Journal: bioRxiv

Article Title: Cardiovascular Disease-Associated Non-Coding Variants Disrupt GATA4-DNA Binding and Regulatory Functions

doi: 10.1101/2024.09.19.613959

Figure Lengend Snippet: Expression and purification of GATA4 ZF. A) SDS-PAGE (left) and western blot (right) of GATA4 ZF (18.8 kDa). Abbreviations: wash fractions (W), eluted fractions (E), concentrated fractions (C), un-induced protein sample (U), IPTG-induced protein sample (I), supernatant fraction (S), and pellet fraction (P). B) Western Blot of full-length GATA4 expressed in HeLA cells (48.6 kDa).

Article Snippet: HeLa Cervical Adenocarcinoma Human (HeLa) cells (ATCC–CCL-2) were grown in Eagle’s Minimum Essential Medium (EMEM) (ATCC - 30-2003) with 10% Fetal Bovine Serum (FBS) at 37 °C and 5% CO 2 .

Techniques: Expressing, Purification, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Cardiovascular Disease-Associated Non-Coding Variants Disrupt GATA4-DNA Binding and Regulatory Functions

doi: 10.1101/2024.09.19.613959

Figure Lengend Snippet: CVD-associated SNPs alter gene expression and are in eQTL in cardiac tissue. A) Relative luciferase activity in HeLa cells transfected with reporter plasmids containing reference (blue with black circles) and alternate (orange with black squares) of variants rs1506537, rs56992000, rs2941506, and rs2301249. B) Cardiac tissue eQTL analysis of MRPL33 , TOP2B , PGAP3 , and CSK expressed in heart atrial appendage or left ventricle when rs1506537, rs56992000, rs2941506, and rs2301249 occur, respectively. C) University of California Santa Cruz (UCSC) Genome Browser tracks of variants rs2941506 and rs56992000 near TAD boundaries and regulatory elements.

Article Snippet: HeLa Cervical Adenocarcinoma Human (HeLa) cells (ATCC–CCL-2) were grown in Eagle’s Minimum Essential Medium (EMEM) (ATCC - 30-2003) with 10% Fetal Bovine Serum (FBS) at 37 °C and 5% CO 2 .

Techniques: Gene Expression, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: Capturing trophectoderm-like stem cells enables step-wisely remodeling of placental development

doi: 10.1101/2025.08.25.672082

Figure Lengend Snippet: (A) The morphology of ESCs, TBLCs and ESCs, TBLCs in TS medium after 3 days of induction. Scale bars, 250 μm. (B) FACS analysis of the percentage of CDX2 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using V6.5 cell line. (C) FACS analysis of the percentage of CD40 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using TC1 cell line. (D) FACS analysis of the percentage of CD40 + TELCs obtained from the TBLCs after induction with different molecules, including FGF4, Activin A, TGFβ1 and BMP4. The corresponding cell morphology is displayed in the lower panel. (E) Scatterplots displaying the transcriptome comparison of TELCs before and after CD40-based FACS using RNA-seq. Upregulated (FC>2) and downregulated (FC<0.5) genes are shown in red and blue, respectively. (F) The morphology of TBLCs of different passages and long-term culture in TX and TS medium, also the morphology of TBLCs after CD40 FACS after induction. Scale bars, 250 μm. (G) Western blotting was used to detect OCT4, CDX2 and EOMES in TELSCs from different passages. β-Tubulin was used as a loading control. (H) The morphology 8C embryos cultured in TX medium. Scale bars, 250 μm. (I) FACS analysis of the percentage of CD40 + cells in TELSC em s at different passages. (J) Immunofluorescence staining of TFAP2C and PEG10 in TBLCs, TELSCs and TELSC em s. Scale bars, 50 μm. (K) Cell cycle analysis of ESCs, TELSCs and TELSC em s. (L) Heatmap indicating the relative expression of TBLCs, TELSCs and TELSC em s. The representative genes and enrichment of GO terms of these genes is shown. (M) Heatmap indicating the relative expression of characteristic genes in TELSCs, TELSC em s and TSCs. Bubble chart showing the relative expression of these genes in mouse embryos. (N) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (O) The scatter plot displays differentially expressed genes between TELSCs and TSCs cultured in various media. The bar graph summarizes the number of differentially expressed genes identified under each comparison condition. (P) GSEA analysis of ESCs, TBLCs, TELCs and TELSCs based on “embryonic placenta development” and “placenta development” geneset. (Q) Heatmap indicating the differentially expressed genes in Hippo pathway of TELSCs and TBLCs. (R) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Bubble chart showing the relative expression of these genes in mouse embryos. (S) Phase contrast images of TBLCs cultured in TS medium for 24h supplemented with Verteporfin at the indicated concentration. Scale bars, 100 µm. (T) Heatmap indicating the differentially expressed genes of TELCs and TBLCs induction in TS medium plus verteporfin. Bubble chart showing the relative expression of these genes in mouse embryos. (U) GSEA analysis of TELCs, TBLCs induction in TS medium and in TS medium plus verteporfin based on TE geneset. (V) The morphology of TELSCs cultured in TS medium, TS medium plus ITS-X and TS medium plus TGFβ1. (W) Heatmap indicating the differentially expressed genes of TELSCs, TBLCs induction in TX medium withdraw ITS-X, in TS medium and in TS medium plus ITS-X. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (X) GSEA analysis of TBLCs induction in TX medium withdraw ITS-X and in TX medium based on “Positive regulation of stem cell proliferation” and “Positive regulation of cell cycle” geneset.

Article Snippet: All TSLs were cultured on Matrigel-coated plates, in 30% TS medium (RPMI 1640 (GIBCO, 11875119), 20% FBS, 1% GlutaMax (GIBCO, 35050061), 1% penicillin-streptomycin (GIBCO, 15140163), 1% sodium pyruvate (GIBCO, 11360070)) and 70% MEF-conditioned TS medium supplemented with 25 ng/ml human recombinant FGF4 (MCE, HY-P7014) and 1 μg/ml heparin (STEMCELL, 7980).

Techniques: Cell Culture, Comparison, RNA Sequencing, Western Blot, Control, Immunofluorescence, Staining, Cell Cycle Assay, Expressing, Concentration Assay

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: Virus and Cell Specific HMGB1 Secretion and Subepithelial Infiltrate Formation in Adenovirus Keratitis

doi: 10.1101/2025.01.07.631509

Figure Lengend Snippet: (A) Cytoplasmic and nuclear extracts prepared from uninfected or HAdV-D37-infected for 2, 12, 24, and 48 hpi. were resolved on 4-20% SDS-PAGE. Western blots for HMGB1 in the nuclear (Nuc), cytoplasmic (Cyt), and supernatant (Sup) extracts with TBP (nuclear) β-actin (cytoplasmic) as loading controls in cell types THE, PHCE, HCF, A549, and HEK293 (left to right). (B) Densitometric analysis of HMGB1 band intensity of cytoplasmic and nuclear extracts in THE, PHCE, HCF, A549, and HEK293 (left to right). p values were determined by t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ( n=3 ). (C) Quantification of extracellular HMGB1 measured from supernatants collected at indicated time points pi in THE and PHCE cells ( n=3 ). (D) Schematic representation (created using Biorender.com ) for HAdV-D37 induced translocation of HMGB1 from the cell nucleus to the cytoplasm and then into the extracellular space.

Article Snippet: A549 (CCL-185), a human lung carcinoma cell line, and HEK293 (CRL-1573), a human embryonic kidney cell line, were purchased from American Type Culture Collection (ATCC).

Techniques: Infection, SDS Page, Western Blot, Translocation Assay

Journal: bioRxiv

Article Title: Virus and Cell Specific HMGB1 Secretion and Subepithelial Infiltrate Formation in Adenovirus Keratitis

doi: 10.1101/2025.01.07.631509

Figure Lengend Snippet: (A) Western blot analysis for HMGB1 expression along with β-actin for load control from whole cell lysates of uninfected (M) or HAdV-D37-infected (V) THE cells for 2, 12, 24, and 48 hpi. qRT-PCR analysis of HMGB1 gene expression for mock and virus infected cells at the same times pi is shown as bar graphs below the Western blot. (B) Bar graph for qRT-PCR of the viral early gene E1A expression, a surrogate marker for viral entry, and normalized to human ACTG gene for quantification. (C) Viral late protein pIIIa expression in cytoplasmic and nuclear extracts prepared from uninfected or HAdV-D37-infected THE, PHCE, HCF, A549 and HEK293 for 2, 12, 24, and 48 hpi show successful infection of all cell types.

Article Snippet: A549 (CCL-185), a human lung carcinoma cell line, and HEK293 (CRL-1573), a human embryonic kidney cell line, were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Control, Infection, Quantitative RT-PCR, Gene Expression, Virus, Marker

Journal: bioRxiv

Article Title: The p53 Protein is a Suppressor of Atox1 Copper Chaperon in Tumor Cells Under Genotoxic Effects

doi: 10.1101/2023.07.25.550476

Figure Lengend Snippet: Dependence of Atox1 and p53 level in HCT116 and A549 cell lines with different TP53 status: A - immunoblotting with antibodies to p53, p21, and Atox1; beta-actin was used as a normalization. A densitometric analysis of the obtained data is shown below. B – RT-qPCR analysis with primers for TP53, CDKN1A, and ATOX1 genes; GAPDH gene was used as a reference. C - immunofluorescence staining with primary antibodies to Atox1, secondary antibodies with AlexaFluor488. DAPI was used for nuclei staining. WT – wild type cells, TP53 -/- – cells without TP53. For all experiments: n = 3, mean +/− SEM, paired Student t-test, p < 0,05.

Article Snippet: Transformed human cell lines used: HCT116 (colon adenocarcinoma) with intact p53; HCT116p53 -/- with a deletion of both alleles of the TP53 genes, as well as the A549 line with wild (A549) and knockout p53 (A549p53 -/- ) by the CRISPR-Cas9 method, acquired at ATCC.

Techniques: Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The p53 Protein is a Suppressor of Atox1 Copper Chaperon in Tumor Cells Under Genotoxic Effects

doi: 10.1101/2023.07.25.550476

Figure Lengend Snippet: Influence of cytotoxic agents on the activity of Atox1 at different status (WT and KO) of the TP53 gene in A549 and HCT116 cell lines, 24h after drugs exposure. A - immunoblotting with antibodies to p53, p21, and Atox1; beta-actin was used as a normalization. A densitometric analysis of the obtained data is shown below. B – RT-qPCR analysis with primers for TP53, CDKN1A and ATOX1 genes; GAPDH gene was used as a reference, C - immunofluorescence staining with primary antibodies to Atox1, secondary antibodies with AlexaFluor488. DAPI was used for nuclei staining. DOX – doxorubicin (0,1μM), CIS – cisplatin (35μM), PMA – phorbol-12-myristate- 13-acetate (80nM), H 2 O 2 – hydrogen peroxide (450μM), BLE – bleomycin (10μM). WT – wild type cells, TP53 -/- – cells without TP53. For all experiments: n = 3, mean +/− SEM, two-way ANOVA, p < 0,05.

Article Snippet: Transformed human cell lines used: HCT116 (colon adenocarcinoma) with intact p53; HCT116p53 -/- with a deletion of both alleles of the TP53 genes, as well as the A549 line with wild (A549) and knockout p53 (A549p53 -/- ) by the CRISPR-Cas9 method, acquired at ATCC.

Techniques: Activity Assay, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The p53 Protein is a Suppressor of Atox1 Copper Chaperon in Tumor Cells Under Genotoxic Effects

doi: 10.1101/2023.07.25.550476

Figure Lengend Snippet: Influence of ionizing radiation on the activity of Atox1 at different status (WT and KO) of the TP53 gene in A549 and HCT116 cell lines, 24h after ionizing irradiation (10Gy) exposure. A - immunoblotting with antibodies to p53, p21, and Atox1; beta-actin was used as a normalization. A densitometric analysis of the obtained data is shown below. B – RT-qPCR analysis with primers for TP53, CDKN1A and ATOX1 genes; GAPDH gene was used as a reference. The value of WT 0Gy (control) was taken as a 1.0 for all genes and is not shown in the graphs. For all experiments: n = 3, mean +/− SEM, two-way ANOVA, p < 0,05.

Article Snippet: Transformed human cell lines used: HCT116 (colon adenocarcinoma) with intact p53; HCT116p53 -/- with a deletion of both alleles of the TP53 genes, as well as the A549 line with wild (A549) and knockout p53 (A549p53 -/- ) by the CRISPR-Cas9 method, acquired at ATCC.

Techniques: Activity Assay, Irradiation, Western Blot, Quantitative RT-PCR, Control