Journal: Nanomedicine : nanotechnology, biology, and medicine

Article Title: Bioresponsive peptide-polysaccharide nanogels - A versatile delivery system to augment the utility of bioactive cargo.

doi: 10.1016/j.nano.2018.10.008

Figure Lengend Snippet: Figure 5. (A) Merged confocal microscopy images of DAPI (blue, cell nuclei) and GFP(green) fluorescence after a 72 h incubation of A549 lung carcinoma cells with free GFP, or GFP-loaded nanogels in the absence (NGGFP) or presence (NGGFP + mAb) of a monoclonal anti-CD44 blocking antibody (60x magnification; scale bar = 10 μm). Fluorescent micrograph of cells co-incubated with NGGFP and an excess of free HA (NGGFP + HA) is shown in Figure S6 of the Supporting Information. (B) Quantitation of average GFP fluorescence per cell for each treatment condition (n = 15; statistical comparison made relative to GFP control, with * indicating a P b 0.01). (C) Fluorescent confocal microscopy images of A549 cells treated with NGGFP at 24 and 72 h. Samples are co-stained with Texas-red labeled transferrin (TransferrinTR) to visualize endosomes. Individual fluorescence channels and merged images shown (60× magnification; scale bar = 10 μm).

Article Snippet: InVivoMAb anti-human CD44 antibody (Hermes-1 clone) was purchased from BioXCell (West Lebanon, NH).

Techniques: Confocal Microscopy, Fluorescence, Incubation, Blocking Assay, Quantitation Assay, Comparison, Control, Staining, Labeling

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: SRGN promotes NSCLC cell migration in a CD44-dependent manner . a-b Wound healing assay was performed by seeding NSCLC-H460 ( a ) and -H1299 ( b ) cells in the Culture-Insert 2 Well μ-Plates. After removing the insert, wound closure was determined at 0, 24, 48, and 72 h. Wound area was assessed by ImageJ and normalized with respect to the area at 0 h. c H460/sh-CTRL and H460/sh-SRGN cells were cultured in serum-free medium for 24 h and subjected to Boyden chamber migration assay. Migrated cells were counted in 3 h. d H1299/Mock and H1299/SRGN cells were sorted based on CD44 expression. The unsorted as well as CD44-negative H1299/Mock and H1299/SRGN cells were cultured in serum-free medium for 24 h, and subjected to Boyden chamber migration assay. The migrated cells were counted in 3 h. e The parental H1299 cells and the Control-KO and CD44-KO cell clones generated using the CRISPR/Cas9 system were subjected to Boyden chamber migration assay as described above. The expression of CD44 was shown by western blot. f CD44 expression in HuTu80/Mock and HuTu80/CD44 cells was assessed by flow cytometry (left panel) and western blot (right panel). g-h HuTu80/Mock and HuTu80/CD44 cells were suspended in conditioned media derived from H1299/Mock and H1299/SRGN cells ( g ) as well as from H460/sh-CTRL and H460/sh-SRGN cells ( h ), and subjected to Boyden chamber migration assay as described above. The Boyden chamber assays were performed by loading the lower chamber with medium containing 5% FBS in ( c ) and ( h ), and 2% FBS in ( d ), ( e ) and ( g ). Data are presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student’s t -test

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Migration, Wound Healing Assay, Cell Culture, Expressing, Clone Assay, Generated, CRISPR, Western Blot, Flow Cytometry, Derivative Assay

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: CS-GAG modification is critical for SRGN-mediated cell migration. a Western blot analysis of SRGN in the total cell lysate and CM of H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells cultured in serum-free medium for 48 h. b Wound healing assay was performed in H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells. After 72 h, wound area (lower panel) was assessed by ImageJ and normalized to 0 h. c Boyden chamber migration assay was performed in H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells. Migrated cells were counted in 3 h. d Boyden chamber migration assay was performed in HuTu80/CD44 cells that were suspended in CM derived from H1299/Mock, H1299/SRGN or H1299/SRGN(S/A) cells, respectively. Migrated cells were counted in 5 h. e CM harvested from H1299/Mock and H1299/SRGN cells were treated with or without ChaseABC for 24 h, and subjected to western blot analysis of SRGN. HuTu80/CD44 cells were suspended in non-treated or ChaseABC-treated CM and subjected to Boyden chamber migration assay. Migrated cells were counted in 5 h. Data are presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student’s t -test

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Modification, Migration, Western Blot, Cell Culture, Wound Healing Assay, Derivative Assay

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: GAG modification is critical for SRGN binding to CD44. CM was harvested from H1299/Mock, H1299/SRGN or H1299/SRGN(S/A) cells, and incubated with CD44-Fc or IgG control for 18 h at 37 °C. Western blot analysis of SRGN in the input proteins and proteins bound to CD44-Fc and IgG using anti-SRGN antibody was shown

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Modification, Binding Assay, Incubation, Western Blot

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: SRGN induces cytoskeleton reorganization and Rho-family GTPase activation. a-c Phalloidin staining of actin. H1299/Mock and H1299/SRGN cells ( a ) were incubated in serum-free medium for 24 h and H460/sh-CTRL, and H460/sh-SRGN cells ( b ) were incubated in 2% FBS-containing medium for 72 h, followed by Alexa Fluor™ 594 Phalloidin staining of filamentous actin. c HuTu80/Mock and HuTu80/CD44 cells were seeded into the Culture-Insert 2 Well on slide and incubated overnight to form the gap for cell migration. After removing the insert, cells were washed with 1X PBS twice and incubated with CM harvested from H1299/SRGN cells that had been treated with or without ChaseABC for 24 h, followed by Alexa Fluor™ 594 Phalloidin staining of filamentous actin. Filopodia (arrows) and lamellipodia (arrowheads) structures were observed using confocal microscopy. The circumscribed rectangular regions are further enlarged for better viewing. d H1299/Mock, H1299/SRGN, H460/sh-CTRL, and H460/sh-SRGN cells were seeded in FN-coated dishes. In 30 min, attached cells were lysed and subjected for pulling down of Rac1-GTP, Cdc42-GTP and RhoA-GTP. Western blot analysis was performed for total and active Rac1, Cdc42, and RhoA as indicated

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Activation Assay, Staining, Incubation, Migration, Confocal Microscopy, Western Blot

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: SRGN promotes cell migration through Src-elicited focal adhesion turnover. a After serum-starvation for 24 h, H1299/Mock and H1299/SRGN cells were plated in FN-coated dishes. In 30 min, cells were lysed and assayed for Src phosphorylation. In parallel, serum-starved cells were subjected to western blot analysis of phosphorylated paxillin and ERK/MAPK, and co-immunoprecipitation assay for paxillin/FAK adhesion complex formation. b H460/sh-CTRL and H460/sh-SRGN cells were cultured in serum-free medium for 24 h and examined for Src phosphorylation and paxillin/FAK complex formation. c H1299/SRGN cells were cultured in serum-free medium for 24 h, treated with PP2 (10 μM) or DMSO for 3 h, and phosphorylation of Src and paxillin, and paxillin/FAK complex formation were assessed. d Src expression was depleted in H1299/SRGN cells by shRNA-mediated knockdown approach, and phosphorylation of paxillin and the formation of paxillin/FAK complex were assessed in the control-knockdown (sh-CTRL) and Src-knockdown (sh-Src) cells. e-f H1299/Mock and H1299/SRGN cells (e) or H460/sh-CTRL and H460/sh-SRGN cells (f) were treated with or without PP2, and subjected to Boyden chamber migration assay. Migrated cells were counted in 3 h. Western blot analysis of phosphorylated Src and total Src was shown. Data are presented as the mean ± SD of three independent experiments. *** P < 0.001 by Student’s t-test. g HuTu80/CD44 cells were incubated with CM harvested from H1299/Mock and H1299/SRGN cells that had been treated with or without ChaseABC for 24 h, followed by western blot analysis of designated proteins. h H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells were cultured in serum-free medium for 24 h, and subjected to western blot analysis of phosphorylation of Src, paxillin and other proteins as indicated. i A working model is proposed for SRGN-mediated cell migration. SRGN in the tumor microenvironment binds to tumor cell surface CD44 via its CS-GAGs, and promotes Src activation and subsequent paxillin phosphorylation and the dissociation of paxillin/FAK adhesion complex, to accelerate focal adhesion turnover. SRGN also promotes the activation of Rac1 and Cdc42 to enhance cytoskeleton reorganization, leading to increased cell migration

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Migration, Western Blot, Co-Immunoprecipitation Assay, Cell Culture, Expressing, shRNA, Incubation, Activation Assay

Journal: International journal of molecular sciences

Article Title: Experimental Evaluation of Quantum Dots and Antibodies Conjugation by Surface Plasmon Resonance Spectroscopy.

doi: 10.3390/ijms232012626

Figure Lengend Snippet: Figure 4. Dependence of Au/CD44/anti-CD44 complex dissociation and surface regeneration efficiency on the nature of the regeneration solution.

Article Snippet: Recombinant human CD44 biomarker (career free, His tag C-Termus), monoclonal mouse IgG clone # 2C5 (anti-CD44) and recombinant human growth hormone from E. coli (hGH) were obtained from R&D Systems (Abingdon, UK).

Techniques:

Journal: Cancer Biology & Therapy

Article Title: Transcription factor 3 promotes migration and invasion potential and maintains cancer stemness by activating ID1 expression in esophageal squamous cell carcinoma

doi: 10.1080/15384047.2023.2246206

Figure Lengend Snippet: TCF3 regulates the expression of cancer stem markers CD44 and CD133 by transcriptionally regulating ID1. a. RNA-seq results show significantly upregulated and downregulated gene signatures. b-c. In KYSE-150 and TE-1, with the knockdown of TCF3 the protein expression level of ID1 is subsequently reduced. d-e. In KYSE-150 and TE-1, with the knockdown of TCF3, the mRNA expression of ID1 is subsequently reduced. f-g. CHIP and Dual luciferase reporter assay suggested TCF3 could transcriptionally regulate ID1.h-i. With knockdown of TCF3 in KYSE-150 and TE-1 CD44, fluorescence intensity decreased. J-K. The protein expression of CD44 and CD133 was reduced with the knockdown of TCF3 in KYSE-150 and TE-1. l -m. With the knockdown of TCF3 in KYSE-150 and TE-1 knockdown increased sensitivity to the chemotherapeutic drug cisplatin. n-o. mRNA expression of ID1 was reduced in KYSE-150 and TE-1 after siRNA knockdown. p-q. The protein expression level of CD44 and CD133 was subsequently reduced in KYSE-150 and TE-1 when ID1 was knocked down. I. KYSE-150 has a significant decrease in the sphere formation efficiency after si-TCF3 was transfected. s. with knockdown of TCF3, CD44+ ESCC cells number were decreased.

Article Snippet: CD44 antibody purchased from R&DSystems (FAB6127G).

Techniques: Expressing, RNA Sequencing Assay, Luciferase, Reporter Assay, Fluorescence, Transfection

Journal: Cancer Biology & Therapy

Article Title: Transcription factor 3 promotes migration and invasion potential and maintains cancer stemness by activating ID1 expression in esophageal squamous cell carcinoma

doi: 10.1080/15384047.2023.2246206

Figure Lengend Snippet: ID1 promotes proliferation, migration, invasion, and drug resistance of ESCC cells. a-b. The fluorescence intensity of ID1 was reduced in KYSE-150 and TE-1 after ID1 was knocked down. c-d. The migration and invasion abilities of KYSE-150 and TE-1 were decreased when ID1 was knocked down. e -f. The wound healing rates of KYSE-150 and TE-1 were decreased when ID1 was knocked down. g-h. Cell proliferation is affected in KYSE-150 and TE-1 after the siRNA knockdown of ID1.I&J. Increased sensitivity to the chemotherapeutic drug cisplatin with knockdown of TCF3 in KYSE-150 and TE-1. k. KYSE150 has a significant decrease in the sphere formation efficiency after si-ID1 was transfected. l. with knockdown of ID1, CD44+ ESCC cells number were decreased.

Article Snippet: CD44 antibody purchased from R&DSystems (FAB6127G).

Techniques: Migration, Fluorescence, Transfection

Journal: Cancer Biology & Therapy

Article Title: Transcription factor 3 promotes migration and invasion potential and maintains cancer stemness by activating ID1 expression in esophageal squamous cell carcinoma

doi: 10.1080/15384047.2023.2246206

Figure Lengend Snippet: The expressions of cancer stem markers CD44 and CD133 were correlated with the progression of ESCC. a. The expression of CD44 is corresponding to the tumor staging of ESCC. b. The expression of CD133 is corresponding to the tumor staging of ESCC. c. The expression of CD44 positively correlates with TCF3 expression.d.CD133 positively correlates with the expression of TCF3. * p < .05, ** p < .01. *** p < .001.

Article Snippet: CD44 antibody purchased from R&DSystems (FAB6127G).

Techniques: Expressing

Journal: Genes & Diseases

Article Title: ROS and Lipid Droplet accumulation induced by high glucose exposure in healthy colon and Colorectal Cancer Stem Cells

doi: 10.1016/j.gendis.2019.09.010

Figure Lengend Snippet: 72 hrs in HG cell culture medium is sufficient to increase CSC markers in CR-CSCs. a) Primary CR-CSC spheroids (#1, #4, #8 and #9) were cultured in ultra-low adhesion flasks and morphology was assessed by phase-contrast microscopy (scale bar: 1000 μm). b) Fold variation of protein expression in CR-CSCs treated with HG versus NG. Data are representative of 3 independent experiments performed with cells derived from 3 different CR-CSC lines. c) Representative Western blot of PI3K, phosphorylated (p-AKT) and total AKT in CR-CSC treated with HG or NG (#9 CR-CSC line is shown). β-Actin has been used as loading control. The uncropped blots are reported in Supplementary Fig. 1 . d) Fold variation of positivity for CD133 or CD44v6 in CR-CSCs treated as in ( b and c ), measured by FACS.

Article Snippet: Cells were dissociated, harvested, washed twice in Phosphate Buffer Solution (PBS) 1X (Thermo Fisher Scientific; #10010023) and stained with conjugated antibodies CD44v6-APC (2F10, mouse IgG1) (R&D systems; #FAB3660A), CD133/2-APC (293C3-APC) (Miltenyi Biotec; #130-090-854), or corresponding isotype-matched control (IMC) CD3e-APC (UCHT1, mouse IgG1) (R&D systems; #FAB100A).

Techniques: Cell Culture, Microscopy, Expressing, Derivative Assay, Western Blot, Control