Journal: Frontiers in Immunology

Article Title: MicroRNA-1 Negatively Regulates Peripheral NK Cell Function via Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (TWEAK) Signaling Pathways During PPRV Infection

doi: 10.3389/fimmu.2019.03066

Figure Lengend Snippet: Regulation of NK cell function by miR-1 is mediated by TWEAK signaling. (A) Goat CD16 + CD14 − NK cells were transfected with control miRNA (MC) or miR-1 mimics, or miR-1 mimics and incubated with recombinant TWEAK at the final concentration of 50 nM. Forty-eight hours later, the cells were infected with PPRV at an MOI of 1, and 24 h later, the protein expression of NKG2D, perforin, IFN-γ, and TNF-α was measured by Western blot. Histograms of the right panel showed densitometric analysis of indicated protein normalized to GAPDH to correct for protein loading. (B) Goat CD16 + CD14 − NK cells were transfected with lentivirus TWEAK, si-TWEAK, or respective negative control. Forty-eight hours later, the cells were infected with PPRV at an MOI of 1, and 24 h later, the protein levels of SOCS-1, MyD88, STAT3, pSTAT3, and NF-κB p65 in NK cells were determined by Western blot and normalized to the levels of GAPDH. Results are expressed as means ± standard error of mean (SEM). P -values were calculated using Student's t -test. An asterisk indicates a comparison with the indicated control. * P < 0.05; ** P < 0.01. n.s., not significant.

Article Snippet: For downstream signaling pathway detection, membranes were probed overnight with rabbit anti-SOCS1 (Sangon Biotech, Shanghai, China), rabbit anti-NF-κB p65 (Bioss, Beijing, China), rabbit anti-MyD88 (Bioss, Beijing, China), rabbit anti-STAT3 (Bioss, Beijing, China), or rabbit anti-pSTAT3 (Bioss, Beijing, China).

Techniques: Cell Function Assay, Transfection, Incubation, Recombinant, Concentration Assay, Infection, Expressing, Western Blot, Negative Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Change in Oxidative Stress and Mitochondrial Dynamics in Response to Elevated Cold-Inducible RNA-Binding Protein in Cardiac Surgery-Associated Acute Kidney Injury

doi: 10.1155/2022/3576892

Figure Lengend Snippet: CPB induced mitochondrial dynamics disorder and increased inflammatory factor secretion. (a–f) Expression of IL-6, IL-1 β , and TNF- α in the serum at 0 and 4 hours after CPB. Compared to the CPB group, C23 administration decreased the expression of IL-6, IL-1 β , and TNF- α by 54.0%, 67.5%, and 45.2% at 4 hours, respectively. (g) Western blot analysis of renal Bax, Bcl-2, and cleaved-caspase-3 expression. (h) CPB upregulated the NADPH oxidase via the TLR-4/MyD88 pathway, which promoted the expressions of mitochondrial fission-related proteins Fis1 and Drp1 and reduced the expression of fusion-related protein Mfn2. ∗ P < 0.05 vs. sham; # P < 0.05 vs. CPB.

Article Snippet: The other antibodies used in this study included TLR-4 (19811-1-AP, Proteintech, 1 : 1000), MyD88 (BA2321, Boster, 1 : 500), and anti- β -actin (4967, Cell Signaling Technology, 1 : 1000).

Techniques: Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Change in Oxidative Stress and Mitochondrial Dynamics in Response to Elevated Cold-Inducible RNA-Binding Protein in Cardiac Surgery-Associated Acute Kidney Injury

doi: 10.1155/2022/3576892

Figure Lengend Snippet: Putative mechanism of CIRP in cardiac surgery-associated acute kidney injury. During cardiac surgery, CIRP is secreted into the circulation in response to hypothermia and hemodynamics change. Extracellular CIRP promotes the expression of NADPH oxidase in renal tubular epithelial cells via the TLR-4/MyD88 pathway and aggravates intracellular oxidative stress. ROS accumulation induces mitochondrial dynamics disorder, which ultimately increases apoptosis and promotes AKI. CIR: cold-inducible RNA-binding protein; NADPH: nicotinamide adenine dinucleotide phosphate; ROS: reactive oxygen species.

Article Snippet: The other antibodies used in this study included TLR-4 (19811-1-AP, Proteintech, 1 : 1000), MyD88 (BA2321, Boster, 1 : 500), and anti- β -actin (4967, Cell Signaling Technology, 1 : 1000).

Techniques: Expressing, RNA Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2–MyD88 signal pathway in dendritic cells and enhances Th2 immune responses

doi: 10.1084/jem.20062027

Figure Lengend Snippet: EDN-induced IL-6 production was dependent on MyD88. (A) Human monocyte–derived DCs were transfected with or without (Chariot II alone) gripNAhMyD88. After 24 h of culture, the DCs (4 × 10 5 cells/ml) were incubated with EDN at the indicated concentrations for 40 h, and the concentration of IL-6 in the culture supernatant was measured by ELISA. Shown are the data of one experiment representative of two. (B) DCs generated from the bone marrow progenitors of WT (MyD88 +/+ ) and MyD88 knockout (MyD88 −/− ) mice were incubated at 10 6 /ml in the presence of 1 μg/ml Pam3, EDN, or LPS for 48 h, and the production of IL-6 in the culture supernatants was measured by ELISA. The results of one experiment representative of two are presented as the average (mean ± SD) of triplicate wells.

Article Snippet: For the transient silencing of DC MyD88, monocyte-derived iDCs were transduced without or with gripNA MyD88 (5′-CCTGCAGCCAYDDCGGGC-3′; Active Motif) at 10 nmol/10 6 cells using Chariot II (Active Motif) according to the manufacturer's instructions.

Techniques: Derivative Assay, Transfection, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Generated, Knock-Out