Journal: bioRxiv

Article Title: Addressing anemia severity in antimony-resistant Leishmania donovani infection at the nexus of oxidative outburst and iron transit

doi: 10.1101/2024.03.04.583250

Figure Lengend Snippet: (A.i.) Giemsa-stained images of LD-S, LD-R, in murine peritoneal MФs with or without the presence of suboptimal dose of SbV (1.52µg/ml). In some experimental condition LD infection was performed in the presence of N-acetyl-L-cysteine (NAC), while in some experimental conditions, LDs are pre-treated with H 2 O 2 + 6-AN. All the infections were performed for 24hrs (A.ii.) Bar graph showing amastigotes/100MФs in all the experimental sets mentioned in A.i. (B.) Western blot of whole cell lysate showing significant high expression of Heme-oxygenase 1 (HO1) in LD-R infected MФs while no significant change in expression level was observed for Ferritin (FTH1) at 4hrs pi. β-actin is used as the positive control. (C.) Schematic representation of HO-1 promoter region with p50 and c-Rel binding sites. Site A (−4/−635) was demarcated as green, and Site B (−636/−1377) was demarcated as red. (D.i.) Western blot of nuclear fraction showing significant high expression of p50 and c-Rel in LD-R infected MФs 4hrs pi with Histone (H3) as loading control. (D.ii.) Confocal images representing the localization of p50 and c-Rel, in MФs, with DAPI representing the nucleus. The right-most panel shows the RGB-profile plot with grey regions demarcating the region where p50 and cRel are colocalized with DAPI. p50 and c-Rel were found to be localized in the nucleus of LD-R-infected MФs. One representative small nucleus of LD has been marked in ( * ) to show the infected MФs. (E.) Fold luciferase activity of RAW264.7 cell lysate transfected either with HO1+PGL3 promoter, or Site A ( −/− ), Site B( −/− ) deleted constructs followed by infection with LD-S and LD-R for 4hrs. PGL3 enhancer empty vector is used for normalization for each transfected set. Each experiment was performed in 3 biological replicates and graphical data are represented as Mean with SEM. P ≤ 0.05 is marked as *, P ≤ 0.01 is marked as **, P ≤ 0.001 is marked as ***, and P ≤ 0.0001 is marked as ****.

Article Snippet: Murine HO-1 promoters (−1385/+137), 1522bp using primers 5’-AAGGTACCTGAGGCTGGAGAGATGGCC-3’ and 3’-TAAAAGCTTCACCGGACTGGGCTAGTTCAG-5’ were PCR amplified and cloned in promoterless PGL3 enhancer empty vector (Promega, E1771) at the upstream of luciferase gene.

Techniques: Staining, Infection, Western Blot, Expressing, Positive Control, Binding Assay, Luciferase, Activity Assay, Transfection, Construct, Plasmid Preparation

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: bioRxiv

Article Title: Improved transfection methods of primary cultured astrocytes for observation of cytoskeletal structures

doi: 10.1101/2025.01.27.635031

Figure Lengend Snippet: (A) Lower magnification view of astrocytes cultured at high density (initial cell density of 0.6 × 10 5 cells/cm 2 ; cultured for 7 days) and low density (initial cell density of 0.3 × 10 5 cells/cm 2 ; cultured for 6 days). Phase contrast images show cells just before trypsin treatment (pre-transfection) and cultured for one day after electroporation. Scale bar = 200 μm. (B-D) Representative images of (B) EGFP-ezrin and mCherry-actin, (C) EGFP-actin and mCherry-lasp-2, and (D) EGFP-ezrin and mCherry-lasp-2 at 2, 3, and 2 days post-transfection, respectively. See supplementary movie 1 for (D); (D’) kymographs of magnified segment of stem process surface from supplemental movie 1 with a sequence interval of 20 sec. Arrows in (C) and (D) indicate examples of elliptical structures of fluorescent-tagged lasp-2. Asterisks in (D’) indicate examples of structures where lasp-2 and ezrin colocalized.

Article Snippet: The regions coding EGFP of the pLenti CMV EGFP Puro (658-5) vector (Addgene) were removed using BamHI and SalI and replaced with PCR products using NEBuilder HiFi DNA Assembly (New England BioLabs, MA, USA).

Techniques: Cell Culture, Transfection, Electroporation, Sequencing

Journal: bioRxiv

Article Title: Improved transfection methods of primary cultured astrocytes for observation of cytoskeletal structures

doi: 10.1101/2025.01.27.635031

Figure Lengend Snippet: (A) Lower magnification view of astrocytes before and after (1d, 2d and 5d) infection. Scale bar = 200 μm. (B-E) Representative images of (B) EGFP, (C) EGFP-lasp-2, (D) mCherry-lasp-2, and (E) mCherry-lasp-2 and added Sara Fluor 497 actin probe in astrocytes at 7, 4, 6, and 5 days post-infection with lentivirus vectors, respectively. (F) Time-lapse imaging of astrocytes infected with lentiviral coding with EGFP or EGFP-lasp-2 every 2 hours. Arrows in (C) to (E) indicate examples of elliptical structures of fluorescent-tagged lasp-2.

Article Snippet: The regions coding EGFP of the pLenti CMV EGFP Puro (658-5) vector (Addgene) were removed using BamHI and SalI and replaced with PCR products using NEBuilder HiFi DNA Assembly (New England BioLabs, MA, USA).

Techniques: Infection, Imaging