Journal: Stem Cell Reports

Article Title: Cell-Cycle Control of Bivalent Epigenetic Domains Regulates the Exit from Pluripotency

doi: 10.1016/j.stemcr.2015.07.005

Figure Lengend Snippet: The MENIN/MLL2 H3K4 Methyl Transferase Is Cell-Cycle Regulated and Controls the Activation of Bivalent Genes in G1 (A) qChIP of Fucci hESC cell-cycle fractions examining levels of MLL2 and WDR5 on the GATA6 and SOX17 promoters. Data are the average of three independent replicates. (B) qChIP assays for MLL2 in untreated or MI-2 (25 μM for 24 hr)-treated WA09 ESCs at the indicated promoters. Data are the average of three independent replicates. (C) qChIP assays for MLL2 in ESCs of pluripotency genes. Data are the average of three independent replicates. (D) qRT-PCR transcript analysis of Fucci-sorted hESCs after treatment with the MLL/MENIN inhibitor, MI-2 (25 μM for 24 hr). Data are the average of three independent replicates. (E) Immunoblot analysis of WA09 hESC and DE (2 days) lysates (20 μg per lane) with or without MI-2 (25 μM). (F) Immunostaining for SOX17 (top) and FOXA2 (bottom) following the infection of GFP-control or MENIN shRNA lentivirus in WA09 hESCs or cells differentiated to DE for 3 days. Cells are co-stained with DAPI to visualize nuclei. Micron bar represents 50 μm. (G) Quantitation of immunostaining represented in (E) for three independent fields, n > 1,000. Data are representative of three independent experiments. (H) CXCR4 flow cytometry analysis of WA09 hESCs and DE (3 days differentiation) transduced with MENIN or GFP shRNA lentivirus. The percentage of CXCR4 + cells in each condition is indicated. (I) qRT-PCR transcript analysis of WA09 hESCs and DE (3 days differentiation) following lentiviral infections with MENIN or GFP (control [C]) shRNA. Data are the average of three independent replicates. ∗ p < 0.05. Error bars in this figure represent the SEM. See also Figure S3 .

Article Snippet: Immunoblotting, immunoprecipitations, and immunostaining were performed as previously described , with antibodies raised against MLL1 (A300-086A), MLL2 (A300-113A, Bethyl Laboratories); MENIN (ab2605), JMJD3 (ab85392, Abcam), WDR82 (kind gift from David Skalnik, IUPUI School of Science); pSMAD2 (3104S), pSerine-CDKs Substrate, P-S-100 (9477), pThreonine-Proline (9391) (Cell Signaling Technology); OCT4 (sc-8628), CDK2 (sc-163) (Santa Cruz Biotechnology); BRACHYURY (AF2085), SOX17 (AF1924) (R&D systems); FOXA2 (07-633, Millipore).

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Immunostaining, Infection, Control, shRNA, Staining, Quantitation Assay, Flow Cytometry, Transduction

Journal: Stem Cell Reports

Article Title: Cell-Cycle Control of Bivalent Epigenetic Domains Regulates the Exit from Pluripotency

doi: 10.1016/j.stemcr.2015.07.005

Figure Lengend Snippet: CDK2 Phosphorylates MLL2 to Regulate Its Binding (A) Cell-cycle Fucci profiles of untreated and CDK2 inhibitor (CDK2I, CVT-313, 20 μM) treated cells for 4 hr. (B) qRT-PCR analysis of transcript levels in ESCs treated with CVT-313 for 4 hr. Data are the average of three independent replicates. (C) qChIP for MLL2 and MENIN in ESCs treated with CVT-313 for 4 hr. Data are the average of three independent replicates. (D) MLL2 immunoprecipitations (IPs) from ESC lysates (200 μg protein; ±CVT-313) were probed with MLL2, phospho-threonine (pTP), or phospho-serine (pSP) antibodies. IgG was used as an IP control, and whole cell lysate was immunoblotted (input) alongside IPs. (E) ESCs were transfected with a construct expressing constitutively active (CA) or inactive (KE mutant) CDK2-CCND1 gene fusion driven by CAGi promoter, and then qChIP assays for MLL2 were performed 36 hr later for GATA6 and SOX17 . Data are the average of three independent replicates. (F) Model depicting the cell-cycle control of bivalent domains. All data are representative of biological replicates. Error bars in this figure represent the SEM. ∗ p < 0.05.

Article Snippet: Immunoblotting, immunoprecipitations, and immunostaining were performed as previously described , with antibodies raised against MLL1 (A300-086A), MLL2 (A300-113A, Bethyl Laboratories); MENIN (ab2605), JMJD3 (ab85392, Abcam), WDR82 (kind gift from David Skalnik, IUPUI School of Science); pSMAD2 (3104S), pSerine-CDKs Substrate, P-S-100 (9477), pThreonine-Proline (9391) (Cell Signaling Technology); OCT4 (sc-8628), CDK2 (sc-163) (Santa Cruz Biotechnology); BRACHYURY (AF2085), SOX17 (AF1924) (R&D systems); FOXA2 (07-633, Millipore).

Techniques: Binding Assay, Quantitative RT-PCR, Control, Transfection, Construct, Expressing, Mutagenesis

Journal: Advanced Science

Article Title: A Micropatterned Human‐Specific Neuroepithelial Tissue for Modeling Gene and Drug‐Induced Neurodevelopmental Defects

doi: 10.1002/advs.202001100

Figure Lengend Snippet: Distinct tissue architectures in ME‐primed and unprimed μNETs were mediated by differences in morphogenetic cellular processes. A) Expression patterns of N‐cadherin (NCAD) and E‐cadherin (ECAD) cell adheren junctions in (i) 56‐h old ME‐primed μNET, (ii) 120‐h old ME‐primed μNET and (iii) 120‐h old unprimed μNET. Images are cross‐sectional views of 3D confocal sections (top panel) and magnified view of single optical sections transversing the μNETs along the dotted white lines (bottom panel). Segregation of ECAD and NCAD are indicated by white arrows. (B) Expression patterns of apical constriction markers ZO1, ppMLC and F‐actin in ME‐primed and unprimed μNETs. Images are magnified cross‐sectional side views of 3D confocal optical sections of μNETs. (C) Merged image of ZO1and ppMLC in ME‐primed μNETs at 34 h, prior to the tissue folding, marking ppMLC cables (arrowheads) along the prospective folding axis (white dotted line). (D) Quantification of angular distribution of ppMLC cables, that linearly extended more than 10 µm across multiple cells, relative to prospective folding axis (white dotted line in (C)). Data are average of ten to 15 cables per image from nine independent samples. E) Expression patterns of planar cell polarity marker, DVL2, and ppMLC in ME‐primed μNETs. Images are (i,ii) cross‐sectioned side view and (iii) magnified view of single optical section transversing at the hinge region in μNETs (along the dotted white lines) displaying apical colocalization of DVL2 and ppMLC. F) F‐actin staining showing prospective NE cell morphologies at (i) magnified cross‐section side view of fold showing different optical sections transversing the ME‐primed μNETs at different z ‐axis, (ii) single optical section transversing the μNETs along section (II) showing elongated pseudostratified columnar like cells on apical (ap) side of the fold, and (iii) magnified view of hinge displaying wedge‐shaped cells at the fold, with shorter cell length at apical (ap) side and longer cell length on the basal (ba) side of NE tissue. G) Quantification of nuclei elongation as measured by the aspect ratio of individual nuclei for optical section transversing the μNETs at I, II, and III. H) Quantification of cell shape as measured by the ratio of basal to apical cell length for side view sections at I, II(hinge), and III (equivalent area for each section as yellow dotted square in D(i). Data are average of ± s.e.m of 20 nuclei per slice from four independent samples (One‐way ANOVA followed by Tukey´s post‐test, ** p < 0.0001). I) Expression patterns of mitotically active phospho‐histone3 (PH3) + cells in 56‐h old ME‐primed μNETs. (i) Cross‐sectional view and (ii) top view of whole μNET structure with μNET; (iii) single optical section showing magnified view of cells at the hinge region, as indicated by white line in (i) tranversing white box region in (ii). J) Quantification of % PH3 + cells at the apical and basal side of in SOX2 + cell layer in μNETs at the hinge region. Data are average of ± s.e.m of 12 slices from three independent samples (Student's t ‐test, ** p < 0.0001). Scale bars = 50 µm, C, Fii, Eiii = 20 µm.

Article Snippet: After overnight incubation, the samples were incubated with primary antibodies diluted in antibody dilution buffer (2% BSA, 0.2% Triton‐X 100 in sterile PBS) at 4 °C for 48 h. The primary antibodies used in this study were SOX2 (mouse, MAB2018, R&D Systems, 1:50), SOX2 (goat, AF2018, R&D Systems, 1:30), Brachyury (goat, AF2085, R&D Systems, 1:50), Brachyury (rabbit, MAB20851, R&D Systems, 1:50), ECAD (rabbit, sc‐7870, Santa Cruz, 1:50), NCAD (mouse, ab19348, Abcam, 1:100), Nestin (rabbit, ab92391, Abcam, 1:200), pSMAD1 (rabbit, 9516S, cell signaling technology, 1:100), SMAD2 (mouse, 610 842, BD biosciences, 1:150), pERK (rabbit, 4695P, Cell signaling technology, 1:500), ppMLC (rabbit, 3674S, cell signaling technology, 1:100), ZO1 (mouse, 33‐9100, Thermofisher scientific, 1:100), SOX17 (goat, AF1924, R&D Systems, 1:50), FOXA2 (mouse, sc‐374375, Santa Cruz, 1:50), GBX2 (mouse, SAB1403854, Sigma, 1:100), NCAM (rabbit, AB5032, Merck, 1:100), β ‐catenin (mouse, sc‐7963, Santa Cruz, 1:50), NANOG (goat, AF1997, R&D Systems, 1:200), OCT4 (mouse, 111 351, Santa Cruz, 1:200), SSEA4 (mouse, MAB4304, Millipore, 1:200), TRA‐1‐81 (mouse, MAB4381, Millipore, 1:200), TRA‐1‐60 (mouse, MAB4360, Millipore, 1:200).

Techniques: Expressing, Marker, Staining

Journal: Advanced Science

Article Title: A Micropatterned Human‐Specific Neuroepithelial Tissue for Modeling Gene and Drug‐Induced Neurodevelopmental Defects

doi: 10.1002/advs.202001100

Figure Lengend Snippet: FMR1 silencing in Fragile X Syndrome resulted in μNET structural dysmorphia that was mediated by cadherin and β ‐catenin dysfunctions. A–C) Localization patterns of germ layer markers: endoderm (FOXA2), mesoendoderm (T), and neuroepithelium (SOX2) in ME‐primed μNETs generated from normal H1 human embryonic stem cell, an isogenic FMR1 knock‐out H1 cell line (FMR1‐KO H1) and a FXS human embryonic stem cell line (FXS‐hESC). A) Maximum intensity projections of 3D confocal sections of 56‐h old (top panel) and 72‐h old (bottom panel) μNETs generated from normal and diseased cell lines. B) Magnified cross‐sectional views of 3D confocal sections. C) Localization map of SOX2 + , T + and FOXA2 + cells along x ‐ z cross‐sectional plane indicated by white dotted lines in (A), illustrate a loss of laminar organization of SOX2 + , T + , and FOXA2 + cells in the FMR1‐KO‐H1 and FXS‐hESC μNETs as compared to normal H1 μNETs. Data are overlay map of >3 optical sections from two independent experiments. D,E) Magnified views of single optical sections transversing 56‐h old ME‐primed μNETs generated using control and diseased cell lines along the dotted white lines in (B) showing the expression patterns of FMRP targets, namely N‐cadherin (NCAD), E‐cadherin (ECAD), and β ‐catenin relative to SOX2. D) In FMR1‐KO‐H1 and FXS‐hESC μNETs, regions with SOX2 + NE cell migration was accompanied by premature ECAD downregulation and NCAD upregulation indicated by white arrows. E) β ‐catenin was preferentially lost from the SOX2 + NE layers in the FMR1‐KO‐H1 and FXS‐hESC μNETs; whereas β ‐catenin was uniformly expressed in the normal H1 μNETs. F,G) Quantification of β ‐catenin expression levels in ME‐primed μNETs generated using normal and diseased cell lines in E) SOX2 + cells, F) SOX2 − cells. Data are average of ± s.e.m of eight optical sections from three independent experiments. Asterisks indicate statistical significance (one‐way ANOVA followed by Tukey´s post‐test, * p < 0.005, ** p < 0.0001). Scale bars in (A) = 100 µm, (B–D) = 50 µm.

Article Snippet: After overnight incubation, the samples were incubated with primary antibodies diluted in antibody dilution buffer (2% BSA, 0.2% Triton‐X 100 in sterile PBS) at 4 °C for 48 h. The primary antibodies used in this study were SOX2 (mouse, MAB2018, R&D Systems, 1:50), SOX2 (goat, AF2018, R&D Systems, 1:30), Brachyury (goat, AF2085, R&D Systems, 1:50), Brachyury (rabbit, MAB20851, R&D Systems, 1:50), ECAD (rabbit, sc‐7870, Santa Cruz, 1:50), NCAD (mouse, ab19348, Abcam, 1:100), Nestin (rabbit, ab92391, Abcam, 1:200), pSMAD1 (rabbit, 9516S, cell signaling technology, 1:100), SMAD2 (mouse, 610 842, BD biosciences, 1:150), pERK (rabbit, 4695P, Cell signaling technology, 1:500), ppMLC (rabbit, 3674S, cell signaling technology, 1:100), ZO1 (mouse, 33‐9100, Thermofisher scientific, 1:100), SOX17 (goat, AF1924, R&D Systems, 1:50), FOXA2 (mouse, sc‐374375, Santa Cruz, 1:50), GBX2 (mouse, SAB1403854, Sigma, 1:100), NCAM (rabbit, AB5032, Merck, 1:100), β ‐catenin (mouse, sc‐7963, Santa Cruz, 1:50), NANOG (goat, AF1997, R&D Systems, 1:200), OCT4 (mouse, 111 351, Santa Cruz, 1:200), SSEA4 (mouse, MAB4304, Millipore, 1:200), TRA‐1‐81 (mouse, MAB4381, Millipore, 1:200), TRA‐1‐60 (mouse, MAB4360, Millipore, 1:200).

Techniques: Generated, Knock-Out, Expressing, Migration