Journal: Journal of Extracellular Vesicles

Article Title: Calcified apoptotic vesicles from PROCR + fibroblasts initiate heterotopic ossification

doi: 10.1002/jev2.12425

Figure Lengend Snippet: Single‐cell RNA‐sequencing (scRNA‐seq) analysis identifies novel apoptotic‐preferential PROCR + fibroblasts in the early stage of HO. (a) The workflow depicting the collection and processing of specimens of sham and HO tendons for scRNA‐seq. (b) Dimension reduction presentation (via tSNE) of combined single‐cell transcriptome data from Achilles tendons of rats from the sham and HO groups after 1 and 3 weeks ( n = 14402). Each dot represents a single cell and is labelled with corresponding cell categories and is coloured according to its cell type identity. Clusters were generated using a resolution of 0.2 before subclustering into major cell types according to the Methods. The Seurat 3 R‐Package segregation grouped the cells into 6 distinct cell clusters. (c and d) Quantitative analysis of clusters based on the combined single‐cell transcriptome data in (b). (e) tSNE of fibroblast clusters (F1–F5). (f) Apoptosis score analyses of fibroblasts based on (e). (g) Bioinformatic analysis of PROCR + cell populations based on (e). (h) Representation analysis of GO categories showing different functions for PROCR + cells. (i) Representation analysis of KEGG categories showing different functions for PROCR + cells.

Article Snippet: To inhibit fibroblast apoptosis in the Achilles tendon, Z‐VAD‐FMK (T7020, TargetMol) dissolved in normal saline was injected into rats at 1 μg/rat, three times a week.

Techniques: Single Cell, RNA Sequencing, Generated

Journal: Journal of Biological Chemistry

Article Title: Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m115.655548

Figure Lengend Snippet: FIGURE 5. NOTCH3 protects smooth muscle cells from cell death. HAoSMCs with lentivirally overexpressed NICD2, NICD3, and GFP, or siRNA knockdown of NOTCH2 (siN2), NOTCH3 (siN3), and control (siCTL) were exposed to 50 J/cm2 UV radiation and collected for protein 9 h later. A and B, Western blots to detect NOTCH2 and NOTCH3 expression, and level of total and cleaved caspase3 with tubulin used as loading control, n 3. C and D, caspase3 enzymatic activity assay using fluorescent caspase substrate AFC-DEVD, n 4. Significance determined by one-way ANOVA; *, p 0.05.

Article Snippet: Lysates were then used in a caspase3 enzymatic activity assay using AFCDEVD substrate (MP Biomedicals) as described previously (43).

Techniques: Knockdown, Control, Western Blot, Expressing, Enzyme Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m115.655548

Figure Lengend Snippet: FIGURE 7. Genetic deletion of Notch3 has unique effects on the survival of mouse aortic smooth muscle cells. A, mRNA expression of pro-survival genes in aortas of wild-type (WT), Notch2-deficient (Notch2fl/fl; MCC/), and Notch3-mutant (Notch3/) adult mice. qPCR with relative expression compared with GAPDH, n 4. B, SMCs were isolated and cultured from aortas of wild-type, Notch2, and Notch3-deficient mice and used in enzymatic caspase3 activity assays, n 4. C, total and phosphorylated (phospho)-ERK was measured in cultured smooth muscle cells following serum challenge, with tubulin used as loading control, n 4. Significance determined by one-way ANOVA, *, p 0.05.

Article Snippet: Lysates were then used in a caspase3 enzymatic activity assay using AFCDEVD substrate (MP Biomedicals) as described previously (43).

Techniques: Expressing, Mutagenesis, Isolation, Cell Culture, Activity Assay, Control

Journal: Oncotarget

Article Title: Sensitization of glycoengineered interferon-β1a-resistant cancer cells by cFLIP inhibition for enhanced anti-cancer therapy

doi: 10.18632/oncotarget.14573

Figure Lengend Snippet: The cells were treated with 100 ng/mL R27T and collected at the indicated times post-treatment. A, B . Caspase-8 activity was assessed by protease activity assays in OVCAR-3 (A) or HeLa (B) cells (*** P < 0.001 compared to the untreated group). C, D . For the detection of caspase-3/8/9, cFLIPL, and cFLIPS, western blot analysis was performed in OVCAR-3 (C) or HeLa cells (D). β-actin was used as the loading control. E . OVCAR-3 cells were treated with R27T (100 ng/mL) alone or in combination with 50 μM zVAD for 72 h, and cell viability was measured by WTS assays. Data are presented as the means ± SD of three independent experiments (*** P < 0.001 compared to the untreated group). F . OVCAR-3 cells transfected with control or cFLIPS vector were treated with 100 ng/mL R27T for 72 h. Cell viability was analyzed by WTS assays (*** P < 0.001 compared to the untreated group).

Article Snippet: After centrifugation, the supernatant was collected and incubated with 1 μg of anti-caspase-8 antibody (Acris Antibodies, Herford, Germany) in the presence of 20 μL Dynabead protein G (Thermo Scientific).

Techniques: Activity Assay, Western Blot, Transfection, Plasmid Preparation