Journal: bioRxiv

Article Title: Sialidases derived from Gardnerella vaginalis remodel the sperm glycocalyx and impair sperm function

doi: 10.1101/2025.02.01.636076

Figure Lengend Snippet: A) Schematic describing lectin flow cytometry and representative glycan epitopes. B, C, D) Sperm were exposed to sialidases (AUS 20 units or GvNanH2 160 units) for 1 hour, fixed in formalin, and stained with MAL-II-biotin/Neutravidin-Alexa Fluor 488, SNA-Cy5, or PSA-FITC respectively. Each point represents the median fluorescence intensity from one donor. Statistics represent a mixed model comparison with post-hoc Holm-Šidák tests. E) Schematic representing change in zeta potential after removal of negatively charged sialic acid on sperm surface. F) Sperm were treated with sialidase at various doses for 1 hour, and zeta potential was measured via dynamic light scattering. Each measurement represents the average of seven technical replicates, and statistics represent a mixed model comparison with post hoc Holm-Šidák tests.

Article Snippet: Biotinylated Maackia Amurensis Lectin II (MAL II, Cat #:B-1265-1) and Cy5-conjugated Sambucus Nigra Lectin (SNA, Cat #: CL-1305-1) were purchased from Vector Labs. Human Contraception Antibody (HCA), was a gift from ZabBio.

Techniques: Flow Cytometry, Staining, Fluorescence, Comparison, Zeta Potential Analyzer

Journal: bioRxiv

Article Title: Single Particle Tracking of Genetically Encoded Nanoparticles: Optimizing Expression for Cytoplasmic Diffusion Studies

doi: 10.1101/2024.11.17.623896

Figure Lengend Snippet: A) Characteristic log-log plots of MSD vs. lag time (τ). Slope α indicates diffusion type: Brownian (α=1), subdiffusive (α<1), superdiffusive (α>1). Plateau indicates confinement. B) Log-log plot of ensemble- and time-averaged MSD ( ate ) for particles longer than 10 frames (50 ms), corrected for static error and fitted to a power law with different lag time Δt values of 5, 10, 15, or 20 ms (τ = nΔt), the corresponding alpha values are given in the legend; C) image of U2OS with lower (fluorescent intensity = 16 r.u.) and higher GEM expression (fluorescent intensity = 147 r.u.). Sample size was 60 cells. D) Density probability function of collected D α , E) α,and F) D eff collected after fit with power-law relationship with σ/Δt = 0.5; G) Plot of D eff vs. fluorescence intensity in cells throughout the U2OS colony. The red line indicates the division between cells with “High GEM expression” (30 cells) and “Low GEM expression” (30 cells); H) Corresponding plot of α vs. fluorescence intensity.

Article Snippet: U2OS cells expressing pCMV-pfv-Sapphire-Ires-DsRed (Addgene #116934) or pCMV-pfv-paGFP were sorted by flow cytometry using a Cell Sorter SH800S, while U2OS cells expressing TRE-pfv-Sapphire were selected with 2 μg/mL puromycin.

Techniques: Diffusion-based Assay, Expressing, Fluorescence

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Ter119 negative cells and Ter119 positive erythroid cells were purified from wild-type mouse bone marrow cells. G4 levels were tested by flow cytometry using the BG4 antibody that specifically recognizes G4. Quantification is on the right. (B) Bone marrow lineage-negative cells were cultured in Epo medium for 2 days. G4 levels were tested on different days using flow cytometry by the BG4 antibody. Quantification is on the right. (C) CD34+ human HSPCs were cultured in Epo medium for 21 days. The levels of G4 were measured by flow cytometry as in B at the indicated time. Cells at day 7, 14, and 21 represent proerythroblasts, polychromatic to orthochromatic erythroblasts, and orthochromatic to mature red blood cells, respectively. (D) Flow cytometric assays of G4 levels in the indicated bone marrow lineage cells purified from wild-type mice. (E) Quantification of D. (F) Gating strategy of various erythroblasts. Populations I to VI represent proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, late orthochromatic to reticulocytes, and mature red blood cells, respectively. (G-H) Flow cytometric assay of G4 level in bone marrow erythroid populations I (G) and V (H) from the indicated mice. Quantification is on the right. (I) Bone marrow lineage negative cells from the indicated mice were cultured in Epo medium for 2 days. G4 levels on different days were measured by flow cytometry using BG4 antibody. Quantification is below the histogram. (J) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (K) Quantitative analyses of G4 levels in cells from J using flow cytometric assays. (L) Quantitative analyses of cell death in cells from J using flow cytometric assays. The dead cells are defined as propidium iodide and annexin V double positive. (M) Quantitative analyses of G4 levels in bone marrow mononuclear cells from the patient with DDX41 mutated MDS. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Purification, Flow Cytometry, Cell Culture, Transduction, Expressing, Western Blot, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Epo medium-cultured mouse bone marrow lineage negative HSPCs were treated with 1 μM PDS for the indicated time. Immunofluorescence assays of γ-H2AX were performed, and representative images of the erythroid cells were presented. Scale bar: 5 μm. (B) Flow cytometry assay of the cells in A. (C) Statistical quantification of γH2AX signals in B. (D) Epo medium-cultured mouse bone marrow lineage negative HSPCs were cultured for 1 day, followed by the treatment of 1 μM PDS for 6 hours. Quantitative RT-PCR analyses of indicated ribosome RNAs were performed using different primer sets. (E) Western blotting assays of indicated in cells from D. Actin was used as a loading control. (F) Same as D except that bone marrow lineage negative HSPCs from HBBCre:Ddx41 fl/fl mouse were cultured for 1 day before the quantitative RT-PCR assays. (G) Western blotting assays of the indicated proteins in F. Cells from both day 1 and day 2 cultured cells were analyzed. (H) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (I) Immunohistochemical stains of p53 in bone marrow core biopsies from the patient in normal individual. Scale bar: 100 μm. (J) Quantification of γ-H2AX in bone marrow mononuclear cells from the patient in I and 2 control individuals. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Cell Culture, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Western Blot, Control, Transduction, Expressing, Immunohistochemical staining, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Representative wide-field picture and H&E stains of bone marrow organoid in culture. (B) Whole-mount 3D imaging of the organoids. Imaris was used for cell surface rendering. Organoids were stained with indicated antibodies and subsequently imaged using a laser scanning confocal platform. (C) Confocal immunofluorescence assays of erythroid islands in the iPSC-derived bone marrow organoids (left) and a primary human bone marrow biopsy (right). CD71 was labeled with green for organoids and magenta for primary bone marrow. DAPI: blue. (D) Flow cytometry assays of the organoids using indicated antibodies for various lineages. (E) 10,000 CellVue-labeled donor CD34+ HSPCs were co-incubated with iPSC-derived bone marrow organoids for 3 days in each well of a 96-well plate, followed by an immunofluorescence assay. Representative pictures show the engraftment of donor hematopoietic cells into the organoid. Green, red, and blue represent CD71, CellVue, and DAPI-positive nuclei, respectively. The arrow points to an engrafted CellVue positive cell expressing CD71. (F) Flow cytometry of the organoids using indicated antibodies for various lineages of the engrafted cells in organoids from E. (G) Same as E, except the donor CD34+ cells were transduced with lentiviral vectors expressing Cas9 and indicated sgRNAs before co-incubation. After 3 days, the cells were collected for flow cytometric assays of erythroid and myeloid differentiation of CellVue-positive donor hematopoietic cells and negative iPSC-derived hematopoietic cells. Each data point represents cells combined from 10 organoids. The comparison was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01. (H) Schematic model of the function of DDX41 during erythropoiesis. The diagram is generated through BioRender.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Imaging, Staining, Immunofluorescence, Derivative Assay, Labeling, Flow Cytometry, Incubation, Expressing, Transduction, Comparison, Generated

Journal: Nanoscale

Article Title: Ultrasmall Prussian blue nanoparticles attenuate UVA-induced cellular senescence in human dermal fibroblasts via inhibiting the ERK/AP-1 pathway.

doi: 10.1039/d1nr04268h

Figure Lengend Snippet: Fig. 3 USPBNPs decreased SA-β-gal activity, attenuated cell cycle arrest and reduced the expression of p16, p21 and p53 in UVA- radiated HDFs. (A) SA-β-gal staining of HDFs at 24 h after UVA radiation with or without USPBNPs pretreatment; scale bar: 100 μm. (B) Percentage of SA-β-gal positive stained cells calculated by Image J. (C) Cell cycle analysis of HDFs at 24 h after UVA irradiation with or without USPBNPs pretreatment by flow cytometry. (D) Cell cycle distribution in each group. (E) The protein level of p16, p21 and p53 in HDFs at 24 h after UVA irradiation with or without USPBNPs pretreatment detected by western blot. (F) Quantification of the western blot band signals of p16, p21 and p53 by Image J. *P < 0.05, **P < 0.01, ***P < 0.001, versus control group; #P < 0.05, ##P < 0.01 and ###P < 0.001, versus UVA group.

Article Snippet: The following primary antibodies were used, including anti-p16 (CST), antip21(CST), anti-p53 (CST), anti-IL-6 (Abcam), anti-TNF-α (Proteintech), anti-MMP-1 (Proteintech), anti-MMP-3 (Proteintech), anti-MMP-9 (Proteintech), anti-γH2AX (CST), anti-p-ERK (CST), anti-ERK (CST), anti-c-Jun (CST), anti-c-Fos (CST) and anti-GAPDH (CST).

Techniques: Activity Assay, Expressing, Staining, Cell Cycle Assay, Irradiation, Flow Cytometry, Western Blot, Control

Journal: Nature

Article Title: Structural basis of human transcription-DNA repair coupling.

doi: 10.1038/s41586-021-03906-4

Figure Lengend Snippet: Fig. 1 | Structure of the Pol II–TCR complex. a, Cryo-EM density (left) and ribbon model (right) of the Pol II–CSB–CSA–DDB1–UVSSA complex. The scheme depicts the domain composition and colour code for proteins. The solid black lines mark residues included in the model.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used F-12 Pol II antibody, Santa Cruz Biotechnology, sc-55492; anti-mouse HRP conjugate, Abcam, ab5870 Validation Mouse monoclonal antibody to RNA polymerase II subunit A.

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: Structural basis of human transcription-DNA repair coupling.

doi: 10.1038/s41586-021-03906-4

Figure Lengend Snippet: Fig. 2 | Formation and structure of ECTCR. a, Electrophoretic mobility shift assay monitors replacement of DSIF by CSB on the Pol II elongation complex. The gel was scanned in three different channels to reveal the elongation complex (via DNA), DSIF and CSB through different fluorescent labels. The experiment was repeated three times. Asterisks indicate the attachment of

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used F-12 Pol II antibody, Santa Cruz Biotechnology, sc-55492; anti-mouse HRP conjugate, Abcam, ab5870 Validation Mouse monoclonal antibody to RNA polymerase II subunit A.

Techniques: Electrophoretic Mobility Shift Assay

Journal: Nature

Article Title: Structural basis of human transcription-DNA repair coupling.

doi: 10.1038/s41586-021-03906-4

Figure Lengend Snippet: Fig. 3 | Complete Pol II–TCR complex and ubiquitylation by CRL4CSA. a, In vitro ubiquitylation of the complete Pol II–TCR complex by CRL4CSA. The experiment was repeated two times. For gel source data, see Supplementary Fig. 1. b, Tandem mass spectrometry fragment spectrum of the RPB1 peptide

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used F-12 Pol II antibody, Santa Cruz Biotechnology, sc-55492; anti-mouse HRP conjugate, Abcam, ab5870 Validation Mouse monoclonal antibody to RNA polymerase II subunit A.

Techniques: In Vitro, Mass Spectrometry

Journal: Cancer cell

Article Title: Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity.

doi: 10.1016/j.ccell.2021.06.017

Figure Lengend Snippet: Figure 4. Phenotypic plasticity of mesenchymal marker expression (A) Flow cytometry analysis of PDPN and CD105 in purified and in-vitro-cultured CD105pos and CD105neg pancreatic fibroblasts (PaFs) after 1 and 7 weeks. Plots are representative of n = 4 experiments. Relative frequencies shown in relevant quadrants. (B) Normalized Eng mRNA expression in purified CD105pos (n = 4) and CD105neg PaFs (n = 4) treated with control (top) or KPC PDA conditioned medium (bottom). Data displayed as mean ± SD. (C) Representative flow cytometry analysis (n = 4) of CD105 on GFPposCD105pos and GFPposCD105neg PaFs in mono- or co-culture with RFPpos KPC PDA tu- mor cells. (D) Representative flow cytometry analysis (n = 3) of CD105 in isolated CD105pos and CD105neg human PaFs after >3 weeks of in vitro culture. (E and F) MC analysis of primary PaFs treated with the indicated ligands for 3 days. Representative plots displaying relative frequencies of CD105pos and CD105neg PaFs. (G and H) Heatmap of median marker intensity (MMI) displayed as column Z scores for each phenotypic marker on CD105pos (G) and CD105neg (H) PaFs after 3 days of treatment as indicated. Boxplots show MMI with upper and lower boundary of the interquartile range and whiskers denoting maximum and minimum values minus outliers, across all conditions. (I and J) Representative flow cytometry analysis (n = 3) of CD105pos (I) and CD105neg (J) PaFs with IFN-g, IFN-g + KPC PDA conditioned medium, or IFN-g + TGF- b1 treatment. Samples are compared using unpaired t tests (B) (top and bottom). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5 and Table S4.

Article Snippet: The first staining round used mouse anti-human CD105 antibody (CST clone 3A9) at 1/200 and TSA570 (FP1488001KT).

Techniques: Marker, Expressing, Flow Cytometry, In Vitro, Cell Culture, Control, Cytometry, Co-Culture Assay, Isolation