Journal: Journal of Hematology & Oncology

Article Title: Electrostatic anti-CD33-antibody–protamine nanocarriers as platform for a targeted treatment of acute myeloid leukemia

doi: 10.1186/s13045-022-01390-5

Figure Lengend Snippet: The αCD33-mAB-P/P nanocarriers only transport siRNA in presence of free SMCC-protamine (SMCC-P). A Coomassie-stained SDS–PAGE showing αCD33-mAB, αCD33-mAB coupled with SMCC-P and HPLC-fractions 29–30 of αCD33-mAB-P/P upon effective depletion of unbound SMCC-P; HC = heavy chain, LC = light chain, -P = SMCC-protamine. B Antibody–protamine conjugates with fluorescent Alexa488-siRNA in cell-free incubation overnight on chamber slides. αCD33-mAB-P/P with free SMCC-P forms visible vesicular structures (left panel), while αCD33-mAB-P after depletion of free SMCC-P (fraction 30, see A) do not form visible structures (right panel). C. DLS and zeta-potential measurement of αCD33-mAB-P/free protamine-scr-siRNA carriers. D. αCD33-mAB-P/P-scr-siRNA nanoparticles were left to form for 2 h and subjected to electron microscopy on copper grids by phosphotungstate negative staining. E–H. Immunostaining of nanocarriers with an anti-human IgG antibody to illustrate the accessibility and location of the αCD33-mAB in the outside rim and the siRNA in the lumen of the αCD33-mAB-P/P-nanocarriers. I. Protamine was chemically coupled to Cy3 and then incubated with αCD33-mAB-P that was depleted from free protamine and with non-fluorescent control-siRNA to form nanocarriers. This complexation was performed for 2 h at RT and nanocarriers were then immobilized o/n on slides for immunostaining as in G. J. αCD33-mAB-P/P-Cy3-control-siRNA show homogeneous Cy3 (blue) micelles. K. The same vesicles as in L show anti-human IgG-Alexa647 (red) fluorescence in ring-like structure around each vesicle (staining as depicted in E). L. Overlay of panels J and K. α, anti

Article Snippet: Protamine was conjugated using Cy3 Mono-Reactive Dye Pack (cat. no. PA23001, Cytiva, Freiburg, Germany) according to the manufacturer’s protocol to achieve protamine-Cy3.

Techniques: Staining, SDS Page, Incubation, Zeta Potential Analyzer, Electron Microscopy, Negative Staining, Immunostaining, Control, Fluorescence

Journal: Journal of Hematology & Oncology

Article Title: Electrostatic anti-CD33-antibody–protamine nanocarriers as platform for a targeted treatment of acute myeloid leukemia

doi: 10.1186/s13045-022-01390-5

Figure Lengend Snippet: Cellular targeting of Bruton’s kinase (BTK) by αCD33-mAB-P/P-ibrutinib-Cy3.5 and inhibition of clonal growth of treated AML-cells. A Schematic overview: spontaneous assembly of the αCD33-mAB-P/P-ibrutinib-Cy3.5 nanocarrier. B The αCD33-mAB-P/P conjugate was incubated for 2 h with anionic ibrutinib-Cy3.5 (left panels) or uncharged ibrutinib (trademark: imbruvica™; right panels) in 1:20 ratio and applied to cell-culture treated glass slides for fluorescence microscopy. Only αCD33-mAB-P/P-ibrutinib-Cy3.5 complexes led to the formation of numerous vesicles, where the larger vesicles showed intense Cy3.5 fluorescence (upper left panel) and vesicle formation in phase contrast (PC, lower left panel). No nanocarrier formation in presence of uncharged ibrutinib (upper and lower right panels, bubbles in lower right panel are mounting air inclusion artifacts). C Electromobility shift assays showing the electrostatic loading capacity of ibrutinib-Cy3.5 to conjugates from A in a molar ratio. One mol of αCD33-mAB-P/P can bind at least 20–50 mol ibrutinib-Cy3.5. D CD33-positive OCI-AML2 cells were treated by the respective conjugates shown for 72 h, lysed and subjected to SDS–PAGE and Western blotting for phospho-BTK (pBTK), total BTK (tBTK) and actin as a loading control. Both, free ibrutinib-Cy3.5 and αCD33-mAB-P/P-ibrutinib-Cy3.5 complexes inhibited the phosphorylation of BTK. E-M Fluorescence microscopy of OCI-AML2 cells treated with targeting conjugates and controls showing a marked intracellular enrichment of Cy3.5-signals (I). Fluorescence microscopy of OCI-AML2 cells pre-treated with ibrutinib-bodipy (green, G and M) do not show intracellular enrichment of Cy3.5-signals after αCD33-mAB-P/P-ibrutinib-Cy3.5 treatment (J compared to G). N Upper panels: Photographs of representative colony formation assays as summarized in the lower panel. In colony formation assays, 1200 nM untargeted ibrutinib-Cy3.5 did not reduce colony growth of OCI-AML2 cells, while the specifically targeted αCD33-mAB-P/P-ibrutinib-Cy3.5 (60 nM nanocarrier: 1200 nM ibrutinib-Cy3.5) reduced the colony growth to below 30% of the PBS controls, more than the treatment with 1200 nM uncharged ibrutinib (right-most bar). Significance: *, p < 0.05, 2-tailed T-test. Means plus SD of 3 independent experiments. α, anti

Article Snippet: Protamine was conjugated using Cy3 Mono-Reactive Dye Pack (cat. no. PA23001, Cytiva, Freiburg, Germany) according to the manufacturer’s protocol to achieve protamine-Cy3.

Techniques: Inhibition, Incubation, Cell Culture, Fluorescence, Microscopy, SDS Page, Western Blot, Control, Phospho-proteomics

Journal: Virology

Article Title: Impact of dynamin 2 on adenovirus nuclear entry

doi: 10.1016/j.virol.2019.01.008

Figure Lengend Snippet: A. E1A expression at 2 hr pi as measured after virus infection alone (V), or infection following bafilomycin A pretreatment (Baf+V) at 250 nM or 500 nM, and in the solvent control (DMSO+V). B. Effect on E1A mRNA expression of pretreatment with cytochalasin D prior to virus infection (Cyto D+V) at 250 nM or 2 μM; solvent control (DMSO+V), virus alone (V); 2 hr pi (DMSO+V vs. Cyto D+V 2 μM; *p<0.05, ANOVA). C. Effect on E1A mRNA expression of pretreatment with nocodazole prior to viral infection (Noc+V) at 10 μM or 30 μM, virus alone (V), and DMSO control (DMSO+V). D. Confocal microscopy representation of viral entry at 2 hr pi in the presence of solvent control (panel 1, DMSO+V) or 30 μM nocodazole (panel 2, Noc+V). Cy3 labelled HAdV-D37 (red), anti-α tubulin staining (green). E. Western blot showing protein expression levels in keratocytes after dynamin 2 (DNM2) knockdown: mock transfected and virus infected (V), scRNA transfected and virus infected (scRNA+V), siDNM2 transfection prior to viral infection (siDNM2+V). Western blot for α-AP2Al, an adaptor protein shown as negative control for specificity of DNM2 knockdown. Actin blot shows load control. Dynamin 2 mRNA expression levels by qRT-PCR are represented in bar graph just below. F. Early gene expression (E1A) as measured by qRT-PCR from same RNA pool as in E: virus alone (V), scrambled RNA (scRNA+V), dynamin 2 knockdown, virus infected cells (siDNM2+V) (*p<0.001, ANOVA). G. Western blot shows expression of mCherry in empty vector prior to virus infection (EV+V) and mCherry-dynamin 2 (arrow) prior to virus infection (OE-DNM2+V). The bar graph just below shows mRNA expression of dynamin 2. H. E1A mRNA expression as measured in cells after dynamin 2 vector pretreatment, performed on same RNA pool from G (*p=.01, Students t test). Error bars represent standard deviation of the mean. Each experiment was repeated at least 5 times with similar results.

Article Snippet: Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, cat. no. PA23031).

Techniques: Expressing, Virus, Infection, Solvent, Control, Confocal Microscopy, Staining, Western Blot, Knockdown, Transfection, Negative Control, Quantitative RT-PCR, Gene Expression, Plasmid Preparation, Standard Deviation

Journal: Virology

Article Title: Impact of dynamin 2 on adenovirus nuclear entry

doi: 10.1016/j.virol.2019.01.008

Figure Lengend Snippet: A. Western blot for dynamin 2, acetylated tubulin, and GAPDH after mock infection (M), virus infection (V), empty vector transfection prior to virus infection (EV+V), and dynamin 2 vector transfection prior to infection (OE-DNM2+V). Blots were cut at the appropriate sizes and probed with specific antibodies. B. Western blot for dynamin 2 protein, acetylated tubulin, and GAPDH in cells after treatment with scRNA or dynamin 2 siRNA prior to viral infection (scRNA+V, siRNA+V, respectively). C. Confocal microscopy of mock and Cy3-labeled (red) virus infected cells, 1 hr pi, stained for acetylated tubulin (green). Mock infected and virus infected cells both show increased perninuclear acetylated tubulin when knocked down for dynamin 2 (row 2, right hand column, arrows, and row 4, right hand column). Virions in cells knocked down for dynamin 2 appear to accumulate in the perinuclear region at 1 hr pi. Each experiment was repeated at least 5 times with similar results.

Article Snippet: Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, cat. no. PA23031).

Techniques: Western Blot, Infection, Virus, Plasmid Preparation, Transfection, Confocal Microscopy, Labeling, Staining

Journal: Virology

Article Title: Impact of dynamin 2 on adenovirus nuclear entry

doi: 10.1016/j.virol.2019.01.008

Figure Lengend Snippet: A. Modified schematic of SLO assay (Suomalainen et al., 2013). In this assay, SLO permeabilizes only cell membranes and not endosomal membranes, so that Cy3-labeled virions within endosomes appear red, while virions in the cytosol appear yellow upon binding to anti-Cy3 antibody with a green chromophore. B. Streptolysin O (SLO)-penetration assay performed 1 hr pi. Panel 1 shows the no-SLO control, where there is no staining for GM-130, indicating absence of antibody penetration. Panels 2-5 show equivalent staining for GM130 (green), indicating that the transfections did not affect pore formation by SLO. C. SLO assay performed in cells transfected prior to viral infection with empty vector (EV), dynamin 2 overexpression construct (OE-DNM2), scRNA, or siRNA (siDNM2). In SLO treated groups (+SLO), virions in endosomes appear red, while virions in cytosol appear yellow. As a control, left panel shows same groups treated with triton X100 without SLO (−SLO), and thus virions in both cytosol and endosomes appear yellow. Each experiment was repeated at least 3 times with similar results.

Article Snippet: Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, cat. no. PA23031).

Techniques: Modification, Labeling, Binding Assay, Control, Staining, Transfection, Infection, Plasmid Preparation, Over Expression, Construct

Journal: Virology

Article Title: Impact of dynamin 2 on adenovirus nuclear entry

doi: 10.1016/j.virol.2019.01.008

Figure Lengend Snippet: A. Confocal microscopy of cells pretreated with empty vector (EV), dynamin 2 expressing vector (OE-DNM2), scRNA, or dynamin 2 siRNA (siDNM2) and infected with Cy34abeled HAdV-D37 (red) for 1 hr, and stained with anti-pericentrin (green), and DAPI (blue). B. Distance from green signals (arrows) to the closest edge of adjacent nuclei (100 cells per experimental group in three experiments) was measured using Leica application suite X (LAS X, Leica Microsystems, Wetzlar, Germany), and graphed in boxplots (*p<0.0001, Kruskal-Wallis). C. Transmission electron microscopy of infected keratocytes from same pretreatment groups, with 3 distinct images shown per group shown. NU: nucleus. Scale bar = 500 nm. D. Measurements taken from each microtubule organizing center to nearest nuclear membrane were performed on 10 cells per group by a masked observer. Data shown reflects means and standard deviation for each group (*p<0.05, Students t test). E. Confocal microscopy showing co-localization (yellow) of nuclear pore complex protein Nup358 (green), and Cy3-labeled HAdV-D37 (red), 2 hr pi. Each experiment was performed three times with similar results.

Article Snippet: Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, cat. no. PA23031).

Techniques: Confocal Microscopy, Plasmid Preparation, Expressing, Infection, Staining, Transmission Assay, Electron Microscopy, Membrane, Standard Deviation, Labeling