Journal: Scientific Reports

Article Title: Nucleoporin insufficiency disrupts a pluripotent regulatory circuit in a pro-arrhythmogenic stem cell line

doi: 10.1038/s41598-019-49147-4

Figure Lengend Snippet: Nucleoporin insufficiency blunts ESC colony mass. ( a , b ) Representative images of alkaline phosphatase staining in mouse ESC colonies in WT (left panel) and nup155 +/− (right panel) conditions showing positive stain in both cell lines. ( c ) Image analysis revealed significant reduction in size of nup155 +/− ESC colonies (52%, p < 0.05) as well as ( d ) lower percent area of colonies per field compared to WT (21% vs 30% respectively, p < 0.05). ( e ) No change was observed in overall colony number between conditions (p = 0.99).

Article Snippet: Image analysis of ESC colonies was done using NIS-Elements AR imaging software v4.20 (Nikon Corporation, Tokyo, Japan).

Techniques: Staining

Journal: Scientific Reports

Article Title: Nucleoporin insufficiency disrupts a pluripotent regulatory circuit in a pro-arrhythmogenic stem cell line

doi: 10.1038/s41598-019-49147-4

Figure Lengend Snippet: Expression of pluripotency factors is decreased in nup155 deficient ESCs. ( a , b ) Representative images of OCT4 (red), Lamin B (green), and DAPI (blue) fluorescence signal depicting OCT4 intranuclear localization with overall decrease in signal profile in nup155 +/− (right panel) compared to ESC colonies. Bottom panel represents individual cell intensity profiles. ( c , d ) Maximum (Max) intensity of OCT4 in nup155 +/− ESC colonies was significantly decreased compared to WT, along with the OCT4:DAPI signal ratio (*p < 0.001). ( e,f ) SOX2 (red), Lamin B (green), and DAPI (blue) triple staining of nup155 +/− and WT ESC colonies show no overall change in intranuclear signal profile, as well as ( g ) no change in SOX2 max signal intensities between conditions (p = 0.42). ( h ) SOX2:DAPI signal ratio is higher in NUP155 deficient cells compared to WT. ( i,j ) NANOG (red) fluorescent signal profile was diminished in nup155 +/− compared to WT. ( k , l ) NANOG (red) max intensity and NANOG:DAPI ratio were significantly decreased in nup155 +/− ESC colonies compared to WT (*p < 0.001).

Article Snippet: Image analysis of ESC colonies was done using NIS-Elements AR imaging software v4.20 (Nikon Corporation, Tokyo, Japan).

Techniques: Expressing, Fluorescence, Staining

Journal: Cell reports

Article Title: Long interspersed nuclear elements safeguard neural progenitors from precocious differentiation

doi: 10.1016/j.celrep.2024.113774

Figure Lengend Snippet: (A) A schema of the retroviral labeling procedure used to manipulate the levels of L1 in RGLs of forebrain organoids. (B) Representative confocal images of VZ-like regions of retrovirally labeled RGL-derived cells that are migrating into the developing cortical plate of forebrain organoids. Sections were stained with anti-GFP (green) and DAPI. Scale bar, 50 μm. (C) Fold change of retrovirally labeled RGLs 3 days post infection (dpi) observed in CP-like over VZ-like regions. Mann-Whitney U test, *p < 0.05. Boxplots with whiskers indicate minimum to maximum values, with box limits for 25 th to 75 th percentiles, and a centerline for the median. (D) Relative normalized position of GFP-positive migratory cells within the evolving cortical plate of forebrain organoids. Mann-Whitney U test, *p < 0.05. Boxplots with whiskers indicate minimum to maximum values, with box limits for 25 th to 75 th percentiles, and a centerline for the median. (E) Distribution of GFP-positive cells in the CP-like structures. Kolmogorov-Smirnov test, NTC (non-targeting control) vs. shL1HS-2: p = 0.0177, NTC vs. shL1HS-1: p < 0.0001. (C–E) N NTC = 56 cells (from n = 4 independent organoids; 2 organoids per iPSC, 2 organoids per ESC line), N shL1HS-2 = 78 cells (from n = 4 independent organoids; 2 organoids per iPSC, 2 organoids per ESC line), and Ns hL1HS-1 = 113 cells (from n = 4 independent organoids; 2 organoids per iPSC, 2 organoids per ESC line). (F) Representative images of electroporated cells in the CP-like layer 2 weeks after the electroporation of shRNA plasmids. Scale bar, 25 μm. (G) Quantification of dendritic length of electroporated cells. Data are presented as mean ± SEM. Mann-Whitney test, ***p = 0.0019. (H) Sholl analysis for dendritic complexity of neurons in human cortical organoids. Two-way ANOVA followed by Holm-Sidak’s multiple comparisons test, ***p < 0.001, **p < 0.01, and *p < 0.05. N NTC = 29 cells (from n = 2 independent organoids), N shL1HS-2 = 69 cells (from n = 2 independent organoids), and N shL1M = 17 cells (from n = 2 independent organoids). Data are presented as mean ± SEM.

Article Snippet: Briefly, human iPSC or ESC colonies were detached with collagenase Type IV (Gibco) and transferred to an Ultra-Low attachment 10-cm plate (Corning Costar) containing hPSC medium that consisted of DMEM:F12 (Invitrogen), 20% Knockout Serum Replacer (Gibco), 1x Non-essential Amino Acids (Invitrogen), 1x β-mercaptoethanol (Gibco), 1x GlutaMAX (Invitrogen), 10 ng/mL FGF-2 (Peprotech) and ROCK inhibitor Y27632 (10 μM).

Techniques: Retroviral, Labeling, Derivative Assay, Staining, Infection, MANN-WHITNEY, Control, Electroporation, shRNA

Journal: Cell reports

Article Title: Long interspersed nuclear elements safeguard neural progenitors from precocious differentiation

doi: 10.1016/j.celrep.2024.113774

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Briefly, human iPSC or ESC colonies were detached with collagenase Type IV (Gibco) and transferred to an Ultra-Low attachment 10-cm plate (Corning Costar) containing hPSC medium that consisted of DMEM:F12 (Invitrogen), 20% Knockout Serum Replacer (Gibco), 1x Non-essential Amino Acids (Invitrogen), 1x β-mercaptoethanol (Gibco), 1x GlutaMAX (Invitrogen), 10 ng/mL FGF-2 (Peprotech) and ROCK inhibitor Y27632 (10 μM).

Techniques: Virus, Recombinant, Knock-Out, Membrane, RNA Sequencing, Software