Journal: Experimental and Therapeutic Medicine

Article Title: Tumor-associated macrophages suppress estrogen receptor-β expression in triple-negative breast cancer through the PI3K/AKT pathway

doi: 10.3892/etm.2026.13072

Figure Lengend Snippet: Tumor-associated macrophages suppress ERβ expression through the PI3K/AKT pathway. (A) Top 10 Kyoto Encyclopedia of Genes and Genomes enrichment pathways. (B) PI3K/AKT pathway activity in high and low breast cancer MSR1 expression groups. (C) Immunohistochemistry for MSR1 and p-AKT levels in the control and clodronate-treated groups. Original magnification, x200, scale bars, 100 µm. (D) 4T1 cells cultured in CM then treated DMSO or 100 nM, 1 µM or 10 µM of LY294002 for 24 h. Relative mRNA levels of ESR2 in different groups (n=4). (E) Co-localization of p-AKT and ER β in 4T1 cells. Representative cellular immunofluorescence images of 4T1 cells treated with CM or CM + LY294002 (1 µM), ERβ (green), p-AKT (red) and DAPI (blue). Original magnification, x400, scale bar, 20 µm. (F) Binding motif of FOXO3a (from JASPAR). (G) ChIP-PCR shows the binding of FOXO3a on the promoter of ESR2. Agarose gel electrophoresis of ChIP-PCR products. (H) ChIP analysis for binding of FOXO3a to the ESR2 promoter in 4T1 cells upon CM, CON or CM + LY294002 treatment for 24 h. Data are expressed as enrichment relative to the input. ** P<0.01 and *** P<0.001; ns, no significant; CM, conditioned medium; ChiP; chromatin immunoprecipitation; Erβ, estrogen receptor-β; CON, control; p-AKT; phosphorylated AKT; MSR1, macrophage scavenger receptor 1; Expr., expression; FDR, false discovery rate.

Article Snippet: LY294002 (MedChemExpress), a selective PI3K inhibitor, was dissolved at 5 mg/ml in 10% DMSO and intraperitoneally injected at 50 mg/kg (200 μl/injection), twice weekly for five weeks ( , ).

Techniques: Expressing, Activity Assay, Immunohistochemistry, Control, Cell Culture, Immunofluorescence, Binding Assay, Agarose Gel Electrophoresis, Chromatin Immunoprecipitation

Journal: Experimental and Therapeutic Medicine

Article Title: Tumor-associated macrophages suppress estrogen receptor-β expression in triple-negative breast cancer through the PI3K/AKT pathway

doi: 10.3892/etm.2026.13072

Figure Lengend Snippet: PI3K/AKT inhibition and ERβ activation inhibit tumor metastasis. (A) Representative images of Transwell migration assays after treatment with LY294002 (1 µM) or a combination of LY294002 and ERB041 (10-500 nM). Graph of Transwell assays (n=3). (B) Representative images of wound healing assay at 0 and 24 h after treatment with CM, LY294002 (1 µM) or a combination of LY294002 and ERB041 (100 nM). (C) Graph of wound healing assay (n=3). (D) 4T1 cells (1x10 6 ) were injected into the fat pads of BALB/c mice, then treated with DMSO, LY294002 (50 mg/kg intraperitoneally twice a week for five weeks) or LY294002 combined with ERB041 (5 mg/kg subcutaneously daily for five weeks). Representative images showing the H&E staining of lung metastatic nodules in 4T1 mice model. Scale bar, 5 mm (E) Lung metastatic nodule counts in different groups (n=4). (F) The volume of the 4T1 tumor across all groups (n=4). (G) Representative immunohistochemical staining images of p-AKT and ERβ in the breast tumors of different treatment groups. Scale bar, 50 µm. Magnification, x200. * P<0.05, ** P<0.01 and *** P<0.001; ns, not significant. Erβ, estrogen receptor-β; CM, conditioned medium; p-AKT, phosphorylated AKT; Con, control.

Article Snippet: LY294002 (MedChemExpress), a selective PI3K inhibitor, was dissolved at 5 mg/ml in 10% DMSO and intraperitoneally injected at 50 mg/kg (200 μl/injection), twice weekly for five weeks ( , ).

Techniques: Inhibition, Activation Assay, Migration, Wound Healing Assay, Injection, Staining, Immunohistochemical staining, Control

Journal: iScience

Article Title: Thrombospondin 1 aggravates cardiac remodeling in heart failure with preserved ejection fraction by inhibiting mitophagy

doi: 10.1016/j.isci.2026.114639

Figure Lengend Snippet: Thbs1 knockdown alleviates cardiomyocyte hypertrophy, oxidative stress, mitochondrial dysfunction, and mitophagy impairment in vitro (A) Schematic diagram of the in vitro pathological stimulation protocol. H9c2 cells were treated with isoproterenol (ISO) and macrophage-conditioned medium (MCM) derived from LPS+ATP-activated macrophages, with or without Thbs1 knockdown. (B and C) mRNA levels of hypertrophic markers ANP (B) and BNP (C) under pathological stimulation ( n = 3 biological replicates per group, each measured in triplicate). (D) Representative fluorescence images of mitochondrial reactive oxygen species (ROS) detected using MitoSOX Red. Scale bars, 20 μm. (E) Quantification of MitoSOX Red fluorescence intensity ( n = 6 biological replicates per group, each measured in triplicate). (F) JC-1 staining to assess mitochondrial membrane potential (MMP), representative images shown. Scale bars, 20 μm. (G) Quantification of JC-1 aggregates/monomers ratio ( n = 6 biological replicates per group, each measured in triplicate). (H) Immunofluorescence images showing LC3 (red) and VDAC (green) colocalization; nuclei were stained with DAPI (blue). Scale bars, 20 μm. (I) Representative western blot images of mitophagy-related proteins LC3, p62, PINK1, and Parkin. (J–M) Quantification of protein expression: LC3-II/I (J), p62 (K), PINK1 (L), and Parkin (M) ( n = 3 biological replicates per group, each measured in duplicate). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, indicating the level of statistical significance for each comparison.

Article Snippet: Pharmacologic modulation of the PI3K/Akt/mTOR pathway was achieved by pretreating H9c2 cells with 10 μM LY294002 (PI3K inhibitor; HY-10108, MedChemExpress) or 10 μM SC79 (an Akt activator; HY-18749, MedChemExpress) for 1 h before stimulation.

Techniques: Knockdown, In Vitro, Derivative Assay, Fluorescence, Staining, Membrane, Immunofluorescence, Western Blot, Expressing, Comparison

Journal: iScience

Article Title: Thrombospondin 1 aggravates cardiac remodeling in heart failure with preserved ejection fraction by inhibiting mitophagy

doi: 10.1016/j.isci.2026.114639

Figure Lengend Snippet: PI3K/Akt/mTOR pathway activation reverses the protective effects of Thbs1 knockdown in H9c2 cells (A) Representative western blot images showing phosphorylation and total levels of Akt and mTOR in H9c2 cells transfected with si- Thbs1 and treated with ISO plus MCM or the Akt activator SC79. (B and C) Quantification of protein phosphorylation: p -Akt/Akt (B) and p -mTOR/mTOR (C) ( n = 3 biological replicates per group, each measured in duplicate). (D and E) Relative mRNA expression of ANP (D) and BNP (E) assessed by RT-qPCR ( n = 3 biological replicates per group, each measured in triplicate). (F and G) Intracellular ROS production measured using MitoSOX Red mitochondrial superoxide indicator; representative fluorescence images are shown (F) and quantified as mean fluorescence intensity (G) ( n = 6 biological replicates per group, each measured in triplicate). Scale bars, 20 μm. (H) Representative confocal images of mitochondria-lysosome colocalization using Mito-Tracker (green) and Lyso-Tracker (red). Scale bars, 20 μm. (I) Representative western blot images of mitophagy-related proteins LC3, p62, PINK1, and Parkin. (J–M) Quantification of mitophagy-related protein expression: LC3-II/I (J), p62 (K), PINK1 (L), and Parkin (M) ( n = 3 biological replicates per group, each measured in duplicate). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, indicating the level of statistical significance for each comparison.

Article Snippet: Pharmacologic modulation of the PI3K/Akt/mTOR pathway was achieved by pretreating H9c2 cells with 10 μM LY294002 (PI3K inhibitor; HY-10108, MedChemExpress) or 10 μM SC79 (an Akt activator; HY-18749, MedChemExpress) for 1 h before stimulation.

Techniques: Activation Assay, Knockdown, Western Blot, Phospho-proteomics, Transfection, Expressing, Quantitative RT-PCR, Fluorescence, Comparison

Journal: iScience

Article Title: Thrombospondin 1 aggravates cardiac remodeling in heart failure with preserved ejection fraction by inhibiting mitophagy

doi: 10.1016/j.isci.2026.114639

Figure Lengend Snippet: Inhibition of PI3K/Akt/mTOR reverses effects of Thbs1 overexpression and Thbs1 binds ITGB1 in H9c2 cells (A) Representative western blot images showing phosphorylation and total levels of PI3K, Akt, and mTOR in H9c2 cells transfected with Thbs1 overexpression plasmid and treated with ISO plus MCM or the PI3K/Akt/mTOR inhibitor LY294002. (B–D) Quantification of protein phosphorylation: p-PI3K/PI3K (B), p -Akt/Akt (C), and p -mTOR/mTOR (D) ( n = 3 biological replicates per group, each measured in duplicate). (E and F) Intracellular ROS production measured using MitoSOX Red; representative fluorescence images are shown (E) and quantified as mean fluorescence intensity (F) ( n = 6 biological replicates per group, each measured in triplicate). Scale bars, 20 μm. (G) Representative confocal images showing mitochondria-lysosome colocalization using Mito-Tracker (green) and Lyso-Tracker (red). Scale bars, 20 μm. (H) Representative western blot images of mitophagy-related proteins LC3, p62, PINK1, and Parkin. (I–L) Quantification of mitophagy-related protein expression: LC3-II/I (I), p62 (J), PINK1 (K), and Parkin (L) ( n = 3 biological replicates per group, each measured in duplicate). (M) Co-immunoprecipitation (Co/IP) assays showing the interaction between Thbs1 and ITGB1 in H9c2 cells. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, indicating the level of statistical significance for each comparison.

Article Snippet: Pharmacologic modulation of the PI3K/Akt/mTOR pathway was achieved by pretreating H9c2 cells with 10 μM LY294002 (PI3K inhibitor; HY-10108, MedChemExpress) or 10 μM SC79 (an Akt activator; HY-18749, MedChemExpress) for 1 h before stimulation.

Techniques: Inhibition, Over Expression, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation, Fluorescence, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Comparison