Journal: The Journal of Biological Chemistry

Article Title: Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells

doi: 10.1016/j.jbc.2022.101950

Figure Lengend Snippet: SCLC-sEVs and NSCLC-sEVs express several integrin subunits. A and B , lectin blot analysis of whole-cell lysates ( C , 20 μg) and sEVs ( E , 5 μg) using SSA ( A ) and WGA ( B ) lectins. C and D , lectin pull-down (LP) of glycoproteins from the detergent-solubilized sEVs using SSA-conjugated beads (for H520-sEVs) or WGA-conjugated beads (for H446-sEVs), followed by SDS-PAGE/silver staining. Arrows indicate integrin subunits detected in the corresponding bands in proteomics. E and F , validation of integrin expression by Western blot analysis. Whole-cell lysates ( C , 20 μg) and sEVs ( E , 5 μg) from H520 ( E ) and H446 ( F ) cells were analyzed. See also and . Lectin blots ( A and B ) are representative images from three independent experiments. Experiments for ( C − F ) were performed once. NSCLC, non–small-cell lung carcinoma; SCLC, small-cell lung carcinoma; sEV, small extracellular vesicle; SSA, Sambucus sieboldiana ; WGA, wheat germ agglutinin.

Article Snippet: Antibodies used for blotting were CD81 (1:10,000, B-11; Santa Cruz Biotechnology), integrin β1 (1:1,000, 4706S; Cell Signaling Technology), integrin β4 (1:1,000, 4707S; Cell Signaling Technology), integrin β5 (1:1,000, 4708S; Cell Signaling Technology), integrin αV (1:5,000, AF1219; R&D Systems), and integrin α6 (1:5,000, AF1350; R&D Systems).

Techniques: SDS Page, Silver Staining, Biomarker Discovery, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells

doi: 10.1016/j.jbc.2022.101950

Figure Lengend Snippet: Integrin α6β4 expression is unique to NSCLC-sEVs. A , Western blot analysis of sEVs (5 μg) prepared from SCC (H520, SK-MES-1, and LK-2), LCC (H1299), LUAD (HCC827), and SCLC (H446 and SBC-3) cells. B , immunoprecipitation (IP) of the integrin α6 subunit from the detergent-solubilized SCC-sEVs. Equal amounts of input, unbound, and bound fractions were analyzed by Western blot. C , Western blot analysis of whole-cell lysates (20 μg) prepared from SCC (H520, SK-MES-1, and LK-2), LCC (H1299), LUAD (HCC827), and SCLC (H446 and SBC-3) cells. Western blots ( A and C ) are representative images from three independent experiments. LCC, large-cell lung carcinoma; LUAD, lung adenocarcinoma; NSCLC, non–small-cell lung carcinoma; SCC, squamous-cell lung carcinoma; SCLC, small-cell lung carcinoma; sEV, small extracellular vesicle.

Article Snippet: Antibodies used for blotting were CD81 (1:10,000, B-11; Santa Cruz Biotechnology), integrin β1 (1:1,000, 4706S; Cell Signaling Technology), integrin β4 (1:1,000, 4707S; Cell Signaling Technology), integrin β5 (1:1,000, 4708S; Cell Signaling Technology), integrin αV (1:5,000, AF1219; R&D Systems), and integrin α6 (1:5,000, AF1350; R&D Systems).

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells

doi: 10.1016/j.jbc.2022.101950

Figure Lengend Snippet: The integrin α6 subunit carries NSCLC-type N-glycans. A , IP of the integrin α6 subunit from detergent-solubilized SCC-sEVs (H520, SK-MES-1, and LK-2), followed by SDS-PAGE/silver staining. Heavy chain (HC) of immunoglobulin G used for immunoprecipitation. Arrows indicate the immunoprecipitated integrin α6 subunit. Asterisks indicate bands that were not detected with the anti-integrin α6 antibody in Western blot analysis. B , LC-ESI-MS analysis of major N-glycans detected in the immunoprecipitated integrin α6 subunit prepared in ( A ). The relative amounts (%, upper panel ) of each glycan structure were calculated by setting the total peak intensities of all detected alditol N -glycans in each extracted-ion chromatogram (EIC) to 100%. Peak intensities of each alditol N -glycan were calculated based on the EIC in Fig. S6 . Bottom panels show the average mass spectrum (28–55 min) in the base peak chromatogram of N -glycan alditols. The deduced structures of the major N -glycans are shown with the theoretical mass and the charge state. The structure number, observed mass, theoretical mass, peak intensities, and retention time are summarized in . C , relative amounts of oligomannose-type (Oligo-Man) and nonfucosylated (Non-Fuc) or monofucosylated (Mon-Fuc) complex-type N-glycans in total N-glycans. D , relative amounts of biantennary (Bi-antenna) and triantennary (Tri-antenna) N-glycans in the total complex-type N-glycans. E , sialylation profiles (Non-Sia, Mono-Sia, Di-Sia, and Tri-Sia) of biantennary and triantennary N-glycans. See also Fig. S6 and . All experiments were performed once. ESI, electrospray ionization; LC, liquid chromatography; MS, mass spectrometry; NSCLC, non–small-cell lung carcinoma; SCC, squamous-cell lung carcinoma; sEV, small extracellular vesicle.

Article Snippet: Antibodies used for blotting were CD81 (1:10,000, B-11; Santa Cruz Biotechnology), integrin β1 (1:1,000, 4706S; Cell Signaling Technology), integrin β4 (1:1,000, 4707S; Cell Signaling Technology), integrin β5 (1:1,000, 4708S; Cell Signaling Technology), integrin αV (1:5,000, AF1219; R&D Systems), and integrin α6 (1:5,000, AF1350; R&D Systems).

Techniques: SDS Page, Silver Staining, Immunoprecipitation, Western Blot, Glycoproteomics, Liquid Chromatography, Mass Spectrometry

Journal: MedComm

Article Title: Hemidesmosome Mutations Contribute to the Onset and Severity of Acquired Autoimmune Bullous Diseases

doi: 10.1002/mco2.70627

Figure Lengend Snippet: Pathogenic variants in hemidesmosome assembly‐related genes in pemphigoid disease spectrum decrease skin‐related protein expression. (A) Pattern diagram showing the location and pattern of pathogenic antibody deposition for different PD subtypes. (B) Representative immunohistochemistry images of BP, LigA, DH, and EBA mutation‐carrying and control patient samples stained for ITGA6 , LAMC2 , EPPK1 , and ITGB4 , n = 10, 14 for mutations and controls. Scale bar: 150 µm. (C) Quantitative analysis of IHC results for BP samples. (D) Quantitative analysis of IHC results for LigA samples, n = 11, 11 for mutations and controls. p‐ values are calculated using a two‐tailed Student's t ‐test. * p ≤ 0.05, ** p ≤ 0.01. Each sample is represented as one dot.

Article Snippet: For RNA sequencing, 1 × 10 6 cells were seeded into 12‐well plates and cultured for 24 h. Stable Ker‐CT cell lines overexpressing wild‐type (WT) ITGA6 or ITGA6 variants were generated by lentivirus‐mediated transduction and selected with hygromycin (50 μg/ml; HY‐K1051, MCE).

Techniques: Expressing, Immunohistochemistry, Mutagenesis, Control, Staining, Two Tailed Test

Journal: MedComm

Article Title: Hemidesmosome Mutations Contribute to the Onset and Severity of Acquired Autoimmune Bullous Diseases

doi: 10.1002/mco2.70627

Figure Lengend Snippet: Hemidesmosome‐related gene ITGA6 homologue ina‐1 mutation affects protein expression in C. elegans models. (A) Diagram of ina‐1 mutation pattern in C. elegans models and positions of residues mutated by CRISPR‐Cas technology. (B) Diagram of the lam‐2 mutation pattern in C. elegans models and positions of residues mutated by CRISPR‐Cas technology. (C) Representative images of homozygous ina‐1 wild‐type and mutation type embryos for CRISPR‐Cas knock‐in ina‐1 ::wrmScarlet and vab‐10a::GFP with the developmental process from L1, L2, L3, and L4 larvae to adult indicated on the left and right, respectively. Scale bar: 50 µm. Panels 1–4 show wrmScarlet, GFP, and merged signals for the turn of wild‐type C. elegans from L1–L4 larvae to adult (Y1 and Y2 are both adult locations), respectively. (D) Representative images of homozygous embryos for CRISPR‐Cas knock‐in lam‐2 ::wrmScarlet and vab‐10a::GFP with the developmental process from L1–L4 larvae to adult. Scale bar: 50 µm. Each sample is represented as one dot. (E) Quantitative analysis of confocal microscopy results for ina‐1 ::wrmScarlet C. elegans samples shows stable expression of fluorescence signals from L1–L4 larvae to adult. (F) Quantitative analysis of confocal microscopy results for lam‐2 ::wrmScarlet mutants shows stable expression of fluorescence signals from L1–L4 larvae to adults.

Article Snippet: For RNA sequencing, 1 × 10 6 cells were seeded into 12‐well plates and cultured for 24 h. Stable Ker‐CT cell lines overexpressing wild‐type (WT) ITGA6 or ITGA6 variants were generated by lentivirus‐mediated transduction and selected with hygromycin (50 μg/ml; HY‐K1051, MCE).

Techniques: Mutagenesis, Expressing, CRISPR, Knock-In, Confocal Microscopy, Fluorescence

Journal: MedComm

Article Title: Hemidesmosome Mutations Contribute to the Onset and Severity of Acquired Autoimmune Bullous Diseases

doi: 10.1002/mco2.70627

Figure Lengend Snippet: ITGA6 homologue ina‐1 mutation mediated the PD‐like phenotype in C. elegans models. (A) Components and structures of mammalian hemidesmosome type in the skin of PD patients. (B) Components and structures of CeHDs in epidermis adjacent to body‐wall muscles. (C) Transmission electron microscopy (TEM) results revealed that hemidesmosome structures were destroyed in patients with different PD forms ( n = 6, including BP = 3, DH = 2, LigA = 1) and healthy controls ( n = 3). Desmosome structures were normal and served as controls. Black arrows indicate abnormal hemidesmosome structures and normal desmosome structures. Pat 2 represents high magnifications of the region highlighted by dashed rectangles in BP1, BP2, BP3, DH1, DH2, and LigA1. Scale bar: 500 nm. (D) Representative image of ina‐1 wild‐type (WT) shows clear epidermal and muscle junction structure border and normal cuticle basal layer in C. elegans . Scale bar: 1 µm. Scale bar: 500 nm. (E) Epidermal and muscle structure border and basal cuticle layer were basically normal with mild separation phenomena in the ina‐1 type (MUT1). Scale bar: 1 µm; 500 nm.

Article Snippet: For RNA sequencing, 1 × 10 6 cells were seeded into 12‐well plates and cultured for 24 h. Stable Ker‐CT cell lines overexpressing wild‐type (WT) ITGA6 or ITGA6 variants were generated by lentivirus‐mediated transduction and selected with hygromycin (50 μg/ml; HY‐K1051, MCE).

Techniques: Mutagenesis, Muscles, Transmission Assay, Electron Microscopy

Journal: Oncology Letters

Article Title: Effects of endometrial stem cell transplantation combined with estrogen in the repair of endometrial injury

doi: 10.3892/ol.2018.8702

Figure Lengend Snippet: Primers used in the study.

Article Snippet: Following blocking, the membranes were incubated with the following primary antibodies: Rabbit anti-human EGF antibody (cat. no. DF2225, 1:2,000; Affinity Biosciences, Cincinnati, OH, USA), rabbit anti-human PDGF-BB (cat. no. orb303833, 1:2,000; Biorbyt, Cambridge, UK,), rabbit anti-human EMA antibody (cat. no. orb31710; 1:2,000; Biorbyt), rabbit anti-human CK antibody (cat. no. BF0141, 1:1,000; Affinity Biosciences), rabbit anti-human CD49f antibody (cat. no. orb39949, 1:1,000; Biorbyt), rabbit anti-human THY-1 (cat. no. orb136416, 1:2,000; Biorbyt), rabbit anti-human Col I antibody (cat. no. orb345868, 1:2,000; Biorbyt), rabbit anti-human 5B5 antibody (cat. no. orb241612, 1:1,000; Biorbyt), rabbit anti-human vimentin antibody (cat. no. AF7013, 1:2,000; Affinity Biosciences), rabbit anti-rat estrogen receptor (ESR1) (cat. no. DF6094, 1:2,000; Affinity Biosciences) and rabbit anti-rat matrix metalloproteinase (MPP)-9 (cat. no. orb100446, 1:1,000; Biorbyt) overnight at 4°C.

Techniques: Sequencing

Journal: Oncology Letters

Article Title: Effects of endometrial stem cell transplantation combined with estrogen in the repair of endometrial injury

doi: 10.3892/ol.2018.8702

Figure Lengend Snippet: Expression of EMA, CK, CD49f, THY-1, Col I. 5B5 and vimentin in EnSCs. (A) mRNA levels of EMA, CK and CD49f in EnSCs. (B) mRNA levels of THY-1, Col I, 5B5 and vimentin in EnSCs. (C) Protein expression of EMA, CK and CD49f in EnSCs. (D) Protein expression of THY-1, Col I, 5B5 and vimentin in EnSCs. *P<0.05 vs. control. EMA, epithelial membrane antigen; CK, cytokeratin; Col I, collagen type 1; CD49F, integrin α-6; THY-1, Thy-1 membrane glycoprotein; EnSCs, endometrial stem cells.

Article Snippet: Following blocking, the membranes were incubated with the following primary antibodies: Rabbit anti-human EGF antibody (cat. no. DF2225, 1:2,000; Affinity Biosciences, Cincinnati, OH, USA), rabbit anti-human PDGF-BB (cat. no. orb303833, 1:2,000; Biorbyt, Cambridge, UK,), rabbit anti-human EMA antibody (cat. no. orb31710; 1:2,000; Biorbyt), rabbit anti-human CK antibody (cat. no. BF0141, 1:1,000; Affinity Biosciences), rabbit anti-human CD49f antibody (cat. no. orb39949, 1:1,000; Biorbyt), rabbit anti-human THY-1 (cat. no. orb136416, 1:2,000; Biorbyt), rabbit anti-human Col I antibody (cat. no. orb345868, 1:2,000; Biorbyt), rabbit anti-human 5B5 antibody (cat. no. orb241612, 1:1,000; Biorbyt), rabbit anti-human vimentin antibody (cat. no. AF7013, 1:2,000; Affinity Biosciences), rabbit anti-rat estrogen receptor (ESR1) (cat. no. DF6094, 1:2,000; Affinity Biosciences) and rabbit anti-rat matrix metalloproteinase (MPP)-9 (cat. no. orb100446, 1:1,000; Biorbyt) overnight at 4°C.

Techniques: Expressing, Control, Membrane