Journal: Biomaterials Research

Article Title: Anti-senescence Effects of Nanovesicles Derived from Cluster of Differentiation-146-Positive Tonsil Mesenchymal Stem Cells via Modulation of the Tumor Protein 53 Pathway

doi: 10.34133/bmr.0371

Figure Lengend Snippet: In vitro analysis of CD146 + TMSC-NVs in a passage-associated cellular senescence model. (A) Morphological changes of human dermal fibroblasts (HDFs) after treatment with CD146 + TMSC-NVs (scale bar = 200 μm). (B) Senescence-associated β-galactosidase (SA-β-gal) assay after treatment with CD146 + TMSC-NVs (scale bar = 200 μm). (C) Cell proliferation test using Cell Counting Kit-8 (CCK-8) assay during treatment with CD146 + TMSC-NVs. (D) Quantitative data following colorization with the SA-β-gal-positive cells. (E to G) Quantitative data of the polymerase chain reaction for the mRNA expression of elastin, COL1, and HMOX1. Statistical significance is shown as the mean ± standard deviation (* P < 0.05, *** P < 0.001, **** P < 0.0001; ns, not significant).

Article Snippet: To demonstrate the components within NVs, miRNA profiling was conducted by Macrogen, Inc. following the manufacturer’s protocol for the TruSeq Stranded mRNA Library Prep Kit.

Techniques: In Vitro, Cell Counting, CCK-8 Assay, Polymerase Chain Reaction, Expressing, Standard Deviation

Journal: Cell Death and Differentiation

Article Title: Membrane-bound TNF mediates microtubule-targeting chemotherapeutics-induced cancer cytolysis via juxtacrine inter-cancer-cell death signaling

doi: 10.1038/s41418-019-0441-3

Figure Lengend Snippet: MTAs upregulate membrane TNF transcription through the JNK/c-Jun axis. a Effect of transcriptional inhibitor actinomycin D (ActD) on MTA-induced cell death in L929 cells. b RNA-sequencing analysis of Tnf and Jun gene expression patterns during MTA treatment. UCSC genome browser images depict calculated FPKM (fragments per kilobase of transcript per million mapped reads) values in RNA-sequencing data. Gene expression levels are provided in DATA SET . c Effect of Jun knockout on MTA-induced necroptosis in L929 cells. For complementation, wild-type c-Jun was expressed in the knockout cells and its expression was detected by immunoblotting. d qRT-PCR analysis of Tnf mRNA level in MTA-treated WT and Jun KO L929 cells. e Immunoblotting analysis of TNF in P100 fractions of NCZ-treated WT and Jun KO L929 cells. f A panel of MAPK and NF-κB inhibitors was tested for necroptosis inhibition effect on MTA-treated L929 cells. All inhibitors were pretreated for 2 h followed by NCZ challenge. i, inhibitor. g Immunoblotting analysis of the JNK/c-Jun activation in whole cell lysates of NCZ-treated L929 cells. h Immunoblotting analysis of TNF accumulation in membrane fraction (P100) in the presence of JNK inhibitor (JNKi, SP600125) for NCZ-treated L929 cells. i Immunoblotting analysis of TACE expression in whole cell lysates of NCZ-treated L929 cells. j , k Fluorimetric assay of measuring TACE activity in both cell lysate (left panel) and membrane fraction (right panel) of NCZ ( j ) or PTX ( k ) treated L929 cells. D, DMSO; NCZ, nocodazole; PTX, paclitaxel. Cell viability was determined by measuring ATP levels. The data are represented as mean ± SEM of duplicate wells ( a , c , and f ). Results are reported from one representative experiment. Experiments were repeated independently for four ( f ), three ( a , c , d , and g ), or two ( e and h – k ) times

Article Snippet: Qualified total RNA was further purified by RNeasy micro kit (Cat#74004, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany). cDNA libraries were constructed with an TruSeq-stranded mRNA kit (Illumina) using Poly-A selection.

Techniques: Membrane, RNA Sequencing, Gene Expression, Knock-Out, Expressing, Western Blot, Quantitative RT-PCR, Inhibition, Activation Assay, Fluorimetry Assay, Activity Assay

Journal: Cell Death and Differentiation

Article Title: Membrane-bound TNF mediates microtubule-targeting chemotherapeutics-induced cancer cytolysis via juxtacrine inter-cancer-cell death signaling

doi: 10.1038/s41418-019-0441-3

Figure Lengend Snippet: MTAs induce memTNF-mediated apoptosis in RIP3-deficient human carcinoma cell lines. a Immunoblotting analysis of apoptosis markers using whole cell lysates from recombinant TNF (soluble TNF, solTNF)-treated HeLa cells in the presence or absence of pan-caspase inhibitor z-VAD (Z). Cells were treated as indicated for 24 h. b Immunoblotting analysis of apoptosis markers using whole cell lysates from 1 µM MTA-treated HeLa cells in the presence or absence of 20 µM TACE inhibitor TAPI-1 (TACEi) or 20 µM pan-caspase inhibitor z-VAD (Z) for 36 h. c Immunoblotting analysis of apoptosis markers using whole cell lysates from 1 µM MTA-treated WT and TNFR1 KO HeLa cells for 36 h. d Effect of TNFR1 knockout on MTA-induced cell death in HeLa cells. Cells were treated as indicated for 48 h. e – g Immunoblotting analysis of apoptosis markers using whole cell lysates of 1 µM MTA-treated HCT116 (colon cancer, e ), MDA-MB-468 (breast cancer, f ), and BT549 (breast cancer, g ) cells for 36 h. h , i qRT-PCR analysis of JUN mRNA level ( h ) and in flow cytometric analysis of memTNF ( i ) in MTA-treated HeLa, HCT116, MDA-MB-468, and BT549 cells for 12 and 20 h respectively. D, DMSO; NCZ, nocodazole; PTX, paclitaxel; Z, z-VAD. Cell viability was determined by measuring ATP levels. The data are represented as mean ± SEM of duplicate wells ( d ). Results are reported from one representative experiment. Experiments were repeated independently three ( c , d , and h ) or two ( a , b , e – g , and i ) times

Article Snippet: Qualified total RNA was further purified by RNeasy micro kit (Cat#74004, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany). cDNA libraries were constructed with an TruSeq-stranded mRNA kit (Illumina) using Poly-A selection.

Techniques: Western Blot, Recombinant, Knock-Out, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A Th17 cell-intrinsic glutathione/mitochondrial-IL-22 axis protects against intestinal inflammation

doi: 10.1101/2023.07.06.547932

Figure Lengend Snippet: (A-J) Naïve T cells were sorted from spleen and lymph nodes of Gclc fl/fl and Cd4Cre Gclc fl/fl mice and induced to differentiate in vitro into pathogenic Th17 cells by culture with anti-CD3, anti-CD28, IL-6, IL-1β and IL-23. Cells were treated with 10mM NAC as indicated. (A-B) Bulk RNA seq was performed. (A) Heatmap representing GO:0022900 Electron transport Chain. (B) Tfam expression. (n=3). (C) Quantification of total ATP levels in Gclc fl/fl and Cd4Cre Gclc fl/fl in vitro -differentiated Th17 cells as measured in RLU. Data are mean±SEM (n=3); 2 trials. (D) Quantification of FCA of p-PI3K in in vitro -differentiated Gclc fl/fl and Cd4Cre Gclc fl/fl Th17 cells. Data are mean±SEM (n=3); 2 trials. (E) IL-22 protein concentrations as measured by ELISA in culture supernatants of in vitro -differentiated C57BL/6J WT Th17 cells treated with PI3K inhibitor (LY294002) for last 24 hours of differentiation. Data are mean±SEM (n=3). (F) Quantification of qPCR determination of IL-22 mRNA expression of in vitro -differentiated Gclc fl/fl and Cd4Cre Gclc fl/fl Th17 cells. Data are mean±SD (n=3–4). (G) IL-22 protein concentrations as measured by ELISA in culture supernatants of in vitro -differentiated Gclc fl/fl and Cd4Cre Gclc fl/fl Th17 cells. Data are mean±SEM (n=3); 2 trials. (H) Quantification of FCA of p-AKT in in vitro -differentiated Gclc fl/fl and Cd4Cre-Gclc fl/fl Th17 cells. Data are mean±SEM (n=3); 2 trials. (I) Left: FCA to detect p-mTOR in in vitro -differentiated Gclc fl/fl and Cd4Cre-Gclc fl/fl Th17 cells. Right: Quantification of left panel results. Data are mean±SEM (n=3); 3 trials. (J) Quantification of FCA of p-4E-BP1 in in vitro -differentiated Gclc fl/fl and Cd4Cre-Gclc fl/fl Th17 cells. Data are mean±SEM (n=3); 2 trials.

Article Snippet: Samples were prepared using an Illumina Stranded mRNA library prep kit (Illumina) with the addition of IDT for Illumina DNA/RNA UD indexes (Illumina).

Techniques: In Vitro, RNA Sequencing Assay, Expressing, Enzyme-linked Immunosorbent Assay