Journal: Frontiers in Immunology

Article Title: CMTM3 regulates vascular endothelial cell dysfunction by influencing pulmonary vascular endothelial permeability and inflammation in ARDS

doi: 10.3389/fimmu.2025.1544610

Figure Lengend Snippet: Mice, cell lines, and reagents.

Article Snippet: Lipopolysaccharide (HUVECs) , Solarbio , IL2020.

Techniques: Over Expression, Knockdown, Cell Culture, Staining, cDNA Synthesis, SYBR Green Assay, Real-time Polymerase Chain Reaction, Membrane

Journal: Frontiers in Immunology

Article Title: CMTM3 regulates vascular endothelial cell dysfunction by influencing pulmonary vascular endothelial permeability and inflammation in ARDS

doi: 10.3389/fimmu.2025.1544610

Figure Lengend Snippet: (A) The expression levels of CMTM3 transcripts in human umbilical vein endothelial cells (HUVECs) treated with 100 ng/ml LPS for 2h, 6h, and 24h ( n ≧ 3). (B) The expression levels of CMTM3 transcripts in HUVECs subjected to stimulation with 4h-hypoxia/2h-reoxygenation and 5h-hypoxia/2h-reoxygenation. Unstimulated HUVECs were used as the control ( n ≧ 3).

Article Snippet: Lipopolysaccharide (HUVECs) , Solarbio , IL2020.

Techniques: Expressing, Control

Journal: Frontiers in Immunology

Article Title: CMTM3 regulates vascular endothelial cell dysfunction by influencing pulmonary vascular endothelial permeability and inflammation in ARDS

doi: 10.3389/fimmu.2025.1544610

Figure Lengend Snippet: CMTM3 overexpression enhances permeability of the human umbilical vein endothelial cells (HUVECs) under in-vitro ADRS inflammatory conditions ( n ≧ 3). (A) CMTM3 expression levels in control HUVECs as well as adsham and adCMTM3-transfected HUVECs. (B) FITC-dextran assay results show the cellular permeability based on relative fluorescence leakage (RFI) in the adsham- and adCMTM3-transfected HUVECs that were untreated or treated with 100 ng/ml LPS for 6h. (C) FITC-Dextran assay results show the cellular permeability based on relative fluorescence leakage (RFI) in the adsham- and adCMTM3-transfected HUVECs that were untreated or treated with 4h-hypoxia/2h-reoxygenation.

Article Snippet: Lipopolysaccharide (HUVECs) , Solarbio , IL2020.

Techniques: Over Expression, Permeability, In Vitro, Expressing, Control, Transfection, Fluorescence

Journal: Frontiers in Immunology

Article Title: CMTM3 regulates vascular endothelial cell dysfunction by influencing pulmonary vascular endothelial permeability and inflammation in ARDS

doi: 10.3389/fimmu.2025.1544610

Figure Lengend Snippet: CMTM3 knockdown reduces human umbilical vein endothelial cell (HUVEC) permeability under in-vitro inflammatory conditions. (A) CMTM3 expression levels in the control, shsham, and shCMTM3 HUVECs. (B) FITC-dextran assay results show the cellular permeability based on relative fluorescence leakage (RFI) in the shsham and shCMTM3-transfected-HUVECs that were untreated or treated with 100 ng/ml LPS for 6h. (C) FITC-dextran assay results show the cellular permeability based on relative fluorescence leakage (RFI) of shsham and shCMTM3-transfected HUVECs that were untreated or treated with 4h-hypoxia/2h-reoxygenation.

Article Snippet: Lipopolysaccharide (HUVECs) , Solarbio , IL2020.

Techniques: Knockdown, Permeability, In Vitro, Expressing, Control, Fluorescence, Transfection

Journal: Frontiers in Immunology

Article Title: CMTM3 regulates vascular endothelial cell dysfunction by influencing pulmonary vascular endothelial permeability and inflammation in ARDS

doi: 10.3389/fimmu.2025.1544610

Figure Lengend Snippet: The expression levels of IL-6 and TNF-α in the adCMTM3- and shCMTM3-transfected HUVECs after (A, C) LPS stimulation for 6 h and (B, D) 4h/2h hypoxia/reoxygenation treatment.

Article Snippet: Lipopolysaccharide (HUVECs) , Solarbio , IL2020.

Techniques: Expressing, Transfection

Journal: Frontiers in Immunology

Article Title: CMTM3 regulates vascular endothelial cell dysfunction by influencing pulmonary vascular endothelial permeability and inflammation in ARDS

doi: 10.3389/fimmu.2025.1544610

Figure Lengend Snippet: RNA sequencing analysis of lipopolysaccharide (LPS)–treated shCMTM3-HUVECs. (A) Top 20 KEGG pathways representing DEGs between shCMTM3-HUVECs and shsham- human umbilical vein endothelial cells (HUVECs). (B) Top 20 KEGG pathways between LPS-treated shCMTM3-HUVECs and LPS-treated shsham-HUVECs. (C) PPI network analysis of DEGs between shCMTM3-HUVECs and shsham-HUVECs. (D) PPI network analysis of DEGs between LPS-treated shCMTM3-HUVECs and LPS-treated shsham -HUVECs.

Article Snippet: Lipopolysaccharide (HUVECs) , Solarbio , IL2020.

Techniques: RNA Sequencing

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: α-Klotho isoforms, sequence, and Western blot. A, Structure of the two isoforms of α-Klotho. Isoform 1 represents the full-length protein and contains a signal sequence domain (SS), two homologous domains (KL1, KL2), a short transmembrane domain (TM), and a short cytoplasmic tail. Shown is the site of the epitope for the antibody used in our experiments: AA 800 to 900, KL2. This epitope is absent from Isoform 2, a soluble, secreted protein that arises from alternative RNA splicing and contains only AA 1–549, and where the terminal 15 residues are replaced by the sequence shown. B, The full-length α-Klotho protein sequence of 1012 AA is shown, with KL1 and KL2 shown in red and green respectively, and TM highlighted (black). The peptides giving rise to the PRM signature are also shown (bold typeface; common to isoforms 1 and 2, red; exclusive to full-length α-Klotho, isoform 1, blue). C and D, Western blot analysis of cell lysates (C) and tissues (D) supports the presence of the full-length α-Klotho. Full-length rh α-Klotho protein (rh-α-Klotho). EC, epithelial cells.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Sequencing, Western Blot

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: α-Klotho protein expression and distribution in human epithelial and reproductive tissues. IHC, positive staining (brown) was found in all the cellular layers of the epidermis (A) and appendage tissue such as hair follicle and sebaceous gland (B). Intestinal expression was primarily found in epithelial cells as illustrated in jejunum (C) and colon (D). In reproductive tissues, positive staining was found in epithelial Sertoli cells (E), testosterone producing Leydig cells (illustrated with white arrows) of the testis (F), and epithelial cells of the prostate gland G). H–K, In mammary tissue (H), endometrium of uterus (I), and endometrium of salpinx (K), the epithelial cell layer was staining strongly for α-Klotho protein; insets are larger magnifications of the epithelial layer. n ≥ 5 for each tissue.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Expressing, Staining

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: Mass spectrometry characterization of the transmembrane α-Klotho protein in human tissues and cells: extracellular α-Klotho peptide GLFYVDFLSQKD (exon 3). A–D, Representative mass spectrometry spectra (left) and Skyline data (right) confirmed the presence of full-length α-Klotho rh full-length α-Klotho protein (rh-α-Klotho) (A), renal proximal tubular epithelial cells (B), kidney tissue (C), and renal artery (D). E–G, The full-length specific (isoform 1) αKlotho peptide LWITMNEPYTR (exon 4). Representative mass spectrometry spectra (left) and Skyline data (right) confirmed the presence of full-length α-Klotho. E), rh full-length α-Klotho protein (rh-α-Klotho); F, kidney tissue; G, renal artery; and H, neuronal cells.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Mass Spectrometry, Targeted Proteomics

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: Confirmation of Transmembrane α-Klotho Protein Expression in Human Tissues and Cells

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Expressing, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: TGF- β controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription in mice

doi: 10.1172/JCI172095

Figure Lengend Snippet: ( A ) To obtain AT1 cells for culture, P5–P10 pup lungs were obtained after lineage labeling with tamoxifen at P0. Whole lung cell suspensions were obtained using a dispase, DNase, and collagenase digestion buffer after which the epithelial cell population was enriched by depleting the CD45 + and CD31 + population. Remaining cells were fluorescently sorted with FACS to obtain a CD326 + and YFP + suspension. Cells were plated onto fibronectin-coated plates with or without TGF-β1 ligand (7.5 ng/mL) or TGF-β inhibitor SB431542 (10μM) and evaluated at days 2, 4, and 6. ( B ) ICC of RAGE + (AGER + ) cells treated with TGF-β ligand or inhibitor in culture at days 2, 4, and 6 with a zoomed image of cells at day 6 appearing at the bottom. Scale bars: 100 μm. ( C ) Quantification of mean AGER + cell area depicted in B by 1-way ANOVA with Holms Šidák’s test for multiple comparisons ( n = 135–231). ( D ) Quantification of mean cell roundness at day 6 depicted in B by 1-way ANOVA with Holms Šidák’s test for multiple comparisons ( n = 148–167). ( E ) Quantification of qPCR RNA transcript expression levels (FC compared with GAPDH, normalized to controls) of the AT2 marker Sftpb , fibronectin-binding integrins ( F ) Itga5 and ( G ) Itgb1 , and ( H ) basement membrane constituents including the collagen IV subtypes Col4a1 , Col4a3 , and Col4a4 and laminin-332 constituents Lama3 and Lamb3 ( n = 3 per group, 1-way ANOVA with Tukey’s multiple comparisons). ( I ) Representative schematic of findings indicating that TGF-β regulates integrin expression to guide ECM binding and cellular spread, which affects cell identity and matrisome expression and impacts lung development. Schematics in A and I were created in BioRender. Results are representative of 3 experiments.

Article Snippet: Lineage-traced AT1 cells were obtained following FACS and were then placed into an organoid growth medium containing DMEM F12 (Thermo Fisher Scientific) and growth factors including bovine pituitary extract, cholera toxin, FBS, gentamicin, retinoic acid, insulin, transferrin (all from Lonza), and human epithelial growth factor (Peprotech), as previously described ( , ).

Techniques: Labeling, Suspension, Expressing, Marker, Binding Assay, Membrane