Journal: Scientific reports

Article Title: Toll-like receptor 4 mediates Lewis lung carcinoma-induced muscle wasting via coordinate activation of protein degradation pathways.

doi: 10.1038/s41598-017-02347-2

Figure Lengend Snippet: Figure 1. LLC cell-conditioned medium activates myotube catabolism through TLR4. C2C12 myoblasts were transfected with indicated siRNAs. After incubation in differentiation medium for 96 h, myotubes were treated with Lewis lung carcinoma cell-conditioned medium (LCM) or control medium. Activation of NF-κB (p65), p38 MAPK and C/EBPβ was evaluated by Western blot analysis of cell lysate in 1 h (A). Levels of UBR2, atrogin1 and LC3-II were evaluated by Western blot analysis of cell lysate in 8 h (B). Autophagy flux increase was demonstrated by measuring LC3-II levels in myotubes pre-treated with 20 μM of lysosome inhibitor chloroquine (CQ, C). Levels of myosin heave chain (MHC) were evaluated by Western blot analysis of cell lysate in 72 h (D). TLR2 and TLR4 knockdown was monitored at each of the time points. Myotubes treated for 72 h were immunofluorescence-stained for MHC and their diameter was measured (E). Data was analyzed by ANOVA. *Denotes a difference (P < 0.05).

Article Snippet: Antibodies to UBR2 and LC3-II were obtained from Novus Biologicals (Littleton, CO).

Techniques: Transfection, Incubation, Control, Activation Assay, Western Blot, Knockdown, Immunofluorescence, Staining

Journal: Molecular Medicine Reports

Article Title: Mouse bone marrow mesenchymal stem cells with distinct p53 statuses display differential characteristics

doi: 10.3892/mmr.2020.11025

Figure Lengend Snippet: Expression of miRNAs, cytokines and proteins in mBM-MSCs with differential p53 statuses. (A) Reverse transcription-quantitative PCR analysis of miRNAs expression levels of in p53 +/+ , p53 +/− and p53 −/− mBM-MSCs; expressions were normalized to U6. (B) Luminex assay of TNFα and IP-10 secretion in the supernatant obtained from p53 +/+ , p53 +/− and p53 −/− mBM-MSCs. (C) Western blotting and densitometric analysis of protein expression levels of UBR2, RNF31 and MMP19 in p53 +/+ , p53 +/− and p53 −/− mBM-MSCs; GAPDH was used as a loading control. *P<0.05, **P<0.01 and ***P<0.001. IP-10, interferon-γ-inducible protein; mBM-MSCs, mouse bone marrow mesenchymal stem cells; miR/miRNA, microRNA; MMP19, matrix metalloproteinase 19; RNF31, RING-finger protein 31; TNFα, tumor necrosis factor α; UBR2, ubiquitin protein ligase E3 component n-recognin.

Article Snippet: The membranes were incubated with the following primary antibodies: Anti-GAPDH (1:1,000; cat. no. KC-5G5; Kangchen BioTech Co., Ltd.), anti-p53 (1:1,000; cat. no. abs130596; Absin Bioscience, Inc.), anti-Sal-like protein 4 (SALL4); (1:500; cat. no. ab29112; Abcam), anti-protein lin-28 homolog B (LIN28B; 1:500; cat. no. 21626; Signalway Antibody LLC), anti-Sox2 (1:500; cat. no. ab5603; EMD Millipore), anti-octamer-binding protein 4 (Oct4; 1:400; cat. no. 21424; Signalway Antibody LLC), anti-c-Myc (1:200; cat. no. 10057-1-AP; ProteinTech Group, Inc.), anti-ubiquitin protein ligase E3 component n-recognin 2 (UBR2; 1:500; cat. no. BS60150; Bioworld Technology, Inc.), anti-matrix metalloproteinase 19 (MMP19; 1:500; cat. no. BS1235; Bioworld Technology, Inc.) and anti-RING-finger protein 31 (RNF31; 1:500; cat. no ab46322; Abcam).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Luminex, Western Blot