Journal: Tissue Engineering. Part A

Article Title: Opposite Spectrum of Activity of Canonical Wnt Signaling in the Osteogenic Context of Undifferentiated and Differentiated Mesenchymal Cells: Implications for Tissue Engineering

doi: 10.1089/ten.tea.2010.0133

Figure Lengend Snippet: (A) QRT-PCR showing the expression profiles of three downstream targets of canonical Wnt signaling, axin2, myc, and cyclin D1. Quantified mRNA values were normalized by the amounts of glyceraldehyde 3-phophate dehydrogenase mRNA, and results are given as fold induction (*p < 0.05). (B) Immunoblotting analysis of β-catenin performed on nuclear fractions revealed higher levels of nuclear β-catenin in ASCs, E16, and FpN1 cells. Membranes were stripped and reprobed with anti-Lamin B1 antibody to assess for equal loading and transfer of nuclear proteins fraction. Histogram represents the densitometric analysis of electrophoresis bands, and the relative intensities of bands were normalized to their respective loading control and set as 100%. The results are presented as the mean ± standard deviation of three independent experiments. (C) Indirect immunofluorescence staining detected the most intense nuclear staining in ASCs, E16, and FpN1. As negative control normal primary (irrelevant) mouse immunoglobulin G was used. Nuclear counterstaining was performed with DAPI. QRT-PCR, quantitative reverse transcription–polymerase chain reaction; mASCs, mouse adipose-derived stem cells; E16, embryonic-stage day 16 calvarial mesenchymal cells; FpN1, postnatal day 1 frontal bones-derived osteoblast; PpN1, postnatal day 1 parietal bone-derived osteoblast; FpN60, postnatal day 60 frontal bone-derived osteoblast; PpN60, postnatal day 60 parietal bone-derived osteoblast; DAPI, 4′,6-diamidino-2-phenylindole; Wnt, wingless. Color images available online at www.liebertonline.com/ten.

Article Snippet: Nuclear counterstaining was performed using Vectashield H-1200 mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories), and a Zeiss Axioplan microscope equipped with an Axiocam HRc digital camera was used for imaging.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Electrophoresis, Standard Deviation, Immunofluorescence, Staining, Negative Control, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Immunity

Article Title: Class Switch Recombination Occurs Infrequently in Germinal Centers

doi: 10.1016/j.immuni.2019.07.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Stained sections were mounted using Vectashield antifade mounting medium (#H-1000 or #H-1200, Vector Laboratories) and visualized using an Olympus IX71 inverted fluorescence microscope or a Zeiss Axio ScanZ1.

Techniques: Purification, Blocking Assay, Recombinant, Staining, Western Blot, Plasmid Preparation, Protease Inhibitor, Isolation, Cell Isolation, SYBR Green Assay, Multiplex Assay, Expressing, Software, In Silico

Journal: Nature Communications

Article Title: Cis P-tau is a central circulating and placental etiologic driver and therapeutic target of preeclampsia

doi: 10.1038/s41467-023-41144-6

Figure Lengend Snippet: a Immunoblots of placental protein extracts from gestational age-matched control (control; n = 3), early onset PE (e-PE; n = 3), and late onset PE (l-PE; n = 3) probed for cis and trans P-tau. b Sarkosyl soluble and insoluble fractions of placental extracts from age-matched controls ( n = 7) and early-onset PE (e-PE; n = 7) were immunoprecipitated with HT7 (t-tau) and IgG antibodies before immunoblots against cis and trans P-tau. c Representative confocal images of human placental tissue sections from e-PE and age match control for double immunofluorescence with cis P-tau (green), trans P-tau (green), and ProteoStat (red). Inserts are magnified images of boxed areas. Cytotrophoblasts: complete arrow; syncytiotrophoblasts: incomplete arrows. Scale bar: 50 μm. Mean fluorescence intensity (MFI) quantification of cis P-tau, trans P-tau and ProteoStat positive protein aggregates in e-PE ( d ) and l-PE ( e ) placental sections (e-PE, n = 17; control, n = 16; l-PE, n = 19; control, n = 21, total 33–40 placental sections were analyzed per condition, two way ANOVA followed by Bonferroni’s post hoc test; mean±s.e.m. f Pearson’s colocalization coefficient (PCC) for cis P-tau and ProteoStat (left) is higher than for trans P-tau and ProteoStat (right); n = 35. Statistics are represented in the figure derived from two-tailed unpaired t test with Mann Whitney test.; mean±s.e.m. g Age-matched (control) and e-PE placental lysates were treated with (+λ) or without (-λ) lambda phosphatase (Ppase), followed by immunoblot with antibodies identifying early ( cis P-tau and trans P-tau) or late (pS396) tau phosphorylation. Loading control was GAPDH. h Hypoxia-reoxygenation (H/R) stress induces robust cis P-tau aggregation. Human primary trophoblast cells were exposed to hypoxia (1% O 2 ) for 72 h followed by 2-3 h incubation in reoxygenation (21% O 2 ) conditions or normoxia environment (21% O 2 ). The merged picture with nuclear DAPI demonstrated co-localization. Inserts were enlargements of boxed portions. Images are representatives of n = 7 independent experiments, scale bar: 50 μm. i Disruption of β-tubulin positive microtubule network due to hypoxia in human primary trophoblast cells. ProteoStat positive protein aggregate puncta accumulated near damaged microtubule structure. Images are representatives of n = 5 independent experiments. Bar: 100 μm.

Article Snippet: To establish the phosphorylation status of tau proteins in the human placenta, we incubated placental protein extracts (120 µg) from the PE and gestational age-matched control pregnancy for 1.5 h with 1200 units of lambda phosphatase (λ PPase) (New England BioLabs Cat# P0753S) .

Techniques: Western Blot, Control, Immunoprecipitation, Immunofluorescence, Fluorescence, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Incubation, Disruption