Journal: iScience

Article Title: PKR deficiency delays vascular aging via inhibiting GSDMD-mediated endothelial cell hyperactivation

doi: 10.1016/j.isci.2022.105909

Figure Lengend Snippet: Interfering PKR reduces the release of IL-1β and HMGB1 (A) Aortic IL-1β and HMGB1 level in WT and PKR knockout mice of different age (n = 5). (B) IL-1β and HMGB1 secretion in siCon and PKR knockdown (siPKR) HUVECs stimulated with H 2 O 2 (n = 3). ∗∗∗ p< 0.001; ## p< 0.01; ### p< 0.001 (two-way ANOVA test). Data are represented as mean ± SEM.

Article Snippet: Serum or cellular supernatant levels of HMGB1 and IL-1b were measured with ELISA kits (HMGB1 ELISA kit, Shino-Test Corp., Kanagawa, Japan, mouse IL-1b ELISA kit, R&D Systems, USA and Human IL-1β ELISA Kit, Abcam, USA).

Techniques: Knock-Out

Journal: iScience

Article Title: PKR deficiency delays vascular aging via inhibiting GSDMD-mediated endothelial cell hyperactivation

doi: 10.1016/j.isci.2022.105909

Figure Lengend Snippet: Inhibiting endothelial cell hyperactivation prevents the release of IL-1β and HMGB1 (A) Representative immunoblots and densitometric analysis of GSDMD, N-GSDMD, PKR and p -PKR levels in HUVECs with glycine priming and then stimulated with H 2 O 2 (n = 3). (B) IL-1β and HMGB1 secretion in HUVECs with glycine priming and then stimulated with H 2 O 2 (n = 3). (C) Representative immunoblots and densitometric analysis of GSDMD, N-GSDMD, PKR and p -PKR level in HUVECs with disulfiram priming and then stimulated with H 2 O 2 (n = 3). (D) IL-1β and HMGB1 secretion in HUVECs with disulfiram priming and then stimulated with H 2 O 2 (n = 3). ∗∗∗ p< 0.001; ## p< 0.01; ### p< 0.001 (one-way ANOVA test). Data are represented as mean ± SEM.

Article Snippet: Serum or cellular supernatant levels of HMGB1 and IL-1b were measured with ELISA kits (HMGB1 ELISA kit, Shino-Test Corp., Kanagawa, Japan, mouse IL-1b ELISA kit, R&D Systems, USA and Human IL-1β ELISA Kit, Abcam, USA).

Techniques: Western Blot

Journal: iScience

Article Title: PKR deficiency delays vascular aging via inhibiting GSDMD-mediated endothelial cell hyperactivation

doi: 10.1016/j.isci.2022.105909

Figure Lengend Snippet:

Article Snippet: Serum or cellular supernatant levels of HMGB1 and IL-1b were measured with ELISA kits (HMGB1 ELISA kit, Shino-Test Corp., Kanagawa, Japan, mouse IL-1b ELISA kit, R&D Systems, USA and Human IL-1β ELISA Kit, Abcam, USA).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Lysis, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Isolation, Transfection, Negative Control, Software, Electron Microscopy

Journal: Journal of Healthcare Engineering

Article Title: Enamel Matrix Derivatives for Periodontal Regeneration: Recent Developments and Future Perspectives

doi: 10.1155/2022/8661690

Figure Lengend Snippet: Recent animal studies and clinical trials on EMD and other therapeutic agents.

Article Snippet: The exact mechanism by which EMD participates in the periodontal regeneration at the cellular and molecular level is still unclear, though Emdogain® (Straumann, Basel, Switzerland), a porcine-derived tooth enamel matrix product, is commercially available with about 15 years of supportive clinical data [ ].

Techniques: Clinical Proteomics, Transplantation Assay

Journal: Scientific Reports

Article Title: LATS kinases and SLUG regulate the transition to advanced stage in aggressive oral cancer cells

doi: 10.1038/s41598-022-16667-5

Figure Lengend Snippet: LATS1 and LATS2 phosphorylate SLUG on T208. ( A ) SAS and SAS-δ cells were co-transfected with siRNAs against LATS1 and LATS2 (siLATS1/2), and then cultured with TGF-β1 for 48 h, followed by WB with the indicated antibodies. ( B ) SAS cells transfected with GL2 (as a negative control) or LATS2 siRNA duplex were treated with cycloheximide (CHX) for the indicated periods, and then subjected to subcellular fractionation. Protein levels of SLUG and LATS2 were determined by WB. Lamin A/C and α-tubulin are nuclear and cytoplasmic fraction markers, respectively. The relative levels of cytoplasmic and nuclear SLUG normalized to the corresponding band intensities of α-tubulin and Lamin A/C, respectively, are shown below top panel. The normalized band intensities of SLUG at 0 h after CHX treatment were defined as 1.0 (lanes 1, 5, 9, and 13). ( C ) WB of parental SAS and SAS-δ, which were cultured in medium containing 10% FBS in the presence (+) or absence (–) of TGF-β1 (48 h), was performed with the indicated antibodies. The relative levels of the indicated proteins normalized to the corresponding band intensity of actin, SLUG, or SMAD2, are shown below panels. ( D ) Immunofluorescence staining with anti-SLUG-pT208 (red) and α-tubulin (green) antibodies. Cells were cultured as in ( C ). DNA was visualized by Hoechst 33258 staining (blue). The cell size were comparable between SAS and SAS-δ in the presence or absence of TGF-β1.

Article Snippet: However, the stabilization of SLUG by LATS1/2 may play only a partial role in regulating the levels of total cellular SLUG protein because the level of SLUG in SAS-δ was higher than that in SAS despite the lower levels of LATS1/2 proteins in SAS-δ.

Techniques: Transfection, Cell Culture, Negative Control, Fractionation, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: LATS kinases and SLUG regulate the transition to advanced stage in aggressive oral cancer cells

doi: 10.1038/s41598-022-16667-5

Figure Lengend Snippet: Downregulation of SLUG enhances in vitro invasiveness of SAS-δ via activation of the LATS1/2–SLUG-pT208 axis. ( A ) Invasion assays of parental SAS and SAS-δ transfected with siRNAs against TAZ, SLUG or siControl (negative control) were performed in the absence of TGF-β1 as in Fig. A. Invading cells on the membrane were stained, and then the membranes were photographed using a digital camera. ( B ) Bar graphs showing the index of invasion in ( A ), calculated by dividing the percent of invasion (% invasion = the average number of invading cells / the average number of migrating cells × 100) of parental SAS or SAS-δ cells transfected with the indicated siRNAs by the percent invasion of cells transfected with siControl. ( C ) WB of lysates from siRNA-treated cells used in the invasion assays. α-tubulin was used as a loading control. ( D ) SAS-δ cells were transfected with siSLUG, siLATS1/2 or siControl, and then cultured in the presence (+) or absence (–) of TGF-β1 (10 ng/ml) for 48 h, followed by WB with the indicated antibodies. Actin was used as a loading control. ( E ) Bar graph showing the ratio of SLUG-pT208 to total SLUG protein in SAS-δ transfected with siSLUG or siLATS1/2 in the presence (+) or absence (–) of TGF-β1 (10 ng/ml) for 48 h. Two experiments (Exp. 1 and 2, corresponding to those shown in ( D ) and Fig. S6A, respectively) were performed independently. ( F ) A speculated model for the molecular mechanism by which downregulation of SLUG promotes accumulation of T208-phosphorylated SLUG via LATS1/2 activation in SAS-δ. Knockdown of SLUG in SAS-δ increases LATS1/2, thereby efficiently phosphorylating residual SLUG protein on T208 for enhancing the invasiveness of the cells.

Article Snippet: However, the stabilization of SLUG by LATS1/2 may play only a partial role in regulating the levels of total cellular SLUG protein because the level of SLUG in SAS-δ was higher than that in SAS despite the lower levels of LATS1/2 proteins in SAS-δ.

Techniques: In Vitro, Activation Assay, Transfection, Negative Control, Membrane, Staining, Control, Cell Culture, Knockdown