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: Breast Cancer Research and Treatment

Article Title: Vitamin D and androgen receptor-targeted therapy for triple-negative breast cancer

doi: 10.1007/s10549-016-3807-y

Figure Lengend Snippet: Effect of AR and/or VDR therapy on cancer stem cell and differentiation phenotype: a the effect of DHT and calcitriol on tumorsphere formation in TNBC HR2-av cell lines MFM-223 and CAL-148. The cells were trypsinized and single cell suspensions were plated into six-well low adhesion plates in 4-ml sphere media (seeding: MFM-223 10,000 and CAL-148 6000) containing DHT (10 nM, black bars ) or calcitriol (MFM-223 25 nM and CAL-148 10 nM, white bars ), or DHT + calcitriol ( hatched bars ). After 2 weeks during which spheres are formed from single cells, the plates were stained with iodo-nitrotetrazolium chloride overnight and the spheres were counted using GelCount. Y -axis represents the TFE, tumorsphere formation efficiency (number of spheres formed per the number of cells seeded in a well) represented as fold change in comparison to control group (no treatment, vehicle only). The statistical significance of the drug treatments was determined using two-tailed Student’s T -test. Error bars represent mean ± SEM. * P < 0.05, ** P < 0.01. b AldeFluor assay on HR2-av cell lines MFM-223 and CAL-148 after AR and VDR treatment. The cells were treated as described in panel a for 8 days with media change containing fresh drug every 3 days and ALDH + population was examined by AldeFluor assay kit using FACS profiles representing ALDH + population before ( top ) and after ( bottom ) co-treatment. The graph shows ALDH + cells plotted as percentage in each treatment group. Gray bar vehicle treatment, black bar DHT, white bar Cal, hatched DHT and Cal combination. Error bars represent mean ± SEM. The statistical significance of the drug treatments was determined using two-tailed Student’s T -test. * P < 0.05, ** P < 0.01. c , d Western blot analysis of differentiation and cancer stem cell markers in HR2-av cell lines MFM-223 and CAL-148 after DHT and calcitriol treatment. The cells were treated with DHT (10 nM) and/or calcitriol (MFM-223: 25 nM and CAL-148: 10 nM) for 8 days with media including drug every 3 days. The cell lysates with 30 µg protein were loaded in each lane of a 4–15 % SDS-PAGE gel and after transfer to nitrocellulose membrane, it was probed with the following antibodies at appropriate dilutions: E-cadherin (BD Biosciences 61081), vimentin (Sigma 5255), cytokeratin 5 (Abcam ab75869), cytokeratin 18 (Thermofisher Scientific MS-142-P), Claudin-4 (Invitrogen 32-9400), ALDH1A1 (Cell Signalling mAb12035), CD326 (Abcam ab32392), CD49f (ABD Serotec MCA1457), CD133 (Sigma C9493), CD24 (Santa Cruz SC-53660), CD166 (Abcam ab49496), Nanog (Abcam ab109250), and Musashi (Abcam ab52865). β-Actin or β-tubulin were used as loading controls. e mRNA expression of stem cell markers. MFM-223 cells were treated with 10 nM DHT and/or 25 nM calcitriol for 96 h. The mRNA was quantified using a PCR-based array (Qiagen). The expression level of each gene was determined by comparing the treatment to vehicle control, and genes with more than 2-fold change are presented here in a Heatmap generated using conditional formatting in Microsoft Excel. Scale bar red represents high expression, green represents low expression

Article Snippet: The cell lysates with 30 μg protein were loaded in each lane of a 4–15 % SDS-PAGE gel and after transfer to nitrocellulose membrane, it was probed with the following antibodies at appropriate dilutions: E-cadherin (BD Biosciences 61081), vimentin (Sigma 5255), cytokeratin 5 (Abcam ab75869), cytokeratin 18 (Thermofisher Scientific MS-142-P), Claudin-4 (Invitrogen 32-9400), ALDH1A1 (Cell Signalling mAb12035), CD326 (Abcam ab32392), CD49f (ABD Serotec MCA1457), CD133 (Sigma C9493), CD24 (Santa Cruz SC-53660), CD166 (Abcam ab49496), Nanog (Abcam ab109250), and Musashi (Abcam ab52865). β-Actin or β-tubulin were used as loading controls. e mRNA expression of stem cell markers.

Techniques: Staining, Comparison, Control, Two Tailed Test, Western Blot, SDS Page, Membrane, Expressing, Generated

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