Journal: Cell Reports Medicine

Article Title: A PD-1-targeted, receptor-masked IL-2 immunocytokine that engages IL-2Rα strengthens T cell-mediated anti-tumor therapies

doi: 10.1016/j.xcrm.2024.101747

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Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat# 130-110-618.

Techniques: Purification, Blocking Assay, Control, Recombinant, Staining, Luciferase, Isolation, Cell Isolation, Biomarker Discovery, Amplification, Knock-In, Plasmid Preparation, Expressing, Software

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Multi-omics analysis reveals potential regulatory roles of SPI1 in EHT (A) Heatmap of 575 expressed TFs during the differentiation of Pluripotent Stem Cells (PSC) into Hematopoietic Progenitor Cells (HPC). Four expression clusters (TC1-TC4) are identified and are denoted by color and number. (B) Line graph of variable accessible chromatin elements during PSC to HPC differentiation, again with four identified clusters (C1-C4). (C) Enriched TF motifs in peaks of clusters C1-C4. (D) Heatmap showing variations in TF activity among the top 50 active transcription factors during differentiation. (E) Pearson correlation analysis of TF activity and gene expression for five key TFs. (F) Visualization of ATAC-seq footprint for motifs of the five representative TFs across different developmental stages. See also Figure S2 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Biomarker Discovery, Expressing, Activity Assay, Gene Expression

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 at the in vitro -generated HECs stage severely impairs HPC generation (A) Schematic illustration of the in vitro differentiation approach. SPI1 siRNA or control siRNA were introduced on D4 of EHT, with evaluations from D5 to D8. (B) Flow cytometric sorting strategy for HECs treated with siRNA. (C) qPCR results showing SPI1 expression from D5 to D8, using two distinct siRNA sequences targeting SPI1 . (D) Western blot analysis of PU.1 expression in siCtrl and siSPI1_1/2 groups from D5 to D8, with GAPDH as the internal normalization control. (E) Representative flow cytometry density plots showing CD34 + CD43 + cells among total cells in siCtrl and siSPI1_1/2 groups, from D5 to D8. (F) Quantification of suspended CD34 + CD43 + HPCs from siCtrl and siSPI1_1/2 groups, from D5 to D8. (G) Microscopy images of cells on D8 for siCtrl and siSPI1 conditions, scale bar = 100 μm. (H) Representative flow cytometry density plots of CD34 + CD43 + HPCs in suspended cells for siCtrl and siSPI1_1/2 groups on D8. Data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S4 , Table S1 and Table S2 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, In Vitro, Generated, Control, Expressing, Western Blot, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 during EHT does not alter HPC proliferation or apoptosis but affects differentiation potential (A) Flow cytometry sorting strategy for D8 suspended CD34 + CD43 + HPCs (FAM +/− ). (B and C) Representative flow cytometry density plots (B) and frequency plot (C) for cell cycle, G0/G1 (Edu − Hoechst low ), S (Edu + Hoechst low/high ) and G2/M (Edu − Hoechst high ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. (D and E) Representative flow cytometry density plots (D) and frequency plot (E) of early apoptosis (Annexin V + DAPI − ) and late apoptosis (Annexin V + DAPI + ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. Significance determined by two-tailed unpaired Student’s t-test. (F) Colony Forming Unit (CFU) assay of FAM − CD34 + CD43 + HPCs generated on D8 under siCtrl and siSPI1_1/2 conditions. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-way ANOVA. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figure S5 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, Flow Cytometry, Two Tailed Test, Colony-forming Unit Assay, Generated

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Transcriptional regulatory network suggests LYL1 and KLF1 as potential downstream targets of SPI1 (A) Heatmap displaying reordered normalized trans -regulation score (TRS) at the HPC stage. Identified modules from nonnegative matrix factorization (NMF) are represented in different colors. (B) Boxplots showing gene expression levels in different modules from A, comparing Endothelial Progenitor Cells (EPC) and HPC. p values, two-tailed paired Student’s t test. (C) All SPI1 Target Genes (TGs) are ordered descendingly according to their TRS, with a focus on the top 10 transcription factors (TFs) within these TGs. (D) Violin plots showing expression levels of the top 10 TFs from SPI1 TGs in both in vivo AGM hematopoiesis (left) and the in vitro system (right). (E) qPCR results showing the expression of the top 10 transcription factors from SPI1 target genes in D8 CD34 + CD43 + HPCs, excluding non-expressed genes SNAI3 and RUNX3 , in siCtrl and siSPI1_1/2 groups. Data are from n = 3 independent experiments, with significance assessed using two-tailed unpaired Student’s t test. (F) Enrichment analysis of Gene Ontology (GO) terms and KEGG signaling pathways of SPI1 ’s TGs that have p -value <0.001. Data are presented as means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance.

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Gene Expression, Two Tailed Test, Expressing, In Vivo, In Vitro, Protein-Protein interactions

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Overexpression of LYL1 and KLF1 can rescue SPI1 knockdown phenotypes (A) Co-transfection strategy of SPI1 siRNA with KLF1 / LYL1 overexpression during EHT to assess D8 HPC differentiation potential. (B) Western blot analysis of KLF1/LYL1 from D6 differentiated cells in the siCtrl+OE-Ctrl, siSPI1, and siSPI1+OE-KLF1/LYL1 groups, normalized to GAPDH. (C and D) Flow cytometry analysis of suspended CD34 + CD43 + HPCs on D8: (C) Representative density plot and (D) quantification in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. n = 3 independent experiments. (E) Colony-forming units (CFU) assay on D8 suspended CD34 + CD43 + HPCs across siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups, with significance assessed using two-way ANOVA. (F) Schematic description of lymphoid differentiation. (G) Representative flow cytometry density plots of the distribution of CD45 -/mid/+ CD5 + CD7 + cells under the normal conditions at D29. (H–I) Analysis of CD45 + CD5 + CD7 + cells: (H) Representative flow cytometry density plots (H) and frequency plot (I) of CD45 + CD5 + CD7 + cells in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figures S6 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Over Expression, Knockdown, Cotransfection, Western Blot, Flow Cytometry, Colony-forming Unit Assay, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

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Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Recombinant, Transfection, Plasmid Preparation, Reverse Transcription, In Vivo, In Vitro, Biomarker Discovery, Software

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Integrated multiomic analysis identifies TRIP13 as a mediator of alveolar epithelial type II cell dysfunction in idiopathic pulmonary fibrosis

doi: 10.1016/j.bbadis.2024.167572

Figure Lengend Snippet: Key resources.

Article Snippet: Mouse IgG1 anti-human CD326 (EpCAM)-conjugated microbeads , Miltenyi Biotec , Cat# 130–061–101 RRID: AB_2832928.

Techniques: Plasmid Preparation, Magnetic Beads, Recombinant, Blocking Assay, Lysis, Protease Inhibitor, Red Blood Cell Lysis, Software, Microscopy, Fluorescence, Imaging

Journal: Journal of visualized experiments : JoVE

Article Title: Retroviral overexpression of CXCR4 on murine B-1a cells and adoptive transfer for targeted B-1a cell migration to the bone marrow and IgM production

doi: 10.3791/61003

Figure Lengend Snippet: Table of Materials

Article Snippet: Incubate on ice for 10 minutes at 4 °C. table ft1 table-wrap mode="anchored" t5 caption a7 Name of Material/Equipment Company Catalogue Number Comments/Description anti-CD16/CD32, or Fc block Life Technologies MFCR00 Ter119 biotin eBioscience 13–5921–82 Clone: Ter119 CD3e biotin eBioscience 13–0033–85 Clone: eBio500A2 Gr-1 biotin eBioscience 13–5931–82 Clone: RB6–8C5 CD23 biotin eBioscience 13–0232–81 Clone: B3B4 NK1.1 biotin BD Biosciences 553163 Clone: PK136 F4/80 biotin Life Technologies MF48015 Clone: BM8 anti-biotin microbeads Miltenyi Biotec 130–090–485 LS columns Miltenyi Biotec 130–042–401 CD5 PE eBioscience 12–0051–83 Clone: 53–7.3 IgM PECF594 BD Biosciences 562565 Clone: R6–60.2 CD23 PECy7 eBioscience 25–0232–82 Clone: B3B4 B220 APC eBioscience 17–0452–83 Clone: RA3–6B2 CD19 APCef780 eBioscience 47–0193–82 Clone: eBio1D3 CD45.1 PerCP-Cy5.5 BD Biosciences 560580 Clone: A20 CD45.2 BV421 BD Biosciences 562895 Clone: 104 CXCR4 APC eBioscience 17–9991–82 Clone: 2B11 Live/Dead Yellow Life Technologies {"type":"entrez-nucleotide","attrs":{"text":"L34968","term_id":"522211","term_text":"L34968"}} L34968 RPMI-1640 Gibco 11875–093 heat-inactivated fetal bovine serum Gibco 16000–044 HEPES Gibco 15630–080 Non-essential amino acids Gibco 11140–050 Sodium pyruvate Gibco 11360–070 Gentamicin Gibco 15710–064 Beta-mercaptoethanol Gibco 21985–023 ODN 1668 InvivoGen tlrl-1668 70 micron filter caps Falcon 352235 Insulin syringes BD Biosciences 329461 Ctl-GFP retrovirus N/A N/A Generated in house using GFP-expressing retroviral plasmid MigR1 provided by Dr. T.P.

Techniques: Blocking Assay, Generated, Retroviral, Plasmid Preparation, Cloning, Software