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: bioRxiv

Article Title: Spatially regulated mRNA decay sharpens expression patterns in the Drosophila embryo

doi: 10.1101/2025.02.08.637258

Figure Lengend Snippet: (A) Heatmaps generated from eve smFISH data showing the number of active transcription sites across the expression domain at progressive time points during nc14 (underlying data in Fig. S1A). (B) Maximum projection of an smFISH image of early mid eve stripe 2, with mRNAs shown in white and DAPI in blue. Scale bars: 5 μm (main image) and 1 μm (inset regions). Green boxes mark cells displayed in the inset regions in the centre and the anterior and posterior edges. Active transcription sites were identified within the nuclear compartment in Z and are marked with a green arrow. (C) As in B for stripe 2 in late mid nc14. (D) (i) Confocal images of fixed nc14 embryos stained with smFISH probes for eve (magenta) and Me31B-GFP P-bodies (green). Scale bar: 5 μm. Images are maximum projections of 7 slices; individual mRNAs (magenta arrowheads), P-bodies (green arrowheads), and colocalised mRNA and P-body signals (white arrowheads) are highlighted. (E) P-body colocalisation index of eve mRNAs in early mid and late mid nc14; n = 3 embryos, p = 0.0034, unpaired t-test used to determine significance with α = 0.05. (F) Schematic illustrating anterior-posterior (AP) binning of a region across stripe 2 used in (G). (G) P-body colocalisation data across stripe 2 for 3 timepoints from mid nc14. Data over the AP axis are fit with a gaussian process (GP). The green shaded region marks the 95% confidence interval. Heatmaps above display the average mRNA per cell over the corresponding AP bins.

Article Snippet: A Rabbit anti-Me31B antibody (1:400, biorbyt, orb1147725) was used to immunostain embryos in and Fig. S5. smFISH staining in , and S1-4 was carried out in the Me31B-GFP line (BDSC Stock #51530).

Techniques: Generated, Expressing, Staining

Journal: bioRxiv

Article Title: Spatially regulated mRNA decay sharpens expression patterns in the Drosophila embryo

doi: 10.1101/2025.02.08.637258

Figure Lengend Snippet: (A) smFISH image of P-bodies (green arrowheads) marked by Me31B-GFP in wt and pcm - mCherry-BLID blue light treated nc14 embryos. (B) (i) P-body volumes in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP embryos, n = 3 embryos, p = 0.0041, nested t-test used to determine significance with α = 0.05. (ii) P-body numbers in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP blue light treated embryos, n = 3 embryos, unpaired t-test used to determine significance with α = 0.05. (C) smFISH images of eve mRNAs (magenta arrowheads), P-bodies (green arrowheads) and colocalised mRNAs (white arrowheads) in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP blue light treated embryos. (D) Number of mRNAs per cell in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP blue light treated embryos, n = 3 embryos, unpaired t-test used to determine significance with α = 0.05. (E) Percentage of mRNAs localised to P-bodies in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP blue light treated embryos; n = 3 embryos, unpaired t-test used to determine significance with α = 0.05. Scale bars: 2 μm.

Article Snippet: A Rabbit anti-Me31B antibody (1:400, biorbyt, orb1147725) was used to immunostain embryos in and Fig. S5. smFISH staining in , and S1-4 was carried out in the Me31B-GFP line (BDSC Stock #51530).

Techniques:

Journal: bioRxiv

Article Title: Spatially regulated mRNA decay sharpens expression patterns in the Drosophila embryo

doi: 10.1101/2025.02.08.637258

Figure Lengend Snippet: (A) smFISH images of eve mRNAs in early nc14 in control ( nos-Cas9 and Me31B-GFP ) and degron ( pcm - mCherry-BLID and pcm-mCherry-BLID; Me31B-GFP ) embryos. All embryos have membrane ingression < 5 μm. (B) Quantitation of wild-type and mutant eve patterns in control ( nos-Cas9 and Me31B-GFP ) degron ( pcm - mCherry-BLID and pcm - mCherry-BLID; Me31B-GFP ) and pcm mutant embryos ( pcm 15 / FM7 and pcm 15 / FM6; Me31B-GFP ) from eve RISH ( n = 106, 26, 57,11, 63 and 94). (C) . Confocal images of mutant eve expression patterns in pcm 15 /FM6; Me31B-GFP embryos. Scale bars: 50 μm.

Article Snippet: A Rabbit anti-Me31B antibody (1:400, biorbyt, orb1147725) was used to immunostain embryos in and Fig. S5. smFISH staining in , and S1-4 was carried out in the Me31B-GFP line (BDSC Stock #51530).

Techniques: Control, Membrane, Quantitation Assay, Mutagenesis, Expressing