Journal: American Journal of Translational Research

Article Title: MiR-199a-5p inhibition protects cognitive function of ischemic stroke rats by AKT signaling pathway

doi:

Figure Lengend Snippet: Expression of miR-199a-5p and AKT signaling pathway in ischemic stroke rats. (A) Bioinformatics prediction of binding sites on the 3’UTR between miR-199a-5p and AKT. (B) Dual luciferase reporter assay; *P < 0.05, compared with the NC mimic group. The mRNA (C) and protein (D, E) expression level of miR-199a-5p, AKT and mTOR mRNA in each group of rats were detected. oP < 0.05, compared to the Normal group; ●P < 0.05, compared with the Sham group; □P < 0.05, compared with the Model group; ■P < 0.05, compared with the miR-199a-5p inhibitor group; ΔP < 0.05, compared with the IGF-1 group.

Article Snippet: Grouping Rats were grouped into Normal group (normal rats), Sham group (rats received sham operation), Model group (MCAO model rats), miR-199a-5p inhibitor group (MCAO model rats transfected with miR-199a-5p inhibitor), IGF-1 group (MCAO model rats treated with AKT signaling pathway activator), miR-199a-5p inhibitor + IGF-1 group (MCAO model rats transfected with miR-199a-5p inhibitor and treated with AKT signaling pathway activator). miR-199a-5p inhibitor and IGF-1 were purchased from MedChemExpress LLC.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay

Journal: American Journal of Translational Research

Article Title: MiR-199a-5p inhibition protects cognitive function of ischemic stroke rats by AKT signaling pathway

doi:

Figure Lengend Snippet: Down-regulation of miR-199a-5p or activation of AKT signaling pathway improves the behaviors of ischemic stroke rats. A. Statistical analysis of the time used by the rats in each group at 180° head down. B. Statistical analysis of the time taken by each group of rats to reach the ground. C. Results of the trial of the rats in each group. oP < 0.05, compared to the Normal group; ●P < 0.05, compared with the Sham group; □P < 0.05, compared with the Model group; ■P < 0.05, compared with the miR-199a-5p inhibitor group; ΔP < 0.05, compared with the IGF-1 group.

Article Snippet: Grouping Rats were grouped into Normal group (normal rats), Sham group (rats received sham operation), Model group (MCAO model rats), miR-199a-5p inhibitor group (MCAO model rats transfected with miR-199a-5p inhibitor), IGF-1 group (MCAO model rats treated with AKT signaling pathway activator), miR-199a-5p inhibitor + IGF-1 group (MCAO model rats transfected with miR-199a-5p inhibitor and treated with AKT signaling pathway activator). miR-199a-5p inhibitor and IGF-1 were purchased from MedChemExpress LLC.

Techniques: Activation Assay

Journal: American Journal of Translational Research

Article Title: MiR-199a-5p inhibition protects cognitive function of ischemic stroke rats by AKT signaling pathway

doi:

Figure Lengend Snippet: Down-regulation of miR-199a-5p or activation of AKT signaling pathway reduce cerebral infarction area in ischemic stroke rats. A. Brain tissues stained by TTC in each group; B. Cerebral infarct volume of each group. oP < 0.05, compared to the Normal group; ●P < 0.05, compared with the Sham group; □P < 0.05, compared with the Model group; ■P < 0.05, compared with the miR-199a-5p inhibitor group; ΔP < 0.05, compared with the IGF-1 group.

Article Snippet: Grouping Rats were grouped into Normal group (normal rats), Sham group (rats received sham operation), Model group (MCAO model rats), miR-199a-5p inhibitor group (MCAO model rats transfected with miR-199a-5p inhibitor), IGF-1 group (MCAO model rats treated with AKT signaling pathway activator), miR-199a-5p inhibitor + IGF-1 group (MCAO model rats transfected with miR-199a-5p inhibitor and treated with AKT signaling pathway activator). miR-199a-5p inhibitor and IGF-1 were purchased from MedChemExpress LLC.

Techniques: Activation Assay, Staining

Journal: American Journal of Translational Research

Article Title: MiR-199a-5p inhibition protects cognitive function of ischemic stroke rats by AKT signaling pathway

doi:

Figure Lengend Snippet: Down-regulation of miR-199a-5p or activation of AKT signaling pathway can inhibit neuronal apoptosis in ischemic stroke rats. A. TUNEL fluorescence staining results of hippocampal CA1 area in each group (200×). B. Apoptosis rate of hippocampal CA1 region in each group of rats. C. Expression of apoptosis related proteins. D. Quantification results of protein expression. oP < 0.05, compared to the Normal group; ●P < 0.05, compared with the Sham group; □P < 0.05, compared with the Model group; ■P < 0.05, compared with the miR-199a-5p inhibitor group; ΔP < 0.05, compared with the IGF-1 group.

Article Snippet: Grouping Rats were grouped into Normal group (normal rats), Sham group (rats received sham operation), Model group (MCAO model rats), miR-199a-5p inhibitor group (MCAO model rats transfected with miR-199a-5p inhibitor), IGF-1 group (MCAO model rats treated with AKT signaling pathway activator), miR-199a-5p inhibitor + IGF-1 group (MCAO model rats transfected with miR-199a-5p inhibitor and treated with AKT signaling pathway activator). miR-199a-5p inhibitor and IGF-1 were purchased from MedChemExpress LLC.

Techniques: Activation Assay, TUNEL Assay, Fluorescence, Staining, Expressing

Journal: Molecular Biomedicine

Article Title: Epithelial membrane protein 1 drives hepatic stellate cell activation via the TLN1/FAK cascade in MASLD donor liver transplantation

doi: 10.1186/s43556-025-00371-7

Figure Lengend Snippet: EMP1 localizes to hepatic non-substantive cells and induces HSC activation. ND: untreated control group; OA/PA: group using oleic acid (OA) + palmitic acid (PA) to simulate MASLD environment; I/R: group treated with hypoxia/reoxygenation incubator to simulate IRI; OA/PA + I/R: group simulating MASLD environment and treated with IRI. a , b . Cellular localization of EMP1 in the liver was resolved using the UCSC CellBrowser single-cell sequencing database, and it was found that EMP1 was mainly localized to hepatic non-substantive cells, especially endothelial cells, stellate cells, and inflammatory macrophages. c , d . Protein ( n = 3/group) and mRNA ( n = 5/group) expression of EMP1 in different LNPCs cell lines under OA/PA + IRI. e , f . Protein ( n = 3/group) and mRNA ( n = 5/group) expression of EMP1 in LNPCs cell lines under different intervention conditions of single (OA/PA or IRI) and combined (OA/PA + IRI) injury. g . By double immunofluorescence staining of hepatic tissues with EMP1, α-SMA, and α-SMA, it was observed that EMP1 co-localized with activated HSC and showed a co-regulation in expression ( n = 3/group). SMA double immunofluorescence staining, it was observed that EMP1 was co-localized with activated HSC and co-expressed with up-regulation of expression ( n = 3/group). Where red is EMP1, green is α-SMA, and blue is DAPI cell nuclei. The scale bar = 50 μM, shown in the lower left corner, and the red box shows the local zoomed image, where the yellow part is red + green co-localized EMP1 in activated HSC. h . Correlation of EMP1 with the expression of HSC activation markers and inflammatory factors in the liver as demonstrated by raw letter analysis of the GEPIA2 database. i. Immunofluorescence staining was performed to detect α-SMA, under the overexpression or silencing of EMP1. α-SMA is shown in green, and the nuclei of the cells in blue with DAPI, which was labeled as a red scale bar due to the high staining luminance. The scale bar = 100 μm ( n = 3/group). j , k . Construction of OE-EMP1, sh-EMP1 by plasmid transfection cell lines, HSC activation was induced by administration of TGF-β, and it was detected whether the expression of EMP1 interfered with HSC activation at the mRNA ( n = 5/group) and protein ( n = 3/group) levels. All data used for statistics above are SD ± mean, n.s > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, and were analyzed by two-way ANOVA or t-test followed by Tukey test

Article Snippet: TGF-β (HY- P70648 ), Defactinib (HY-12289A), CHX (HY-12320), and MG132 (HY-13259) were purchased from MedChemExpress.

Techniques: Activation Assay, Control, Sequencing, Expressing, Double Immunofluorescence Staining, Immunofluorescence, Staining, Over Expression, Labeling, Plasmid Preparation, Transfection

Journal: Molecular Biomedicine

Article Title: Epithelial membrane protein 1 drives hepatic stellate cell activation via the TLN1/FAK cascade in MASLD donor liver transplantation

doi: 10.1186/s43556-025-00371-7

Figure Lengend Snippet: EMP1 mediates HSC activation indirectly through FAK phosphorylation. a , b . Using NC and OE-EMP1 cell lines again transcriptome sequencing ( n = 3/group), heatmaps and volcano plots showed significant differences. c . Enrichment analysis of the differential genes based on the KEGG database was performed and shown in bubble plots, again enrichment to FA signaling pathway genes with a large number of significant differences. d . Correlation of EMP1 expression in liver with key genes in FA signaling pathway, such as PTK2, PIK3C, AKT3, etc., was analyzed by using GEPIA2. e . Detection of the role of EMP1 expression modulation in FAK and its downstream AKTs in the FA signaling pathway in HSC cell lines under TGF-β-induced activation ( n = 4/group). f . Under the intervention of FAK phosphorylation inhibitor Defactinib intervention, the effect of EMP1 expression modulation on FAK-AKT phosphorylated protein levels was again examined ( n = 4/group). g . Detection of HSC activation and inflammatory protein expression under OE-EMP1, sh-EMP1 or FAK phosphorylation inhibition intervention ( n = 3/group). h . Detection of HSC activation and mRNA expression of inflammatory markers under FAK phosphorylation inhibition ( n = 5/group). i . CoIP assay of EMP1 with FAK by constructing GFP-EMP1-Flag cell line. All data used for statistics above are SD ± mean, n.s > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, and were analyzed by two-way ANOVA or t-test followed by Tukey test

Article Snippet: TGF-β (HY- P70648 ), Defactinib (HY-12289A), CHX (HY-12320), and MG132 (HY-13259) were purchased from MedChemExpress.

Techniques: Activation Assay, Phospho-proteomics, Sequencing, Expressing, Inhibition, Co-Immunoprecipitation Assay

Journal: Molecular Biomedicine

Article Title: Epithelial membrane protein 1 drives hepatic stellate cell activation via the TLN1/FAK cascade in MASLD donor liver transplantation

doi: 10.1186/s43556-025-00371-7

Figure Lengend Snippet: EMP1 selective silencing inhibition attenuates MASLD-IRI-induced liver injury by inhibiting HSC activation via TLN1/FAK. Figure 7 The same 4 groups of animal model treatments were performed as in Fig. . The difference is that AAV-shNC and AAV-shEMP1 injections were given to the donors in each group separately, and the area was divided into 8 groups. a , b . Detection of liver injury markers ALT and AST in serum to check the overall level of liver injury ( n = 5/group). c , d . Detection of oxidative products SOD and MDA in liver to reveal the level of oxidative stress-related injury ( n = 5/group). e , f . Pathological examination of rat liver tissues to observe the type of liver injury, H&E showed tissue edema, structural disorders, and necrosis in liver. Tunel staining demonstrated the degree of liver cell death in different models, both scale bar = 50 μm(n = 3/group). g . By double immunofluorescence staining of liver tissues with EMP1 and α-SMA, it was observed that EMP1 co-localized with activated HSC, and there was a common up-regulation of the expression in terms of the expression amount. Where red is EMP1, green is α-SMA, and blue is DAPI cell nuclei. The scale bar = 100 μm ( n = 3/group). h . Detection of mRNA expression of HSC activation, and level of inflammation in ND-sham and HFD-LT hepatic tissues after EMP1-specific silencing (n = 5/group). i. Detection of protein expression ( n = 3/group) of EMP1/TLN1/FAK phosphorylation, degree of HSC activation, and level of inflammation after EMP1-specific silencing. j . AAV-shNC, AAV-shEMP1 primary hepatic stellate cells were extracted and grouped by whether or not they were transfected with si-TLN1 and whether or not they were given a FAK phosphorylation inhibitor, and TGF-β was given to stimulate hepatic stellate cell activation, and the effect of EMP1 or TLN1 silencing on the phosphorylation levels of the EMP1/TLN1/FAK/AKT proteins was examined ( n = 4/group). All data used for statistics above are SD ± mean, n.s > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, and were analyzed by two-way ANOVA or t-test followed by Tukey test

Article Snippet: TGF-β (HY- P70648 ), Defactinib (HY-12289A), CHX (HY-12320), and MG132 (HY-13259) were purchased from MedChemExpress.

Techniques: Inhibition, Activation Assay, Animal Model, TUNEL Assay, Staining, Double Immunofluorescence Staining, Expressing, Phospho-proteomics, Transfection