Journal: bioRxiv

Article Title: AMH regulates ovary size by counteracting ovarian follicle cluster effects

doi: 10.1101/2024.08.15.607694

Figure Lengend Snippet: AMH diffusion and proteolytic activation of proAMH in ovarian stroma. (A) Microdialysis probes were inserted adjacent into ovary stroma where dialysis buffer was pumped through the porous probe allowing molecules up to 1000 kDa in molecular weight to enter the dialysate. The dialysate was collected for AMH assay. Needles were placed adjacent to an antral follicle as shown in the hematoxylin and eosin-stained sheep ovary section (Scale bar = 1 mm). AMH concentrations in follicular fluid or adjacent stroma were quantified in sheep and human ovaries. (B) 2D-PAGE separating recombinant AMH protein by size on the vertical dimension and isoelectric focusing (IEF) on the horizontal direction. Western blot was conducted on the dashed area using an antibody against the AMH N-terminal fragment (AMH N ). The recombinant AMH preparation contains predominantly proAMH (arrow) and lesser quantities of AMH N . (C) Western blot of recombinant proAMH treated with (+) or without (−) recombinant PCSK3 in artificial extracellular fluid or artificial secretory vesicle fluid. ProAMH appears as a 72 kDa band (arrowhead) and the cleaved AMH N fragment as a 64 kDa band (double arrowhead). (D) PCSK activity in mouse ovary fragments was visualised with a fluorogenic reagent that releases rhodamine 110 when exposed to protease activity (green: rhodamine 110 and blue: Hoescht 33342 DNA-stain, scale bar = 100 µm). A yellow ring denotes the basal lamina that separates the granulosa and theca layers. The upper graph shows the relative fluorescence intensity units (RFU) along the straight-line trace shown in the rhodamine 110 image and the lower graph shows mean (±SE) fluorescence intensity in the antrum, granulosa, theca and stroma was compared with 1-way ANOVA (p < 0.001) and Tukey’s B post-hoc test (regions that share the same letter were not significantly different, n = 9).

Article Snippet: Recombinant PCSK3 (8 units) (New England Biolabs, Cat# P8077L) was preincubated in 10 µL of 1.2 mmol/L CaCl , 5 mmol MES (brand), pH 6.0 to activate the enzyme.

Techniques: Diffusion-based Assay, Activation Assay, Molecular Weight, Staining, Recombinant, Western Blot, Activity Assay, Fluorescence

Journal: Communications Chemistry

Article Title: A Hybrid compound H93 treats prostate cancer by directly binding UHRF1 and promoting protein dimerization

doi: 10.1038/s42004-025-01744-3

Figure Lengend Snippet: a DU145 (Left) or VCaP (Right) cells were treated with indicated concentrations of H93 for 48 h, and the total DNA methylation level was measured by 5-methylcytosine (5mC) DNA dot-blot assays. Experiments were performed in triplicate. b DU145 (Left) or VCaP (Right) cells were treated with 0 (black dots), 10 (black squares) or 20 μM (black triangles) of H93 for 48 h, and the total DNA methylation level was measured by 5mC ELISA assays. The data are represented as the mean ± SD, n = 3 independent experiments. c Quantitated gene expression changes of representative tumor suppressor genes in DU145 (Upper) and VCaP (Lower) cells treated with 0 (empty black circles), 2.5 (black squares), 5 (black triangles), 10 (black rhombus) or 20 μM (black dots) of H93. n = 3 independent experiments. d–f DU145 subcutaneous xenografts were induced in immune-deficient nude mice, and then treated with vehicle (20 ml kg −1 day -1 ) or H93 (50 or 100 mg kg −1 day −1 ) by oral administration. n = 8 per group. The tumor nodes were harvested 28 days after treatment. d The tumor volumes (Left) and weights (Right) were compared with vehicle 20 ml kg −1 day −1 group depicted as black dots, H93 50 mg kg −1 day −1 group as black squares, H93 100 mg kg −1 day −1 group as black triangles. n = 8 animals. e The body weights of nude mice were continuously monitored and vehicle 20 ml kg −1 day −1 group was depicted as black line, H93 50 mg kg −1 day −1 group as red line, H93 100 mg kg −1 day −1 group as glaucous line. n = 8 animals. f Representative images of Hematoxylin-Eosin (HE), Ki67 immunochemistry (IHC) and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining in DU145 xenografts subjected to different concentrations of H93 (Left), and the quantitative data of Ki67 (Middle) and TUNEL (Right) staining, with vehicle 20 ml kg −1 day −1 group depicted as black dots, H93 50 mg kg −1 day −1 group as black squares, H93 100 mg kg −1 day −1 group as black triangles, and n = 40 views. The length of scale bars is 200 μm (×100) or 50 μm (×400). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The data are shown as mean ± SD.

Article Snippet: Colorimetric TUNEL Apoptosis Assay Kit (Cat#C1098) was purchased from Beyotime Biotech.

Techniques: DNA Methylation Assay, Dot Blot, Enzyme-linked Immunosorbent Assay, Gene Expression, End Labeling, TUNEL Assay, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Donepezil Ameliorates Pulmonary Arterial Hypertension by Inhibiting M2-Macrophage Activation

doi: 10.3389/fcvm.2021.639541

Figure Lengend Snippet: DON reverses PASMC proliferation and apoptosis resistance in MCT-induced PAH rats. Immunohistochemical staining for Ki67 and TUNEL in lung tissue to show the effect of DON on PASMC proliferation and apoptosis in MCT-induced rats in vivo (×400 magnification). Relative positive cells of the pulmonary artery were counted. To further test PASMC proliferative ability in vitro , PASMCs in the Ctrl, MCT, DON groups were isolated and cultured. The proliferative and apoptotic abilities of PASMCs were, respectively, assayed by immunofluorescence staining with EdU and TUNEL. The ratio of EdU and TUNEL positive cells to total cell numbers per high power field (×200 magnification) were calculated. (A) Representative immunohistochemical staining for Ki67 and TUNEL of pulmonary artery samples. (B) Quantitative analysis of Ki67 positive rate. (C) Quantitative analysis of TUNEL positive rate. (D) Statistical analysis of the EdU+ cell rate. (E) Statistical analysis of the apoptosis rate of PASMCs. (F) Representative immunofluorescent staining for EdU of PASMCs. (G) Representative immunofluorescent staining for TUNEL of PASMCs. Ctrl, control; DON, donepezil; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-20-deoxyuridine; MCT, monocrotaline; OD, optical density; PASMCs, pulmonary artery smooth muscle cells; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. * P < 0.05, vs. Ctrl group, # P < 0.05, vs. MCT group ( n = 5).

Article Snippet: One Step TUNEL Apoptosis Assay Kit (cat. no. C1086; Beyotime Institute of Biotechnology) was used to evaluate the apoptosis rate of PASMCs according to the manufacturer's protocol ( ).

Techniques: Immunohistochemical staining, Staining, TUNEL Assay, In Vivo, In Vitro, Isolation, Cell Culture, Immunofluorescence, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Donepezil Ameliorates Pulmonary Arterial Hypertension by Inhibiting M2-Macrophage Activation

doi: 10.3389/fcvm.2021.639541

Figure Lengend Snippet: M2-macrophages inhibited PASMC proliferation and promoted its apoptosis. PASMCs in MCT-induced rats were isolated and cultured, which were co-cultured with the supernatant of BMDMs isolated from the Ctrl, MCT and DON group, respectively. After 24 h cultivation, the proliferative and apoptotic abilities of PASMCs were, respectively, assayed by EdU and TUNEL Assay Kit. The ratio of EdU and TUNEL positive cells to total cell numbers per high power field (×200 magnification) were calculated. (A) EdU stain to test proliferation of PASMCs. (B) Statistical analysis of EdU+ cell rate. (C) TUNEL assay to detect apoptosis of PASMCs. (D) Statistical analysis of apoptosis rate. BMDMs, bone marrow-derived macrophages; Ctrl, control; DON, donepezil; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-20-deoxyuridine; MCT, monocrotaline; PASMCs, pulmonary artery smooth muscle cells; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling. * P < 0.05, vs. Ctrl group, # P < 0.05, vs. MCT group ( n = 5).

Article Snippet: One Step TUNEL Apoptosis Assay Kit (cat. no. C1086; Beyotime Institute of Biotechnology) was used to evaluate the apoptosis rate of PASMCs according to the manufacturer's protocol ( ).

Techniques: Isolation, Cell Culture, TUNEL Assay, Staining, Derivative Assay, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Donepezil Ameliorates Pulmonary Arterial Hypertension by Inhibiting M2-Macrophage Activation

doi: 10.3389/fcvm.2021.639541

Figure Lengend Snippet: Schematic illustration of the effect of DON on ameliorating MCT-induced PAH by regulating M2 macrophage activation. In the MCT-induced PAH rat model, parasympathetic activity is reduced, inflammatory response and M2-macrophages are activated, and the expression of pro-inflammatory factors and pro-proliferation factor Fizz1 is increased, and subsequently increase PASMC proliferation and apoptosis resistance. However, DON enhances parasympathetic nerve activity by reducing AchE activity, and suppresses the inflammatory response and M2-macrophage activation, thereby inhibiting PASMC proliferation and promoting its apoptosis to reverse PAH, which is speculated to be related to decreased expression of Fizz1. AchE, acetylcholinesterase; Fizz1, found in inflammatory zone 1; IL-6, interleukin-6; MCT, monocrotaline; PAH, pulmonary arterial hypertension; PASMCs, pulmonary artery smooth muscle cells; TNF-α, tumor necrosis factor-α.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (cat. no. C1086; Beyotime Institute of Biotechnology) was used to evaluate the apoptosis rate of PASMCs according to the manufacturer's protocol ( ).

Techniques: Activation Assay, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Rapid multiplex small DNA sequencing on the MinION nanopore sequencing platform

doi: 10.1101/257196

Figure Lengend Snippet: Comparison of MinION library preparation workflows. 2D library is a previously reported workflow ( W ei and W illiams 2016 ); 1D multiplex library is manufacturer’s workflow using a native barcoding kit and a 1D genomic sequencing kit on the current MinION platform; Rapid 1D multiplex library is a new rapid barcoding MinION library preparation workflow reported in this study developed to sequence short reads (<1000 bp) on the current platform. The length of each bar indicates the time needed. The steps are color-coded (Yellow: fragmentation; Red: end preparation including end-repair and dA-tail; blue: size selection and purification; dark blue: MyOne C1 bead purification; purple: sequencing)

Article Snippet: Using Dynabeads TM MyOne TM Streptavidin C1 bead-based 2D library purification (Invitrogen, Cat. 65001), the library was eluted from the beads in ELB buffer at 37 °C for 10 min ( ) ( W ei and W illiams 2016 ).

Techniques: Comparison, Multiplex Assay, Genomic Sequencing, Sequencing, Size Selection, Purification

Journal: Nature Communications

Article Title: Open-source personal pipetting robots with live-cell incubation and microscopy compatibility

doi: 10.1038/s41467-022-30643-7

Figure Lengend Snippet: a Fixed and cleared murine bone and cerebrum samples were sectioned before automated PHIL immunostaining and imaging. PHIL immunostaining preserved normal tissue morphology with intact macroscopic (left) and subcellular (right) features. Compare Supplementary Fig. for comparison to a manually pipetted sample ( n = 2 experiments). b Murine myeloid progenitors were sorted into GATA2VENUS-negative, -middle, and -high populations, plated and fixed with 4% formalin, and PHIL immunostained against VENUS with and without primary antibodies and imaged. VENUS quantification of PHIL immunostained samples showed Venus negative, mid and high in both microscopy and FACS analyses ( n = 2 experiments) and showed no antibody cross-contamination. The relative brightness of the different populations followed the same trend when observed via FACS or microscopy and was consistent with previously conducted manual immunostainings on the same populations . Box plot features, from top to bottom, represent maximum, 3rd quartile, median, 1st quartile, and minimum values. c Automated washing improves cell loss for flow sensitive samples over manual pipetting. Human umbilical cord CD34 + hematopoietic stem and progenitor cells, cultured for 1 day in a μ-Slide VI 0.4 prior to fixation, were washed once with 100 μL manually or with 2, 5, and 10 µL/s flow. Culture channels were imaged and counted before and after washing. Mean cell loss for manual versus 2, 5, and 10 µL/s were 12.6 versus 3.6%, 4.3%, and 6.1% respectively ( p -values from top to bottom: 0.0094, 0.0009, 0.0004, two-tailed t -tests with Benjamin–Hochberg multiple test correction, n = 2 experiments). Box plot features, from top to bottom, represent maximum, 3rd quartile, median, 1st quartile, and minimum values.

Article Snippet: Human cord blood cells were processed by density gradient centrifugation, and CD34 + cells were isolated using EasySep TM CD34 positive selection kit II (samples from Basel, Cat# 17896, Stemcell Technologies, Vancouver, BC, Canada).

Techniques: Immunostaining, Imaging, Comparison, Microscopy, Cell Culture, Two Tailed Test