Journal: The Journal of Clinical Investigation

Article Title: TGF- β controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription in mice

doi: 10.1172/JCI172095

Figure Lengend Snippet: ( A ) To obtain AT1 cells for culture, P5–P10 pup lungs were obtained after lineage labeling with tamoxifen at P0. Whole lung cell suspensions were obtained using a dispase, DNase, and collagenase digestion buffer after which the epithelial cell population was enriched by depleting the CD45 + and CD31 + population. Remaining cells were fluorescently sorted with FACS to obtain a CD326 + and YFP + suspension. Cells were plated onto fibronectin-coated plates with or without TGF-β1 ligand (7.5 ng/mL) or TGF-β inhibitor SB431542 (10μM) and evaluated at days 2, 4, and 6. ( B ) ICC of RAGE + (AGER + ) cells treated with TGF-β ligand or inhibitor in culture at days 2, 4, and 6 with a zoomed image of cells at day 6 appearing at the bottom. Scale bars: 100 μm. ( C ) Quantification of mean AGER + cell area depicted in B by 1-way ANOVA with Holms Šidák’s test for multiple comparisons ( n = 135–231). ( D ) Quantification of mean cell roundness at day 6 depicted in B by 1-way ANOVA with Holms Šidák’s test for multiple comparisons ( n = 148–167). ( E ) Quantification of qPCR RNA transcript expression levels (FC compared with GAPDH, normalized to controls) of the AT2 marker Sftpb , fibronectin-binding integrins ( F ) Itga5 and ( G ) Itgb1 , and ( H ) basement membrane constituents including the collagen IV subtypes Col4a1 , Col4a3 , and Col4a4 and laminin-332 constituents Lama3 and Lamb3 ( n = 3 per group, 1-way ANOVA with Tukey’s multiple comparisons). ( I ) Representative schematic of findings indicating that TGF-β regulates integrin expression to guide ECM binding and cellular spread, which affects cell identity and matrisome expression and impacts lung development. Schematics in A and I were created in BioRender. Results are representative of 3 experiments.

Article Snippet: Lineage-traced AT1 cells were obtained following FACS and were then placed into an organoid growth medium containing DMEM F12 (Thermo Fisher Scientific) and growth factors including bovine pituitary extract, cholera toxin, FBS, gentamicin, retinoic acid, insulin, transferrin (all from Lonza), and human epithelial growth factor (Peprotech), as previously described ( , ).

Techniques: Labeling, Suspension, Expressing, Marker, Binding Assay, Membrane

Journal: Nature structural & molecular biology

Article Title: MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation

doi: 10.1038/nsmb.2903

Figure Lengend Snippet: Top functional annotations enriched among macroH2A1-regulated genes

Article Snippet: Human prostate epithelial cells (Lonza) were cultured in CloneticsTM Prostate Epithelial Cell Growth Medium.

Techniques: Functional Assay

Journal: Nature structural & molecular biology

Article Title: MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation

doi: 10.1038/nsmb.2903

Figure Lengend Snippet: MacroH2A1 directly regulates the acetylation of H2B at K12 and K120 in primary cell types but not in cancer cells. (a) Immunoblots for macroH2A1 (mH2A1), histone H3 (H3) and the indicated histone PTMs in IMR90 cells expressing an shRNA against either Luciferase (Luc) as a control or macroH2A1. (b) Immunoblots for macroH2A1, H3 and the indicated histone marks in primary lung and skin fibroblasts, primary hepatocytes, primary prostate and mammary epithelial cells, A549 lung cancer cells, HeLa cervical carcinoma cells, HEK293T transformed embryonic kidney cells, HepG2 hepatocellular carcinoma cells and MCF-7 breast cancer cells expressing an shRNA against either Luciferase (L, as a control) or macroH2A1 (M). (c) ChIP-qPCR for H2BK12ac, H2BK120ac, macroH2A1 and histone H3 from IMR90 cells expressing an shRNA directed against either Luciferase (Luc, as a control) or macroH2A1 at H2BK12ac and H2BK120ac positive regions who are either positive (left) or negative (right) for macroH2A1 chromatin incorporation. The horizontal dotted line indicates upper limit of the 95% confidence interval of the signal from no-antibody negative control ChIPs. Error bars, +/− s.e.m. (n = 3 independent cell passages). *p < 0.05 from two-tailed Student’s t-tests. (d) Immunoprecipitation with antibodies directed against the indicated PTMs or no antibody (NA) as a control of mononucleosomes from either IMR90 primary lung fibroblasts or A549 lung cancer cells. Immunoblots were performed for both macroH2A1 and histone H3.

Article Snippet: Human prostate epithelial cells (Lonza) were cultured in CloneticsTM Prostate Epithelial Cell Growth Medium.

Techniques: Western Blot, Expressing, shRNA, Luciferase, Control, Transformation Assay, ChIP-qPCR, Negative Control, Two Tailed Test, Immunoprecipitation

Journal: Cell reports

Article Title: Differential Functions of Splicing Factors in Mammary Transformation and Breast Cancer Metastasis

doi: 10.1016/j.celrep.2019.10.110

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human Epithelial Growth Factor , Peprotech , #100-15.

Techniques: Recombinant, Cell Recovery, SYBR Green Assay, MTT Assay, Control, Sequencing, shRNA, Software