHDA (10μM) for 24 hours or seeded with α-Syn PFFs (5μg/ml) for an additional 9 days. The levels of p-mTOR (S2448), p-S6 (S235/236), p-AKT (S473) and p- FOXO3a (S253) and corresponding total protein was assessed by western blot and quantified by densitometry. The ratio of phosphorylated protein/total protein is reported relative to untreated control levels for each shRNA vector (n=5; 2-way-ANOVA, *p<0.05). B. Mesencephalic neuron cultures were transduced with lenti-shRNA and after 5 days treated with MPP+ (25μM) or 6-OHDA (5μM) for 48 hours or seeded with α-Syn PFFs (5μg/ml) for an additional 12 days. The number of TH+ neurons was quantified and is reported relative to TH+ counts in vehicle treated neurons from the same culture. Data represents the mean ± SEM and statistical differences were determined by ANOVA (n=4; *p<0.05).

Article Snippet: Lentiviruses expressing shRNAs specifically targeting Trib3, SESN2 and DDIT4 were purchased from Vectorbuilder (shCTRL VB# VB010000-009mxc, shTrib3 VB# VB230127- 1198jed, shSESN2 VB# VB230127-1195msp, shDDIT4 VB# VB230127-1197gke).

Techniques: Transduction, shRNA, Western Blot, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: ATF4 activates a transcriptional program that chronically suppresses mTOR activity promoting neurodegeneration in Parkinson’s disease models

doi: 10.1101/2025.06.09.658667

Figure Lengend Snippet: A. Cortical or neurons were transduced with recombinant adenoviral vectors (50-MOI) expressing GFP alone or co-expressing GFP and either SESN2, DDIT4 or Trib3 alone or indicated combinations for 24 hours and p-mTOR(S2448)/total mTOR was assessed by western blot and quantified by densitometry (n=3; ANOVA, *p<0.05). B. PUMA+/+ and PUMA-/- cortical neurons were co-transduced with Ad-Trib3/Ad- SESN2/Ad-DDIT4 or Ad-EGFP (3X) for 24 hours and p-mTOR (S2448), p-S6 (S235/236), p-AKT (S473) and p-Foxo3a (S253) levels were determined by western blot and quantified by densitometry (n=3; 2-way ANOVA *p<0.05). C. Mesencephalic neuron cultures were transduced with indicated vectors and dopamine neuron survival was assessed by TH+ counts at 48 hours. Survival is reported relative to untransduced neurons from the same culture (n=3; ANOVA *p<0.05). D. Midbrain neurons were treated with the dual mTOR inhibitor OSI-027 (5μM) for 24 hours and then co-immunostained for tyrosine hydroxylase (TH) and PUMA. Confocal images were acquired and the fluorescence intensity of PUMA was quantified in dopaminergic (TH+) neurons from 3 independent experiments (n=17-23 cells per group; t- test, *p<0.05). E. PUMA+/+ and PUMA-/- cortical neurons were treated with OSI-027 (5μM) and PUMA protein levels were assessed by western blot and quantified by densitometry and normalized to the loading control Actin (n=3; ANOVA *p<0.05). F. Midbrain neurons derived from PUMA+/+ and PUMA-/- mice were treated with OSI-027 for 48 hours and the number of TH+ cells was counted and reported relative to TH+ counts in vehicle treated neurons from the same culture. Data represents the mean ± SEM and statistical differences were determined by 2-way-ANOVA (n=4; *p<0.05). G. PUMA+/+ and PUMA-/- midbrain neuron cultures were transduced with Ad-Trib3/Ad-SESN2/Ad-DDIT4 or Ad-EGFP (3X) and DA neuron survival was assessed by TH+ cell counts after 48 hours (n=3; ANOVA *p<0.05).

Article Snippet: Lentiviruses expressing shRNAs specifically targeting Trib3, SESN2 and DDIT4 were purchased from Vectorbuilder (shCTRL VB# VB010000-009mxc, shTrib3 VB# VB230127- 1198jed, shSESN2 VB# VB230127-1195msp, shDDIT4 VB# VB230127-1197gke).

Techniques: Transduction, Recombinant, Expressing, Western Blot, Fluorescence, Control, Derivative Assay

Journal: Biomedical Reports

Article Title: Resveratrol ameliorates high‑fat diet‑induced insulin resistance via the DDIT4/mTOR pathway in skeletal muscle

doi: 10.3892/br.2025.1977

Figure Lengend Snippet: Expression of DDIT4, mTOR, GLUT4, PI3K, and AKT in MHY1485-treated IR C2C12 cells. (A) Protein expression levels of DDIT4, mTOR, GLUT4, PI3K, AKT after the activation of mTOR. (B) Semi-quantification of p-protein/t-protein ratios for mTOR, PI3K, AKT and protein expression levels of DDIT4, GLUT4 after MHY1485 treatment. (C) Glucose levels in culture medium after MHY1485 treatment. (D) Cell TG levels after MHY1485 treatment. Data are presented as the mean ± SD (n=3). * P<0.05 vs. the CON group; # P<0.05 vs. the PA group; & P<0.05 vs. the PA + RSV group. DDIT4, DNA-damage-inducible transcript 4; mTOR, mammalian target of rapamycin; GLUT4, glucose transporter 4; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; IR, insulin resistance; p-, phosphorylated; t-, total; TG, triglyceride; CON, control; PA, palmitic acid; RSV, resveratrol.

Article Snippet: Some of the PA + RSV group cells were again divided into two groups: The PA + RSV + MHY1485 group C2C12 cells were treated with 10 μM MHY1485 (cat. no. HY-B0795; MedChemExpress) to activate mTOR, and the PA + RSV + DDIT4-siRNA group C2C12 cells were transfected with a small interfering (si)RNA against DDIT4 (DDIT4-siRNA; Shanghai Genechem Co. Ltd.), all incubated at 37 ̊C for 24 h. The cells were collected for western blotting and cellular TG content determination.

Techniques: Expressing, Activation Assay, Control

Journal: Biomedical Reports

Article Title: Resveratrol ameliorates high‑fat diet‑induced insulin resistance via the DDIT4/mTOR pathway in skeletal muscle

doi: 10.3892/br.2025.1977

Figure Lengend Snippet: Expression levels of DDIT4, mTOR, GLUT4, PI3K, and AKT in DDIT4-siRNA-treated IR C2C12 cells. (A) DDIT4-siRNA-1 had the most significant silencing effect. (B) Protein expression levels of DDIT4, mTOR, GLUT4, PI3K, and AKT after DDIT4-siRNA treatment. (C) Semi-quantification of p-protein/t-protein ratios for mTOR, PI3K, and AKT and protein expression levels of DDIT4 and GLUT4 after DDIT4-siRNA treatment. (D) Glucose levels in culture medium after DDIT4-siRNA treatment. (E) TG levels of C2C12 cells after DDIT4-siRNA treatment. Data are presented as the mean ± SD (n=3). * P<0.05 vs. the CON group; # P<0.05 vs. the PA group; & P<0.05 vs. the PA + RSV group. DDIT4, DNA-damage-inducible transcript 4; mTOR, mammalian target of rapamycin; GLUT4, glucose transporter 4; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; siRNA, small interfering RNA; IR, insulin resistance; p-, phosphorylated; t-, total; TG, triglyceride; CON, control; PA, palmitic acid; RSV, resveratrol.

Article Snippet: Some of the PA + RSV group cells were again divided into two groups: The PA + RSV + MHY1485 group C2C12 cells were treated with 10 μM MHY1485 (cat. no. HY-B0795; MedChemExpress) to activate mTOR, and the PA + RSV + DDIT4-siRNA group C2C12 cells were transfected with a small interfering (si)RNA against DDIT4 (DDIT4-siRNA; Shanghai Genechem Co. Ltd.), all incubated at 37 ̊C for 24 h. The cells were collected for western blotting and cellular TG content determination.

Techniques: Expressing, Small Interfering RNA, Control