Journal: Kobe Journal of Medical Sciences

Article Title: Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells

doi:

Figure Lengend Snippet: HEK293 cells transiently transfected with a vector encoding either HA-SphK1 or HA-SphK2 together with control, SphK1-siRNA or SphK2-siRNA were cultured for 72 hr. After cell lysis, the lysates were subjected to SDS-PAGE followed by immunoblot analysis using anti-HA and anti-β-tubulin antibodies (A). K562 cells transfected with control, SphK1-siRNA or SphK2-siRNA were cultured for 48 hr. Cell medium was then changed to exosome-depleted one and cultured for 12 hr. Exosomes were purified from the media and analyzed for cargo contents by immunoblot analysis using anti-TfR1, anti-HSP70, and anti-flotillin 2 antibodies. Cell lysates were also subjected to immunoblot analysis with anti-flotillin 2 antibody (B).

Article Snippet: Anti-EEA1 antibody (catalog number 610456) was purchased from BD Biosciences; anti-TfR1 (catalog number HM2134) from Hycult Biotech; anti-HSP70 (catalog number SPA-815) from Stressgen Biotechnologies; anti-flotillin 2 (catalog number sc-48398) from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Lysis, SDS Page, Western Blot, Purification

Journal: Kobe Journal of Medical Sciences

Article Title: Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells

doi:

Figure Lengend Snippet: K562 cells transiently transfected with a vector encoding either siRNA-resistant HA-SphK2 or HA-SphK2(G248D) together with control or SphK2-siRNA were cultured for 48 hr. Exosomes were prepared as in Fig. 2B and analyzed for cargo contents by immunoblot analysis using anti-flotillin 2 antibody (A). K562 cells were cultured in exosome-depleted medium for 12 hr in the absence or presence of either 10 μM DMS or 50 μM HACPT. Exosomes were purified from the media and analyzed for cargo contents by immunoblot analysis using anti-TfR1, anti-HSP70, and anti-flotillin 2 antibodies (B).

Article Snippet: Anti-EEA1 antibody (catalog number 610456) was purchased from BD Biosciences; anti-TfR1 (catalog number HM2134) from Hycult Biotech; anti-HSP70 (catalog number SPA-815) from Stressgen Biotechnologies; anti-flotillin 2 (catalog number sc-48398) from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Western Blot, Purification

Journal: Pharmaceutics

Article Title: Current and Emerging Strategies for Enhancing Antibody Delivery to the Brain

doi: 10.3390/pharmaceutics13122014

Figure Lengend Snippet: Current and emerging delivery strategies to enhance antibody penetration in the brain. The blood–brain barrier (BBB), composed of endothelial cells (BECs), the acellular basement membrane, pericytes and astrocytic end-feet, shields the brain microenvironment from foreign materials in the blood, including administered biotherapeutics. Strategies to overcome the BBB and enhance the delivery of biotherapeutics, particularly mAbs, include: (1) Bi-specific antibodies. Conjugation of a therapeutic antibody to an antibody or ligand specific to an RMT receptor (e.g., transferrin receptor (TfR) or insulin receptor (IR)) results in endocytosis, allowing the antibody to traverse across the BBB and then engage its CNS target within the brain parenchyma. (2) Focused ultrasound in combination with the intravenous injection of gas-filled lipid shell microbubbles (MBs) imparts a mechanical force upon the brain endothelium, inducing increased vesicle formation within BECs and disruption of tight junctions between BECs, allowing peripherally administered antibodies to cross the BBB through vesicle-mediated transcytosis and paracellular transport, respectively. (3) Nanoparticles describe a range of nanoscale drug delivery vehicles including liposomes (20–1000 nm), gold nanoparticles (Au NPs) (1–150 nm) and dendrimers (2–10 nm). Antibodies can be conjugated to the outer surface of the nanoparticle. In addition, nanoparticles can be conjugated with targeting ligands and polyethylene glycol (PEG) to mediate efficient transcytosis across the BBB and site-specific targeting. These nanoparticles have been demonstrated to cross the BBB via receptor-mediated transcytosis, transmembrane diffusion, and carrier-mediated transport. (4) Exosomes can be loaded with proteins and have been shown to cross the BBB through its interaction with heparan sulfate proteoglycans on the surface of BECs, resulting in endocytosis and subsequent transcytosis across the BBB. This is an emerging strategy and could be used to encapsulate therapeutic antibody fragments or nanobodies in the future; (5) Adeno-associated virus (AAV). Viral vectors encoding protein/antibody genes and packaged into BBB-crossing AAV serotypes likely cross the BBB through glycoprotein-mediated transcytosis (akin to RMT), subsequently resulting in neuronal transduction and expression of protein/antibody genes. This approach would be especially useful for intracellular protein targets. Created with BioRender.com.

Article Snippet: Bi-specific antibody (TfR) , DNL310 (Enzyme transport vehicle-Iduronate-2-sulfatase fusion, ETV:IDS) , Mucopolysaccharidosis Type II (Hunter syndrome) , Denali Therapeutics Inc. , Phase 1/2 , Recruiting , NCT04251026.

Techniques: Conjugation Assay, Injection, Diffusion-based Assay, Transduction, Expressing

Journal: Pharmaceutics

Article Title: Current and Emerging Strategies for Enhancing Antibody Delivery to the Brain

doi: 10.3390/pharmaceutics13122014

Figure Lengend Snippet: Strategies that have enhanced therapeutic antibody delivery to the brain.

Article Snippet: Bi-specific antibody (TfR) , DNL310 (Enzyme transport vehicle-Iduronate-2-sulfatase fusion, ETV:IDS) , Mucopolysaccharidosis Type II (Hunter syndrome) , Denali Therapeutics Inc. , Phase 1/2 , Recruiting , NCT04251026.

Techniques: In Vitro

Journal: Pharmaceutics

Article Title: Current and Emerging Strategies for Enhancing Antibody Delivery to the Brain

doi: 10.3390/pharmaceutics13122014

Figure Lengend Snippet: Current delivery strategies in clinical trials.

Article Snippet: Bi-specific antibody (TfR) , DNL310 (Enzyme transport vehicle-Iduronate-2-sulfatase fusion, ETV:IDS) , Mucopolysaccharidosis Type II (Hunter syndrome) , Denali Therapeutics Inc. , Phase 1/2 , Recruiting , NCT04251026.

Techniques:

Journal: bioRxiv

Article Title: CD44 regulates epigenetic plasticity by mediating iron endocytosis

doi: 10.1101/693424

Figure Lengend Snippet: ( A ) Molecular structure (top) and 1 H NMR spectra of Hyal (bottom). Data showing that Hyal interacts with Fe(II) in near-physiological conditions. These interactions are reversed upon acidification of the media using trifluoroacetic acid (TFA). Functional groups that can interact with iron are highlighted in red. ( B – G ) Parental HMLER CD44 high and corresponding ko cell lines were used throughout these panels. ( B ) Western blot analysis of CD44 and TfR1 levels in CD44 and TFRC ko clones generated using CRISPR-Cas9. ( C ) Fluorescence microscopy analysis of internalized Hyal-FITC. Data showing that knocking out CD44 reduces endocytosis of Hyal. n = 3 biological replicates. ( D ) Fluorescence microscopy analysis of RhoNox-M-positive vesicles colocalizing with internalized Hyal-FITC or Tf-647. Data showing that cells expressing CD44 exhibit RhoNox-M/Hyal-positive vesicles that are free of Tf. The proportion of these vesicles is significantly reduced by knocking out CD44. n = 3 biological replicates. ( E ) Fluorescence microscopy analysis of RhoNox-M-positive vesicles colocalizing with Hyal-FITC in a CD44 ko clone complemented with CD44 . Data showing that CD44 complementation restores a higher proportion of RhoNox-M/Hyal-positive vesicles. n = 3 biological replicates. ( F ) ICP-MS measurements of cellular iron. Data showing that supplementing CD44-expressing cells with Hyal increases iron uptake. n = 5 biological replicates. ( G ) Western blot analysis of ferritin levels representative of 8 biological replicates. Data showing that supplementing CD44-expressing cells with Hyal increases levels of the iron storage protein ferritin. DAPI, 4′,6-diamidino-2-phenylindole. Unt., untreated. Scale bars, 10 μm. Bars and error bars, mean values ± s.d. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; n.s., not significant; unpaired t -tests. See also .

Article Snippet: CD109 (Santa Cruz Biotechnology, #sc-271085, WB: 1:500), CD24-APC (Sony Biotechnology Inc., #2155590, FC: 1 μL/10 6 cells), CD44 (Abcam, #ab189524, WB: 1:30000, FM: 1:200), CD44 (Abcam, #ab25340, FC, blocking: 10 μg/mL, 1 h), CD44 Alexa-Fluor-647 (Novus Biologicals, #NB500-481AF647, FC: 1 μL/10 6 cells), CD44 (R&D Systems, #FAB4948P, FC: 10 μL/10 6 cells), CDCP1 (Cell Signaling, #4115, WB: 1:1000), Drosophila spike-in antibody (Active Motif, #61686, ChIP-seq: 1 µg), E-cadherin (Cell Signaling, #20023195, WB: 1:1000, IF: 1:200), Ferritin (Abcam, #ab75973, WB: 1:1000), Fibronectin (Sigma-Aldrich, #F0791, WB: 1:1000), FSCN1 (Abcam, #ab126772, WB: 1:1000), H3 (Cell Signaling, #9715S, WB: 1:10 6 ), H3K9me2 (Cell Signaling, #4658S, WB: 1:1000, ChIP-seq: 6 μL), MGLL (Abcam, #ab24701, WB: 1:1000), PHF8 (Active Motif, #39711, WB: 1:1000), Transferrin receptor 1 (TfR1, Life Technologies, #13-6800, WB: 1:1000), Transferrin receptor 1 (TfR1, Merck Millipore, #GR09L-100UG, FC, blocking: 10 μg/mL, 1 h), TfR1-PE (R&D Systems, #FAB2474P, FC: 5 μL/10 6 cells), β-Tubulin (Sigma-Aldrich, #T4026-100UL, WB: 1:2000), γ-Tubulin (Sigma-Aldrich, #T5326, WB: 1:2000), Vimentin (Cell Signaling, #3932, WB: 1:500), Zeb1 (Santa Cruz Biotechnology, #sc-81428, WB: 1:500).

Techniques: Functional Assay, Western Blot, Clone Assay, Generated, CRISPR, Fluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: CD44 regulates epigenetic plasticity by mediating iron endocytosis

doi: 10.1101/693424

Figure Lengend Snippet: MDA-MB-468 cells were used throughout the figure and were treated with EGF for 72 h, unless stated otherwise. ( A ) Fluorescence microscopy analysis of E-cadherin and CD44 (scale bars, 10 μm). Bright field microscopy analysis of cell morphology (scale bars, 100 μm). Data showing induction of EMT upon EGF treatment. ( B ) Time course western blot analysis of levels of iron homeostasis and EMT proteins. Data showing that, in contrast to TfR1, CD44 levels increase during EMT. ( C ) Time course flow cytometry analysis of CD44 and TfR1 loading at the plasma membrane. Data showing that, in contrast to TfR1, CD44 loading increases steadily during EMT. ( D ) Fluorescence microscopy analysis of RhoNox-M-positive vesicles colocalizing with internalized Hyal-FITC or Tf-647. Data showing that EMT induction is associated with an increased proportion of RhoNox-M/Hyal-positive vesicles. n = 3 biological replicates. Scale bar, 10 μm. ( E ) ICP-MS measurements of cellular iron. Data showing that levels of cellular iron increase during EMT. n = 11 biological replicates. ( F ) Western blot analysis of ferritin levels in CD44 knock down conditions. Data showing that knocking down CD44 antagonizes the production of the iron storage protein ferritin in cells treated with EGF. ( G ) Time course RT-qPCR analysis of CD44, TFRC and FTH1 mRNA levels. Data indicating that, in contrast to TFRC , the RNA transcript of CD44 increases concomitantly with that of FTH1 during EMT. n = 3 biological replicates. Bars and error bars, mean values ± s.d. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; n.s., not significant; unpaired t -tests. See also .

Article Snippet: CD109 (Santa Cruz Biotechnology, #sc-271085, WB: 1:500), CD24-APC (Sony Biotechnology Inc., #2155590, FC: 1 μL/10 6 cells), CD44 (Abcam, #ab189524, WB: 1:30000, FM: 1:200), CD44 (Abcam, #ab25340, FC, blocking: 10 μg/mL, 1 h), CD44 Alexa-Fluor-647 (Novus Biologicals, #NB500-481AF647, FC: 1 μL/10 6 cells), CD44 (R&D Systems, #FAB4948P, FC: 10 μL/10 6 cells), CDCP1 (Cell Signaling, #4115, WB: 1:1000), Drosophila spike-in antibody (Active Motif, #61686, ChIP-seq: 1 µg), E-cadherin (Cell Signaling, #20023195, WB: 1:1000, IF: 1:200), Ferritin (Abcam, #ab75973, WB: 1:1000), Fibronectin (Sigma-Aldrich, #F0791, WB: 1:1000), FSCN1 (Abcam, #ab126772, WB: 1:1000), H3 (Cell Signaling, #9715S, WB: 1:10 6 ), H3K9me2 (Cell Signaling, #4658S, WB: 1:1000, ChIP-seq: 6 μL), MGLL (Abcam, #ab24701, WB: 1:1000), PHF8 (Active Motif, #39711, WB: 1:1000), Transferrin receptor 1 (TfR1, Life Technologies, #13-6800, WB: 1:1000), Transferrin receptor 1 (TfR1, Merck Millipore, #GR09L-100UG, FC, blocking: 10 μg/mL, 1 h), TfR1-PE (R&D Systems, #FAB2474P, FC: 5 μL/10 6 cells), β-Tubulin (Sigma-Aldrich, #T4026-100UL, WB: 1:2000), γ-Tubulin (Sigma-Aldrich, #T5326, WB: 1:2000), Vimentin (Cell Signaling, #3932, WB: 1:500), Zeb1 (Santa Cruz Biotechnology, #sc-81428, WB: 1:500).

Techniques: Fluorescence, Microscopy, Western Blot, Flow Cytometry, Quantitative RT-PCR

Journal: bioRxiv

Article Title: CD44 regulates epigenetic plasticity by mediating iron endocytosis

doi: 10.1101/693424

Figure Lengend Snippet: ( A ) Time course flow cytometry analysis of CD44 and TfR1 levels at the plasma membrane. Data showing that, in contrast to TfR1, CD44 loading increases steadily during EMT. ( B ) Fluorescence microscopy analysis of RhoNox-M vesicles colocalizing with internalized Hyal-FITC or Tf-647. Data showing that EMT induction is characterized by an increased proportion of RhoNox-M/Hyal-positive vesicles. n = 3 biological replicates for MCF7 cells and n = 1 for primary cells. Scale bars, 10 μm. Bars and error bars, mean values ± s.d. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; n.s., not significant; unpaired t -tests.

Article Snippet: CD109 (Santa Cruz Biotechnology, #sc-271085, WB: 1:500), CD24-APC (Sony Biotechnology Inc., #2155590, FC: 1 μL/10 6 cells), CD44 (Abcam, #ab189524, WB: 1:30000, FM: 1:200), CD44 (Abcam, #ab25340, FC, blocking: 10 μg/mL, 1 h), CD44 Alexa-Fluor-647 (Novus Biologicals, #NB500-481AF647, FC: 1 μL/10 6 cells), CD44 (R&D Systems, #FAB4948P, FC: 10 μL/10 6 cells), CDCP1 (Cell Signaling, #4115, WB: 1:1000), Drosophila spike-in antibody (Active Motif, #61686, ChIP-seq: 1 µg), E-cadherin (Cell Signaling, #20023195, WB: 1:1000, IF: 1:200), Ferritin (Abcam, #ab75973, WB: 1:1000), Fibronectin (Sigma-Aldrich, #F0791, WB: 1:1000), FSCN1 (Abcam, #ab126772, WB: 1:1000), H3 (Cell Signaling, #9715S, WB: 1:10 6 ), H3K9me2 (Cell Signaling, #4658S, WB: 1:1000, ChIP-seq: 6 μL), MGLL (Abcam, #ab24701, WB: 1:1000), PHF8 (Active Motif, #39711, WB: 1:1000), Transferrin receptor 1 (TfR1, Life Technologies, #13-6800, WB: 1:1000), Transferrin receptor 1 (TfR1, Merck Millipore, #GR09L-100UG, FC, blocking: 10 μg/mL, 1 h), TfR1-PE (R&D Systems, #FAB2474P, FC: 5 μL/10 6 cells), β-Tubulin (Sigma-Aldrich, #T4026-100UL, WB: 1:2000), γ-Tubulin (Sigma-Aldrich, #T5326, WB: 1:2000), Vimentin (Cell Signaling, #3932, WB: 1:500), Zeb1 (Santa Cruz Biotechnology, #sc-81428, WB: 1:500).

Techniques: Flow Cytometry, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: CD44 regulates epigenetic plasticity by mediating iron endocytosis

doi: 10.1101/693424

Figure Lengend Snippet: Iron bound to Tf or Hyal enters cells by means of TfR1-or CD44-mediated endocytosis, respectively. Excess cellular iron inhibits the canonical TfR1 pathway. Nuclear iron, αKG produced in mitochondria and iron-dependent enzymes mediate oxidative demethylation of chromatin marks to unlock the expression of specific genes including CD44 . CD44 regulates its own expression at the transcriptional level by mediating iron endocytosis and this pathway prevails in the mesenchymal state of cells. Iron homeostasis can be targeted at the plasma membrane, the endosomal/lysosomal compartment, the mitochondria and the nucleus using specific antibodies or small molecules.

Article Snippet: CD109 (Santa Cruz Biotechnology, #sc-271085, WB: 1:500), CD24-APC (Sony Biotechnology Inc., #2155590, FC: 1 μL/10 6 cells), CD44 (Abcam, #ab189524, WB: 1:30000, FM: 1:200), CD44 (Abcam, #ab25340, FC, blocking: 10 μg/mL, 1 h), CD44 Alexa-Fluor-647 (Novus Biologicals, #NB500-481AF647, FC: 1 μL/10 6 cells), CD44 (R&D Systems, #FAB4948P, FC: 10 μL/10 6 cells), CDCP1 (Cell Signaling, #4115, WB: 1:1000), Drosophila spike-in antibody (Active Motif, #61686, ChIP-seq: 1 µg), E-cadherin (Cell Signaling, #20023195, WB: 1:1000, IF: 1:200), Ferritin (Abcam, #ab75973, WB: 1:1000), Fibronectin (Sigma-Aldrich, #F0791, WB: 1:1000), FSCN1 (Abcam, #ab126772, WB: 1:1000), H3 (Cell Signaling, #9715S, WB: 1:10 6 ), H3K9me2 (Cell Signaling, #4658S, WB: 1:1000, ChIP-seq: 6 μL), MGLL (Abcam, #ab24701, WB: 1:1000), PHF8 (Active Motif, #39711, WB: 1:1000), Transferrin receptor 1 (TfR1, Life Technologies, #13-6800, WB: 1:1000), Transferrin receptor 1 (TfR1, Merck Millipore, #GR09L-100UG, FC, blocking: 10 μg/mL, 1 h), TfR1-PE (R&D Systems, #FAB2474P, FC: 5 μL/10 6 cells), β-Tubulin (Sigma-Aldrich, #T4026-100UL, WB: 1:2000), γ-Tubulin (Sigma-Aldrich, #T5326, WB: 1:2000), Vimentin (Cell Signaling, #3932, WB: 1:500), Zeb1 (Santa Cruz Biotechnology, #sc-81428, WB: 1:500).

Techniques: Produced, Expressing

Journal:

Article Title: A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide

doi: 10.1002/jgm.1345

Figure Lengend Snippet: (a) Schematic representation of chimeric Sindbis virus envelope proteins. 2.2 1L1L was derived from the chimeric 2.2 envelope protein [18]. 2.2 1L1L has two flexible linkers (Gly-Gly-Gly-Gly-Ser) and AVR II-Bst E II cloning sites at amino acid 71 of the E2 protein. BAP Sindbis contains a biotin acceptor peptide (BAP) derived from Escherichia coli biotin holoenzyme synthetase between the two flexible linkers. BBAPH contains BAP at the first amino acid of the E2 protein, and BAP II SINDBIS contains two BAP at both positions of the E2 protein. (b) The schematic strategy to conjugate virus with anti-human or rat transferrin receptor 1 and transferrin. BAP II SINDBIS envelope proteins were covalently conjugated with biotin. Anti-human or rat transferrin receptor antibodies were fused with avidin, designated anti-huTfR IgG-Av or anti-ratTfR IgG3-Av, respectively. Anti-huTfR IgG-Av or anti-ratTfR IgG3-Av can be conjugated with the BAP II SINDBIS envelope protein through the interaction of avidin and the biotin of the envelope protein. Neutravidin has four biotin binding sites. Thus, one neutravidin can bind both the biotinylated BAP II SINDBIS envelope protein and biotinlynated transferrin, which results in bridging the pseudotyped virus with transferrin

Article Snippet: Jurkat and Y3 cells (5 × 10 5 ) were incubated with 100 µl (5 µg/ml) of either mouse anti-rat transferrin receptor 1 (BD Biosciences, San Diego, CA, USA) or mouse antihuman transferrin receptor 1 (BD Biosciences) for 1 h at 4 °C, followed by incubation with 100 µl (200-fold dilution in PBS) of rabbit anti-mouse IgG conjugated with Alexa 488 (Invitrogen) for 1 h at 4 °C.

Techniques: Derivative Assay, Clone Assay, Avidin-Biotin Assay, Binding Assay

Journal:

Article Title: A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide

doi: 10.1002/jgm.1345

Figure Lengend Snippet: Gene transduction mediated by fusion proteins between avidin and anti-human or transferrin receptor antibody fusion proteins. (a) Expression of human and rat transferrin receptor 1 on Jurkat (human T-cell line) and Y3 (rat myeloma cell line) cells. Each cell type was stained either with isotype control control antibody (black line), mouse anti-human (red line), or rat (blue line) transferrin receptor 1 antibody, followed by staining with Alexa 488-conjugated secondary antibody. (b) Jurkat, was infected with the BAP II SINDBIS pseudotype in the presence or absence of fusion protein between avidin and either anti-human (anti-huTfR IgG-Av) or anti-rat (anti-ratTfR IgG3-Av) transferrin receptor 1 antibody. EGFP expression was analysed by flow cytometry 3 and 10 days post-transduction. Representive flow cutometric profiles are shown. Percent EGFP-positive is shown as the mean ± SD of triplicate experiments. (c) Y3-Ag 1.2.3, was infected with the BAP II pseudotype in the presence or absence of anti-huTfR IgG-Av or anti-ratTfR IgG3-Av. EGFP expression was analysed by flow cytometry 3 and 10 days post-transduction. Representive flow cutometric profiles are shown. Percent EGFP-positive is shown as the mean ± SD of triplicate experiments

Article Snippet: Jurkat and Y3 cells (5 × 10 5 ) were incubated with 100 µl (5 µg/ml) of either mouse anti-rat transferrin receptor 1 (BD Biosciences, San Diego, CA, USA) or mouse antihuman transferrin receptor 1 (BD Biosciences) for 1 h at 4 °C, followed by incubation with 100 µl (200-fold dilution in PBS) of rabbit anti-mouse IgG conjugated with Alexa 488 (Invitrogen) for 1 h at 4 °C.

Techniques: Transduction, Avidin-Biotin Assay, Expressing, Staining, Infection, Flow Cytometry