Journal: Cancer Discovery

Article Title: Aged and BRCA -Mutated Stromal Cells Drive Epithelial Cell Transformation

doi: 10.1158/2159-8290.CD-24-0805

Figure Lengend Snippet: hrMSCs are enriched in the stroma underlying and adjacent to fallopian tube STIC lesions. A, Representative H&E-stained tissue sections of normal fallopian tubes and fallopian tubes harboring STIC lesions. Histology is paired with multispectral IF images of each respective group shown. Black and white images denote cells with the phenotypes of interest. Red x’s indicate hrMSCs (WT1 + /CD73 + /CD90 + /CD105 + /CD45 − ), whereas yellow x’s indicate nMSCs (WT1−/CD73 + /CD90 + /CD105 + /CD45 − ). B, Schematic showing the criteria for the Vectra spatial quantification. C, hrMSC abundance normalized to the area of ROI. D, hrMSC to nMSC ratios. E, hrMSC abundance (normalized to the area of ROI) in the expanded normal FT patient cohorts consisting of WT ( n = 10 young, n = 9 aged), BRCA1 mutant ( n = 9), or BRCA2 mutant ( n = 6) fallopian tubes lacking STIC/HGSOC. F, Ratio of hrMSC/nMSC in normal FTs. P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparisons analysis. Data points for C – F are reflective of individual ROIs. Statistics were determined on field ROIs for C and D . Statistics were determined with patient averages for E and F . G, Heatmap with unsupervised clustering of STIC stroma vs. normal stroma. H, Enrichment score of DEGs between STIC stroma vs. normal stroma (left: all, right: top 30 genes) applied to stromal location [from normal to STIC distal (dist) to STIC adjacent that contains both STIC contiguous and adjacent regions (STIC adj) to directly underlying STIC to invasive stroma]. I, Volcano plot of significantly differentially expressed genes in STIC stroma vs. normal stroma. J, Enrichment score of WT1 targets in stroma applied to stromal locations as in I . K, Correlation of stromal WT1 enrichment score with epithelial WT1 enrichment score.

Article Snippet: Anti-human antibodies used included: WT1 (Santa Cruz Biotechnology, Cat# sc-393498, RRID:AB_2905496), CD73 (Abcam, Cat# ab133582, RRID:AB_3674653), CD105 (Abcam, Cat# ab114052, RRID:AB_10900113), CD45 (Atlas Antibodies, Cat# AMAb90519, RRID:AB_2665572), CD90 (Novus, Cat# NBP1-43379G, RRID:AB_3209185), and panCK (Santa Cruz Biotechnology, Cat# sc-81714, RRID:AB_2191222).

Techniques: Staining, Mutagenesis

Journal: Journal of nanobiotechnology

Article Title: Cerium oxide nanoparticles-carrying human umbilical cord mesenchymal stem cells counteract oxidative damage and facilitate tendon regeneration.

doi: 10.1186/s12951-023-02125-5

Figure Lengend Snippet: Fig. 1 Characterization of hUCMSCs. A Primary hUCMSCs were spindle-shaped under light microscopy. B Flow cytometry analysis showed that 98.5% of cells expressed CD29, 97.4% expressed CD44, 98.6% of cells expressed CD90, and 98.1% of cells expressed CD105. Meanwhile, only 0.48% and 0.28% expressed CD45 and CD34. C Lipid droplets were stained by oil red O after adipogenic differentiation induction of hUCMSCs. Mineralized nodules were stained by Alizarin Red after osteogenic differentiation induction of hUCMSCs. Acid mucopolysaccharide was stained by Alcian Blue after chondrogenic differentiation induction of hUCMSCs

Article Snippet: Briefly, 107 hUCMSCs were subjected to analysis for surface markers CD34 (E-AB-F1143D, Elabscience), CD44 (E-AB-F1100D, Elabscience), CD45 (E-AB-F1137D, Elabscience), CD29 (E-AB-F1049D, Elabscience), CD90 (E-AB-F1167D, Elabscience), and CD105 (E-AB-F1310D, Elabscience) by flow cytometry following the manufacturer’s protocol.

Techniques: Light Microscopy, Flow Cytometry, Staining

Journal: Nature Communications

Article Title: A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis

doi: 10.1038/ncomms14017

Figure Lengend Snippet: ( a ) Time course of IL-9 production at various days post infection (dpi) in mice (six per group) infected intranasally with live A. fumigatus conidia. ( b ) Detection of CD90.2 + CD25 + , CD90.2 + ST2 + and CD90.2 + IL-9 + lung type 2 ILCs by flow cytometry (numbers refer to percentages of positive cells) and immunofluorescence staining. ( c ) Absolute number of lung ILC2; ( d ) ILC2–specific transcript on lineage negative lung cells; ( e , f ) ILC2 effector and activating cytokines; ( g ) Il9 and Th9-cell specific transcripts on lung CD4 + T cells and ( h ) immunofluorescence staining of lung CD4 + IL-9 + T cells. Photographs were taken with a high-resolution microscope (Olympus DP71) equipped with a × 40 objective; scale bar, 100 μm. Mean values±s.d. cytokines were determined on lung homogenates by ELISA, Il9 and transcripts assessed by PCR with reverse transcription. 0, uninfected mice. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, knockout versus C57BL/6 mice (data represent pooled results or representative images from three experiments, Two-way ANOVA, Bonferroni post test). Gata3 , GATA binding protein 3; Irf4 , interferon regulatory factor 4; Pu.1 , purine-rich box 1; Rora , RAR-related orphan receptor alpha.

Article Snippet: For immunofluorescence, lungs were incubated at 4 °C with phycoerythrin-conjugated (PE) anti-CD25 (Miltenyi Biotec clone 7D4, 1:60), anti-T1-ST2 (BioLegend clone DIH9, 1:400), anti-IL-9 (Milenyi Biotec clone RM9A4, 1:60) and fluorescein isothiocyanate-conjugated (FITC) anti-mouse CD90.2 (Miltenyi Biotecclone 30-H12, 1:60) and anti-CD4 (BioLegend clone GK1.5, 1:1,000).

Techniques: Infection, Flow Cytometry, Immunofluorescence, Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Knock-Out, Binding Assay

Journal: Nature Communications

Article Title: A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis

doi: 10.1038/ncomms14017

Figure Lengend Snippet: C57BL/6 and Il9R −/− mice (six per group) were intranasally infected with live Aspergillus fumigatus conidia or subjected to ABPA and assessed for ( a ) lung fungal growth (log 10 cfu, mean±s.d.); ( b ) lung histology (periodic acid−Schiff staining); ( c ) expression of CD90.2 + CD25 + , CD90.2 + ST2 + lung ILC2 by immunofluorescence; ( d , e ) Th-cell specific transcripts and cytokine production. ( f ) Lung histology (periodic acid–Schiff and, in the inset, Masson's trichrome staining) and ( g ) TGF-β production in C57BL/6 or Cftr −/− mice infected as above and treated with IL-9 neutralizing antibody for a week. Days post infection (dpi). ( h ) Th-cell specific transcripts and IL-9 production of lung CD4 + T cells from naive mice co-cultured with lung lineage negative (Lin − ) cells in the presence of A. fumigatus conidia, IL-2 or IL-33. Photographs were taken with a high-resolution microscope (Olympus DP71) equipped with a × 20 objective; scale bars, 200 μm and a × 40 objective (insets of f , scale bars, 100 μm). Results are mean values±s.d., ELISA was done on lung homogenates and culture supernatants for cytokines and PCR with reverse transcription on CD4 + lung cells. * P <0.05, ** P <0.01, *** P <0.001, Il9R −/− , Cftr −/− versus C57BL/6 mice; IL-9-treated versus control isotype-treated mice; stimulated versus unstimulated (none) cells and Il9R −/− versus C57BL/6 or Cftr −/− CD4 + T cells. Naive, uninfected mice. Data represent pooled results or representative images from three experiments, Two-tailed Student's t -test ( a ) or Two-way ANOVA ( d , e ) Bonferroni post test. Gata3 , GATA binding protein 3; Irf4 , interferon regulatory factor 4; Pu.1 , purine-rich box 1.

Article Snippet: For immunofluorescence, lungs were incubated at 4 °C with phycoerythrin-conjugated (PE) anti-CD25 (Miltenyi Biotec clone 7D4, 1:60), anti-T1-ST2 (BioLegend clone DIH9, 1:400), anti-IL-9 (Milenyi Biotec clone RM9A4, 1:60) and fluorescein isothiocyanate-conjugated (FITC) anti-mouse CD90.2 (Miltenyi Biotecclone 30-H12, 1:60) and anti-CD4 (BioLegend clone GK1.5, 1:1,000).

Techniques: Infection, Staining, Expressing, Immunofluorescence, Cell Culture, Microscopy, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Two Tailed Test, Binding Assay

Journal: Nature Communications

Article Title: A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis

doi: 10.1038/ncomms14017

Figure Lengend Snippet: C57BL/6 or Cftr −/− mice (six per group) infected intranasally with live A. fumigatus conidia were evaluated at different days after infection (dpi) for ( a ) deposition of DNA on lung epithelial cells by TUNEL, resulting in bright DNA staining; ( b ) detection of c-Kit + FcɛR + and c-Kit + IL-9 + lung mast cells (MC) by flow cytometry (numbers refer to percentages of positive cells); ( c ) toluidine blue, relative MC number mm −2 and, in the inset, immunohistochemical staining for chymase- and tryptase-positive MC in lung section. Photographs were taken with a high-resolution microscope (Olympus DP71) equipped with a × 40 objective and (in the inset) a × 100 objective and with EVOS FL Color Imaging System with a × 60 objective (immunohistochemical staining). ( d ) Toluidine blue stain and transcription factors expression (PCR with reverse transcription) of c-Kit + cells magnetically isolated from lung of uninfected C57BL/6 mice and pulsed with live A. fumigatus conidia. ( e ) Cytokine production (mean values±s.d., ELISA on culture supernatants) by purified lung c-Kit + cells, pulsed with A. fumigatus and stimulated with IgE, IL-9 and IL-33; ( f ) detection of c-Kit + IL-2 + , CD90.2 + IL-2 + and CD4 + IL-2 + lung cells by flow cytometry (numbers refer to percentages of positive cells). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, conidia-pulsed versus unpulsed c-Kit + cells, stimulated versus unstimulated c-Kit + cells and Cftr −/− versus C57BL/6 c-Kit + cells (data represent pooled results or representative images from three experiments, Two-way ANOVA, Bonferroni post test). Mcpt1 , mast cell protease 1; Mcpt6 , tryptase beta 2; Rorc , retinoic acid receptor–related orphan receptor C; Rora , RAR-related orphan receptor alpha; Tbet , T box expressed in T cells; Tph1 , tryptophan hydroxylase 1.

Article Snippet: For immunofluorescence, lungs were incubated at 4 °C with phycoerythrin-conjugated (PE) anti-CD25 (Miltenyi Biotec clone 7D4, 1:60), anti-T1-ST2 (BioLegend clone DIH9, 1:400), anti-IL-9 (Milenyi Biotec clone RM9A4, 1:60) and fluorescein isothiocyanate-conjugated (FITC) anti-mouse CD90.2 (Miltenyi Biotecclone 30-H12, 1:60) and anti-CD4 (BioLegend clone GK1.5, 1:1,000).

Techniques: Infection, TUNEL Assay, Staining, Flow Cytometry, Immunohistochemical staining, Microscopy, Imaging, Expressing, Reverse Transcription, Isolation, Enzyme-linked Immunosorbent Assay, Purification

Journal: Nature Communications

Article Title: A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis

doi: 10.1038/ncomms14017

Figure Lengend Snippet: MC-deficient C57BL6- Kit W/W-v mice (six per group) were infected intranasally with live A. fumigatus conidia, engrafted intravenously with wild-type bone marrow-cultured mast cells (BMMC) or treated intraperitoneally with IL-2 for a week and assessed for ( a ) c-Kit + FcɛR + lung mast cells (MC) and CD90.2 + CD25 + lung ILC2 by flow cytometry (numbers refer to percentages of positive cells) with relative cell number and ( b ) IgE and cytokine production. ( c ) Lung histology (periodic acid–Schiff and Masson's trichrome staining, in the insets); ( d ) cytokine production and ( e ) Th9-cell specific transcripts expression in Cftr −/− mice infected as above and treated with imatinib intraperitoneally for a week. Photographs were taken with a high-resolution microscope (Olympus DP71) equipped with a × 20 objective, scale bars, 200 μm and a × 40 objective (insets of c , scale bars, 100 μm). Results are mean values±s.d., ELISA on lung homogenates for cytokines and PCR with reverse transcription on lung CD4 + T cells. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, MC-deficient C57BL6- Kit W/W-v versus C57BL/6 mice or imatinib-treated versus untreated (none) mice (data represent pooled results or representative images from three experiments, Two-way ANOVA, Bonferroni post test). Irf4 =interferon regulatory factor 4; Pu.1 =purine-rich box 1.

Article Snippet: For immunofluorescence, lungs were incubated at 4 °C with phycoerythrin-conjugated (PE) anti-CD25 (Miltenyi Biotec clone 7D4, 1:60), anti-T1-ST2 (BioLegend clone DIH9, 1:400), anti-IL-9 (Milenyi Biotec clone RM9A4, 1:60) and fluorescein isothiocyanate-conjugated (FITC) anti-mouse CD90.2 (Miltenyi Biotecclone 30-H12, 1:60) and anti-CD4 (BioLegend clone GK1.5, 1:1,000).

Techniques: Infection, Cell Culture, Flow Cytometry, Staining, Expressing, Microscopy, Enzyme-linked Immunosorbent Assay, Reverse Transcription

Journal: Nature Communications

Article Title: A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis

doi: 10.1038/ncomms14017

Figure Lengend Snippet: C57BL/6, Cftr −/− and chimeric C57BL/6 and Cftr −/− mice (10 per group) received 10 × 10 6 viable bone marrow cells 4 weeks before the intranasal infection with A. fumigatus. Chimeric mice were evaluated 7 days after the infection for ( a ) lung histology (periodic acid–Schiff and, in the insets, Masson's trichrome and TUNEL staining); ( b ) cytokines and IgE levels (mean values±s.d., ELISA on lung homogenates); ( c ) detection of c-Kit + FcɛR + mast cells, CD90.2 + CD25 + and CD90.2 + ST2 + type 2 ILCs by flow cytometry (numbers refer to percentages of positive cells in the lung). Photographs were taken with a high-resolution microscope (Olympus DP71) equipped with a × 20 objective; scale bars, 200 μm and a × 40 objective (insets of panel a , scale bars, 100 μm). * P <0.05, ** P <0.01, *** P <0.001, Cftr −/− versus C57BL/6, chimeric C57BL/6 versus C57BL/6, chimeric Cftr −/− versus Cftr −/− mice, Two-way ANOVA, Bonferroni post test.

Article Snippet: For immunofluorescence, lungs were incubated at 4 °C with phycoerythrin-conjugated (PE) anti-CD25 (Miltenyi Biotec clone 7D4, 1:60), anti-T1-ST2 (BioLegend clone DIH9, 1:400), anti-IL-9 (Milenyi Biotec clone RM9A4, 1:60) and fluorescein isothiocyanate-conjugated (FITC) anti-mouse CD90.2 (Miltenyi Biotecclone 30-H12, 1:60) and anti-CD4 (BioLegend clone GK1.5, 1:1,000).

Techniques: Infection, TUNEL Assay, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest

doi: 10.1073/pnas.1707316114

Figure Lengend Snippet: Culture of hCO in MM does not alter cellular composition. (A) DNA intensity in hCO indicates fewer cells in MM; n = 70–74 from 14 experiments. (B) Single hPSC-CMs dissociated from hCO. (Scale bars: 10 µm.) (C) Flow cytometry profiling of hPSC-CMs (α-actinin) dissociated from hCOs reveals a similar purity in both CTRL medium and MM (not significant). (D) Flow cytometry profiling of stromal cells (CD90+) dissociated from hCO reveals a very modest decrease in CD90+ cells in hCOs cultured in MM vs. CTRL medium (P < 0.05); n = 5. (E) CTRL medium hCOs have endothelial tubular structures (CD31) and stromal cells (CD90) present through the hPSC-CMs (MLC2v). There are also epicardial cells (WT-1) present on the outer surface of the hCO. Low magnification images of the entire hCO are shown on the left for each stain, and high magnification confocal images are shown on the right. (F) MM hCOs have endothelial tubular structures (CD31) and stromal cells (CD90) present through the hPSC-CMs (MLC2v). There are also epicardial cells (WT-1) present on the outer surface of the hCO. Low magnification of the entire hCO is shown on the left for each stain, and high-magnification confocal images are shown on the right. Data are presented as mean ± SEM. (Scale bars: low magnification, 200 µm; high magnification, 20 µm.) ***P < 0.001 using t test (A); statistics analyzed using t test (C and D).

Article Snippet: They were washed in Blocking Buffer two times for 2 h and imaged in situ or mounted on microscope slides using Fluoromount-G (Southern Biotech). table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Species Company Catalog no. Dilution α-Actinin (clone EA-53) Mouse IgG 1 Sigma A7811 1:1,000 Ki-67 (D3B5) Rabbit IgG Cell Signaling Technology 9129 1:400 Antiphospho-Histone H3 (Ser10; pH3) Rabbit polyclonal Millipore 06–570 1:200 Active β-catenin (PY489) Mouse IgM Developmental Studies Hybridoma Bank PY489-B-catenin 5 μg/mL Titin Mouse IgM Developmental Studies Hybridoma Bank 9 D10 5 μg/mL MLC2v Rabbit IgG Protein Tech Group 10906–1-AP 1:200 CD31 Mouse IgG 1 Dako M082329-2 1:200 CD90 Mouse IgG 2A RnD Systems MAB2067 1:200 (After reconstitution using manufacturer’s guidelines) WT-1 Rabbit IgG Abcam AB89901 1:200 Caveolin 3 Mouse IgG 1 BD Transduction Laboratories 610421 1:200 pATM Mouse IgG 1 Santa Cruz Biotechnology sc-47739 1:100 8-OxoG Mouse IgM Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab206461","term_id":"91064839","term_text":"AB206461"}} Ab206461 1:100 Pancadherin Rabbit antiserum Sigma C3678 1:200 Connexin 43 Rabbit IgG Abcam ab11370 1:200 GFP Chicken polyclonal Abcam Ab13970 1:200 Alexa Fluor 488 goat anti-rabbit IgG (H+L) NA Life Technologies A-11034 1:400 Alexa Fluor 488 goat anti-mouse IgG (H+L) NA Life Technologies A-11029 1:400 Alexa Fluor 488 goat anti-mouse IgM (µ chain) NA Life Technologies A-21042 1:400 Alexa Fluor 555 goat anti-rabbit IgG (H+L) NA Life Technologies A-21428 1:400 Alexa Fluor 555 goat anti-mouse IgG (H+L) NA Life Technologies A-21422 1:400 Alexa Fluor 633 goat anti-rabbit IgG (H+L) NA Life Technologies A-21070 1:400 Alexa Fluor 488 goat anti-chicken IgG (H+L) NA Life Technologies A-11039 1:400 Open in a separate window NA, not applicable.

Techniques: Flow Cytometry, Cell Culture, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest

doi: 10.1073/pnas.1707316114

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: They were washed in Blocking Buffer two times for 2 h and imaged in situ or mounted on microscope slides using Fluoromount-G (Southern Biotech). table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Species Company Catalog no. Dilution α-Actinin (clone EA-53) Mouse IgG 1 Sigma A7811 1:1,000 Ki-67 (D3B5) Rabbit IgG Cell Signaling Technology 9129 1:400 Antiphospho-Histone H3 (Ser10; pH3) Rabbit polyclonal Millipore 06–570 1:200 Active β-catenin (PY489) Mouse IgM Developmental Studies Hybridoma Bank PY489-B-catenin 5 μg/mL Titin Mouse IgM Developmental Studies Hybridoma Bank 9 D10 5 μg/mL MLC2v Rabbit IgG Protein Tech Group 10906–1-AP 1:200 CD31 Mouse IgG 1 Dako M082329-2 1:200 CD90 Mouse IgG 2A RnD Systems MAB2067 1:200 (After reconstitution using manufacturer’s guidelines) WT-1 Rabbit IgG Abcam AB89901 1:200 Caveolin 3 Mouse IgG 1 BD Transduction Laboratories 610421 1:200 pATM Mouse IgG 1 Santa Cruz Biotechnology sc-47739 1:100 8-OxoG Mouse IgM Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab206461","term_id":"91064839","term_text":"AB206461"}} Ab206461 1:100 Pancadherin Rabbit antiserum Sigma C3678 1:200 Connexin 43 Rabbit IgG Abcam ab11370 1:200 GFP Chicken polyclonal Abcam Ab13970 1:200 Alexa Fluor 488 goat anti-rabbit IgG (H+L) NA Life Technologies A-11034 1:400 Alexa Fluor 488 goat anti-mouse IgG (H+L) NA Life Technologies A-11029 1:400 Alexa Fluor 488 goat anti-mouse IgM (µ chain) NA Life Technologies A-21042 1:400 Alexa Fluor 555 goat anti-rabbit IgG (H+L) NA Life Technologies A-21428 1:400 Alexa Fluor 555 goat anti-mouse IgG (H+L) NA Life Technologies A-21422 1:400 Alexa Fluor 633 goat anti-rabbit IgG (H+L) NA Life Technologies A-21070 1:400 Alexa Fluor 488 goat anti-chicken IgG (H+L) NA Life Technologies A-11039 1:400 Open in a separate window NA, not applicable.

Techniques: Transduction