Journal: Molecular Medicine Reports

Article Title: ER-α36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells

doi: 10.3892/mmr.2025.13649

Figure Lengend Snippet: ER-α36 knockdown suppresses the malignant proliferation of liver cancer cells and induces lysosomal membrane permeabilization. (A) Representative images of liver tumors from nude mice intrahepatically injected with HepG2 cells expressing different ER-α36 levels, harvested at 28 days post-injection. (B) Quantitative analysis of liver tumor volume, liver weight, body weight and the ratio of liver weight to body weight. (C) Representative H&E staining images showing pathological karyomitosis changes. Scale bar, 50 µm. (D) Immunohistochemical staining of LC3, p62, LAMP1 and Ki67 with corresponding IOD values. Scale bar, 50 µm. (E) Immunofluorescence staining of Gal-3 in the tumors formed from the transfected HepG2 cells. Scale bar, 10 µm. (F) Western blotting results showing the levels of LC3-II and LC3-II, p62, LAMP1, p-AKT and t-AKT in the tumors, and (G) quantitative analyses of the LC3-II/LC3-I and p-AKT/t-AKT ratios, and p62 and LAMP1 expression levels. β-actin was used as the internal loading control. Data are presented as the mean ± SEM. **P<0.01. ER, estrogen receptor; H&E, hematoxylin and eosin; LC3, microtubule-associated protein 1 light chain 3; LAMP1, lysosome-associated membrane protein 1; Gal-3, galectin-3; IOD, integrated optical density; p-, phosphorylated; t-, total; Sh36, transfected with ER-α36 specific short hairpin RNA expression vector; Vector, transfected with empty vector.

Article Snippet: Antibodies against p62 (also known as sequestosome 1; cat. no. 18420-1-AP), galectin-3 (Gal-3; cat. no. 60207-1-Ig), lysosome-associated membrane protein 1 (LAMP1; cat. no. 21997-1-AP), AKT (cat. no. 10176-2-AP), phosphorylated-(p-)AKT (cat. no. 66444-1-Ig) and β-actin (cat. no. 60008-1-IG) were purchased from Proteintech Group, Inc.

Techniques: Knockdown, Membrane, Injection, Expressing, Staining, Immunohistochemical staining, Immunofluorescence, Transfection, Western Blot, Control, shRNA, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: ER-α36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells

doi: 10.3892/mmr.2025.13649

Figure Lengend Snippet: ER-α36 knockdown decreases AKT phosphorylation and influences lysosomal localization in liver cancer cells. Stably transfected liver cancer cells with different levels of ER-α36 expression were treated with or without MK-2206 (100 nM) for 6 h. (A) Representative western blots of p-AKT and t-AKT, and (B) quantitative analysis. (C) Immunofluorescence analysis of LAMP1. Cell peripheries and the juxtanuclear region are indicated with dashed lines and solid lines, respectively. Scale bar, 10 µm. (D) Quantification of the juxtanuclear percentage of LAMP1. **P<0.01. ER, estrogen receptor; p-, phosphorylated; t-, total; LAMP1, lysosome-associated membrane protein 1; Sh36, transfected with ER-α36 specific short hairpin RNA expression vector; Vector, transfected with empty vector.

Article Snippet: Antibodies against p62 (also known as sequestosome 1; cat. no. 18420-1-AP), galectin-3 (Gal-3; cat. no. 60207-1-Ig), lysosome-associated membrane protein 1 (LAMP1; cat. no. 21997-1-AP), AKT (cat. no. 10176-2-AP), phosphorylated-(p-)AKT (cat. no. 66444-1-Ig) and β-actin (cat. no. 60008-1-IG) were purchased from Proteintech Group, Inc.

Techniques: Knockdown, Phospho-proteomics, Stable Transfection, Transfection, Expressing, Western Blot, Immunofluorescence, Membrane, shRNA, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: ER-α36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells

doi: 10.3892/mmr.2025.13649

Figure Lengend Snippet: AKT is involved in the lysosomal localization and LMP induced by ER-α36 knockdown. Transfected liver cancer cells with different levels of ER-α36 expression were treated with MK-2206 (100 nM) for 6 h, followed by E2 (1 nM) for 30 min, and the same volume of alcohol was used as a vehicle control. (A) Representative western blots of ER-α36, p-AKT and AKT, and (B) quantitative analysis of ER-α36 expression and the p-AKT/t-AKT ratio. In subsequent assays, the transfected liver cancer cells were treated with MK-2206 (100 nM) for 6 h, followed by E2 (1 nM) for 24 h. (C) Representative western blots of LAMP1 and (D) quantitative analysis of LAMP1 expression. (E) Representative images of colony formation by the liver cancer cells and (F) quantitative analysis of colony forming ability. Data are presented as the mean ± SEM. **P<0.01. (G) Fluorescence microscopy images of the cells following staining with Lyso-Tracker Red. Scale bar, 20 µm. (H) Co-localization of Gal-3 (green) and LAMP1 (red) in the cells as revealed by immunofluorescence staining. Scale bar, 10 µm. ER, estrogen receptor; E2, 17β-estradiol; Gal-3, galectin-3; p-, phosphorylated; t-, total; LAMP1, lysosome-associated membrane protein 1; Sh36, transfected with ER-α36 specific short hairpin RNA expression vector; Vector, transfected with empty vector.

Article Snippet: Antibodies against p62 (also known as sequestosome 1; cat. no. 18420-1-AP), galectin-3 (Gal-3; cat. no. 60207-1-Ig), lysosome-associated membrane protein 1 (LAMP1; cat. no. 21997-1-AP), AKT (cat. no. 10176-2-AP), phosphorylated-(p-)AKT (cat. no. 66444-1-Ig) and β-actin (cat. no. 60008-1-IG) were purchased from Proteintech Group, Inc.

Techniques: Knockdown, Transfection, Expressing, Control, Western Blot, Fluorescence, Microscopy, Staining, Immunofluorescence, Membrane, shRNA, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: Recombinant chromosome 6 open reading frame 120 protein promotes angiogenesis and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells via the PI3K/Akt signaling pathway

doi: 10.3892/mmr.2025.13596

Figure Lengend Snippet: rC6ORF120 modulates the PI3K/Akt signaling pathway in HUVECs (A) Western blot analysis of PISK, p-PI3K, Akt and p-Akt in the HUVECs treated with rC6ORF120. Bar graph representing relative expression of (B) p-PI3K/PI3K and (C) p-Akt/Akt. **P<0.01; ***P<0.001. rC6ORF120, recombinant C6ORF120 protein; HUVECs, human umbilical vein endothelial cells; p-, phosphorylated.

Article Snippet: After blocking with 5% skimmed milk for 1 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against CD31 (cat. no. 28083-1-AP; 1:1,000; Proteintech Group, Inc.), VE-cadherin (cat. no. 66804-1-IG; 1:1,000; Proteintech Group, Inc.), Vimentin (cat. no. 10366-1-AP; 1:5,000; Proteintech Group, Inc.), α-smooth muscle actin (α-SMA; cat. no. 67735-1-IG; 1:5,000; Proteintech Group, Inc.), AKT (cat. no. 60203-2-Ig; 1:1,000; Proteintech Group, Inc.), phosphorylated (p-)AKT (cat. no. 66444-1-Ig; 1:1,000; Proteintech Group, Inc.), PI3K (cat. no. 60225-1-Ig; 1:1,000; Proteintech Group, Inc.), p-PI3K (cat. no. ab182651; 1:1,000; Abcam) and GAPDH (cat. no. 60004-1-Ig; 1:5,000; Proteintech Group, Inc.).

Techniques: Western Blot, Expressing, Recombinant

Journal: Molecular Medicine Reports

Article Title: Recombinant chromosome 6 open reading frame 120 protein promotes angiogenesis and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells via the PI3K/Akt signaling pathway

doi: 10.3892/mmr.2025.13596

Figure Lengend Snippet: Involvement of the PI3K/Akt pathway in rC6ORF120-induced EndMT in HUVECs. Cells were treated with rC6ORF120, the PI3K inhibitor LY294002, or a combination of both. (A) Representative blots depicting the PI3K/Akt signaling pathway and proteins indicative of EndMT. (B-G) Quantitative assessment of protein expression levels. ns indicates no statistically significant difference; *P<0.05; **P<0.01; ***P<0.001. rC6ORF120, recombinant C6ORF120 protein; EndMT, endothelial-mesenchymal transition; HUVECs, human umbilical vein endothelial cells; p-, phosphorylated.

Article Snippet: After blocking with 5% skimmed milk for 1 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against CD31 (cat. no. 28083-1-AP; 1:1,000; Proteintech Group, Inc.), VE-cadherin (cat. no. 66804-1-IG; 1:1,000; Proteintech Group, Inc.), Vimentin (cat. no. 10366-1-AP; 1:5,000; Proteintech Group, Inc.), α-smooth muscle actin (α-SMA; cat. no. 67735-1-IG; 1:5,000; Proteintech Group, Inc.), AKT (cat. no. 60203-2-Ig; 1:1,000; Proteintech Group, Inc.), phosphorylated (p-)AKT (cat. no. 66444-1-Ig; 1:1,000; Proteintech Group, Inc.), PI3K (cat. no. 60225-1-Ig; 1:1,000; Proteintech Group, Inc.), p-PI3K (cat. no. ab182651; 1:1,000; Abcam) and GAPDH (cat. no. 60004-1-Ig; 1:5,000; Proteintech Group, Inc.).

Techniques: Expressing, Recombinant

Journal: World Journal of Gastroenterology

Article Title: Kushenol I combats ulcerative colitis via intestinal barrier preservation and gut microbiota optimization

doi: 10.3748/wjg.v31.i26.105656

Figure Lengend Snippet: Effects of kushenol I on the expression of inflammation-related proteins phosphorylated phosphoinositide 3-kinase (PI3K), PI3K, phosphorylated protein kinase B (AKT), AKT, forkhead box O1, interleukin 1β, Toll-like receptor 4, phosphorylated p38 mitogen-activated protein kinase (MAPK), p38 MAPK, nuclear factor kappa B phosphorylated p65, nuclear factor kappa B p65, and NOD-like receptor thermal protein domain associated protein 3 in the colonic tissues of mice with ulcerative colitis. A: Image of a gel showing protein expression; B: Bar graph depicting protein expression. Values are presented as the mean ± standard error of the mean ( n = 3). a P < 0.05. b P < 0.01. c P < 0.001. P compared with the model group. AKT: Protein kinase B; FOXO1: Forkhead box O1; IL-1β: Interleukin 1β; PI3K: Phosphoinositide 3-kinase; p-p38: Phosphorylated p38; p38 MAPK: p38 mitogen-activated protein kinase; p-p65: Phosphorylated p65; p-PI3K: Phosphorylated phosphoinositide 3-kinase; p-AKT: Phosphorylated protein kinase B; NF-κB: Nuclear factor kappa B; NLRP3: NOD-like receptor thermal protein domain associated protein 3; TLR4: Toll-like receptor 4.

Article Snippet: Antibodies targeting forkhead box O1 (FOXO1), p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 MAPK (p-p38 MAPK), protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), and β-actin were sourced from Proteintech (Proteintech, Rosemont, IL, United States).

Techniques: Expressing