Journal: Cell reports

Article Title: Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis

doi: 10.1016/j.celrep.2021.108991

Figure Lengend Snippet:

Article Snippet: Alexa Fluor® 647 Mouse anti-MAP2B (clone 18) , BD Biosciences , Cat#: 560382; RRID: AB_1645423.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell Reports Medicine

Article Title: Genomic and transcriptomic profiling of peripheral T cell lymphoma reveals distinct molecular and microenvironment subtypes

doi: 10.1016/j.xcrm.2024.101416

Figure Lengend Snippet: Synergy of targeted agents and exploration of potential monotherapy in T3.1 and T3.2 subtypes (A) Pathway enrichment analysis in T3.1 and T3.2 according to the KEGG and Reactome databases. (B) GSEA enriched differentially expressed genes of DNA replication in T3.1 and the VEGF signaling pathway in T3.2. (C) CI curve calculated by Compusyn software in KMT2C mut Jurkat cells treated with chidamide (Chid) and decitabine (Deci) for 48 h. (D) Pathway analysis of differentially downregulated genes in each group upon treatment with Chid and/or Deci. (E) Survival of zPDX upon Chid and/or Deci treatment alone or in combination (n = 11 for each group). (F) Ki-67 positivity of Jurkat cells transfected with PTPN13 WT and PTPN13 mut upon nivolumab (Nivo) and apatinib (Apa) treatment after 72 h. Peripheral blood mononuclear cells (PBMCs) and Jurkat cells were co-cultured at a ratio of 5:1. Nivo was at a concentration of 10 μg/mL, and Apa was at 10 μM. Data are presented as the mean ± SD (n = 3). (G) Multiflow cytometry analysis of Th2 cell markers (CD4 and GATA3) in PBMCs, co-cultured with PTPN13 mut Jurkat cells with or without Nivo (10 μg/mL) and Apa treatment for 72 h. Data are presented as the mean ± SD (n = 3). (H) Normalized mRNA expression of GATA3 across 4 molecular subtypes as revealed by RNA-seq. (I) Cell viability ratio of KMT2C mut Jurkat cells and KMT2C WT cells after treatment with 7,316 compounds at 0.4 μM for 72 h (left). Compounds that specially suppressed the growth of KMT2C mut Jurkat cells are listed on the right. (J) Cell viability of KMT2C mut Jurkat cells after treatment with different concentrations of KDM4D-IN-1 for 48 h. ∗p < 0.05 compared with KMT2C WT cells. Data are presented as the mean ± SD (n = 3). (K) Cell viability ratio of PTPN13 mut Jurkat cells and PTPN13 WT cells after treatment with 7,316 compounds at 0.4 μM for 72 h (left). Compounds that specially suppressed the growth of PTPN13 mut Jurkat cells are listed on the right. (L) Cell viability of PTPN13 mut Jurkat cells after treatment with different concentrations of anlotinib for 48 h. ∗p < 0.05 compared with PTPN13 WT cells. Data are presented as the mean ± SD (n = 3).

Article Snippet: Alexa Fluor® 647 Mouse anti-Ki-67 , BD , Cat# 561126; RRID: AB_10611874.

Techniques: Software, Transfection, Cell Culture, Concentration Assay, Cytometry, Expressing, RNA Sequencing Assay

Journal: Cell Reports Medicine

Article Title: Genomic and transcriptomic profiling of peripheral T cell lymphoma reveals distinct molecular and microenvironment subtypes

doi: 10.1016/j.xcrm.2024.101416

Figure Lengend Snippet:

Article Snippet: Alexa Fluor® 647 Mouse anti-Ki-67 , BD , Cat# 561126; RRID: AB_10611874.

Techniques: Recombinant, DNA Purification, RNA HS Assay, Cell Viability Assay, Sequencing, Software

Journal: JCI Insight

Article Title: Caspase-4/11 promotes hyperlipidemia and chronic kidney disease–accelerated vascular inflammation by enhancing trained immunity

doi: 10.1172/jci.insight.177229

Figure Lengend Snippet: ( A and B ) Flow cytometry analysis demonstrated that HFD+CKD increased the recruitment of CD45 + CD11b + monocytes and CD45 + CD11b + F4/80 + macrophages into the WT aorta, which were significantly suppressed in HFD+CKD casp11 –/– aortas. ( C and D ) Flow cytometry analysis demonstrated that casp11 –/– decreased blood CD45 + CD11b + monocytes in HFD-sham mice compared with WT HFD-sham mice; and HFD+CKD mice did not significantly change blood CD45 + CD11b + monocytes and CD45 + CD11b + F4/80 + macrophages in HFD+CKD mice ( n = 6–8). Flow cytometry analysis showed the infiltrated monocytes (CD11b + CD45 + ) and macrophages (CD11b + CD45 + F4/80 + ) in the blood of WT and casp11 –/– HFD+CKD and HFD-Sham mice. ( E ) Cytokine array showed that casp11 –/– decreased HFD+CKD-induced chemokines and cytokines, including CCL2 and CCL22, MMP-3, chinitianase 3-like 1 (CHIL3L1), IL-12p40, myeloperoxidase, TNFRSF11b, and PCSK9 in plasma. Each sample was pooled from 3 mice in each group ( n = 3). ImageJ was used to quantify the bands, and the significantly changed proteins were indicated. The Kruskal-Wallis test with Benjamini and Hochberg multiple-comparison method was used to control the overall FDR of 5% ( A – E ).

Article Snippet: Flow cytometry antibodies were bought from BD Biosciences except for anti–GSDMD-PE (Santa Cruz, H11), anti–VCAM-1 PECy7 (BioLegend, 105720), anti-CD11b BUV395 (BD Biosciences, 563553), anti-CD45 APC Cy7 (BioLegend, catalog 103116), anti-CD31 BUV737 (BD Biosciences, 612802), and anti-F4/80 Alexa Fluor 647 (BD Pharmingen, catalog 565853).

Techniques: Flow Cytometry, Comparison, Control

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Comparison of cell dissociation protocols. For the first condition, 1.5 mL of 0.25% Trypsin-EDTA was added after collagenase digestion and the mixture was digested for an additional 15 min at 37 °C. The optimal condition (boxed) yields the highest number of viable cells in two independent experiments. NA, not available. (B) Sorting plots of an adult (>8 week-old) mouse lung with genetically labeled ECs. Left two panels: comparison of antibody staining with native fluorescence from tdT (red contour), which colocalizes mostly with CD31 and ICAM2, but also a subset of CD45 cells, perhaps due to inadvertent labeling of hematopoietic cells by Cdh5-CreER . Right two panels: for all sorting experiments, CD45 negative selection was first performed and followed by ICAM2 positive selection (red box), which labels the same ECs as CD31 but has better separation from the corresponding negative ECs.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Labeling, Staining, Fluorescence, Selection

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Representative en face view of immunostaining images from at least 5 mice. Boxed region is magnified as a section view in the first three images of the bottom row. CAR4 staining covers, whereas PLVAP staining surrounds, alveolar islands (dash). Perinuclear CAR4 and PLVAP staining allows assignment of ERG nuclei to CAR4 (cyan arrowhead) versus PLVAP (magenta arrowhead) ECs, which is automatically identified, as shown in the lower rightmost image (grey nuclei are ambiguous). Scale: 10 um. (B) Wholemount immunostaining of lungs with sparsely-labeled ECs, representative of at least 5 mice, viewed as a stack (40 um), a slab (top 20 um), or a section (1 um). Accumulation of tdT to ERG nuclei allows cell numeration (1 through 5). Cell #1 is a Car4 EC and cells #2-5 are non-Car4 ECs. Line profile analysis shows aligned versus shifted peaks for Car4 versus non-Car4 ECs, respectively. For shifted peaks, Car4 ECs are closer to the airspace than non-Car4 ECs (e.g. cell #3). Asterisk, avascular tissue surrounded by a single net-like Car4 EC. Open arrow, CDH5 junction overlapping with a single Car4 EC. Cell perimeter is measured by connecting protrusions that are visible in a projection view. Tam, 0.25 ug tamoxifen. Scale: 10 um. (C) Quantification of cell perimeter and comparison using Student’s t-test.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Immunostaining, Staining, Labeling