Journal: Biomolecules & biomedicine

Article Title: Exogenous spermidine alleviates diabetic cardiomyopathy via suppressing reactive oxygen species, endoplasmic reticulum stress, and Pannexin-1-mediated ferroptosis.

doi: 10.17305/bb.2022.8846

Figure Lengend Snippet: Figure 5. SPD suppresses oxidative stress and endoplasmic reticulum stress in vivo and in vitro. Mice and primary neonatal mouse cardiomyocytes were treated with or without SPD and underwent the following detections: (A) DHE staining of myocardial tissue; (B) mRNA expression of GRP94, GRP78, ATF-4 and Chop; (C) Intracellular ROS level detection was determined by fluorescence microscopy; (D) Representative western blot of Prdx-1, TfR-1, p-p38, and p-JNK in cardiomyocytes; (E) The ER-related protein expression of GRP94, GRP78, ATF-4, and Chop was determined by western blot. *P < 0.05 vs Control group; #P < 0.05 vs HG group (n ≥3). SPD: Spermidine; DHE: Dihydroethidium; ROS: Reactive oxygen species; ER: Endoplasmic reticulum; HG: High glucose

Article Snippet: Non-specific proteins on membranes were blocked with 5% non-fat dried milk for 2 h at room temperature, the membranes were incubated overnight with the following primary antibodies (at a 1:1000 dilution, 4 °C): P2X7, ACSL-4, GPX-4, FTH-1, HO-1, NCOA-4, SSAT (Cat NO. A10511, A6826, A1933, A1144, A1346, A5695, A2506, ABclonal Technology, Wuhan, China); Prdx-1, TfR-1, p-p38, t-p38, p-JNK, t-JNK, GRP94, GRP78, ATF-4, Chop (Cat NO. 15816-1-AP, 10084-2-AP, 28796-1-AP, 14064-1-AP, 80024-1-RR, 24164-1-AP, 60012-2-Ig, 11587-1-AP, 10835-1-AP, 15204-1-AP, Proteintech, Wuhan, China); and

Techniques: In Vivo, In Vitro, Staining, Expressing, Fluorescence, Microscopy, Western Blot, Control

Journal: Scientific Reports

Article Title: DC-SIGN and Toll-like receptor 4 mediate oxidized low-density lipoprotein-induced inflammatory responses in macrophages

doi: 10.1038/s41598-017-03740-7

Figure Lengend Snippet: DC-SIGN participated in the TLR4-NFκB pathway. Negative control (NC) or DC-SIGN siRNA was transfected into macrophages treated or not treated with oxLDL (50 μg/ml) or LPS (62.5 ng/ml) for 60 min. Western blot analysis detected the knockdown efficiency of DC-SIGN and the phosphorylation of p38, JNK, IKKε and NFκB ( A and B ), which was quantified by densitometry in 3 independent experiments and presented as relative units (DC-SIGN/α-tubulin, p38, JNK, IKKε and NFκB phosphorylated protein/total protein). The data are expressed as the mean ± SD from 3 independent tests. * P < 0.05, ** P < 0.01 compared with the macrophages not treated with oxLDL or LPS, ## P < 0.01 compared with the NC in the same group. ( C ) Negative control (NC) or DC-SIGN siRNA was transfected into macrophages treated or not treated with oxLDL (50 μg/ml) or LPS (62.5 ng/ml) for 60 min. Nuclear extracts were then prepared and assayed for p65 activation by EMSA.

Article Snippet: Primary antibodies for DC-SIGN, TLR4, α-tubulin, anti-FLAG, anti-His (Abcam, USA), p65 (t-p65), phosphorylated-p65 (p-p65), IKKε (t- IKKε), phosphorylated-IKKε (p- IKKε), p38 MAPK (t-p38), phosphorylated-p38 MAPK (p-p38), c-Jun N-terminal kinase (t-JNK), and phosphorylated-JNK (p-JNK) were purchased from Cell Signaling Technology (MA, USA).

Techniques: Negative Control, Transfection, Western Blot, Knockdown, Phospho-proteomics, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: The Protective Effects of Ivabradine in Preventing Progression from Viral Myocarditis to Dilated Cardiomyopathy

doi: 10.3389/fphar.2016.00408

Figure Lengend Snippet: Expression of p38 MAPK in the myocardial tissues of mice on days 35 and 65. (A) Western blot analysis for phospho-p38 MAPK (P-p38) and total-p38 MAPK (T-p38). (B) Densitometric analysis of relative protein levels for P-p38/ T-p38. ∗∗ P < 0.001, versus CON-35; ∗∗∗ P < 0.001 versus untreated CVMC- 65; ∗ P < 0.001 versus CON-65.

Article Snippet: For the Western blot, proteins were separated on polyacrylamide gels and transferred to a PVDF membrane for detection with various antibodies, including primary monoclonal antibodies for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), phospho-p38 MAPK (P-p38) and total-p38 MAPK (T-p38) (Cell Signaling Technology Corporation, USA) and a polyclonal antibody for collagen I, collagen III (Biorbyt Corporation, USA).

Techniques: Expressing, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: β-HB treatment reverses sorafenib resistance by shifting glycolysis-lactate metabolism in HCC.

doi: 10.1016/j.biopha.2023.115293

Figure Lengend Snippet: Fig. 5. β-HB treatment reversed sorafenib resistance and enhanced regorafenib sensitivity by inhibiting the B-raf/MAPK pathway in Huh7-SR and Sk-Hep-1-SR cells. (A) Huh7-SR and SK-Hep-1-SR cells were treated with sorafenib (0 and 10 μM) and β-HB (0, 2.5, and 5 mM) for 48 h, and cell viability was then analyzed using an MTT assay. (B) Huh7-SR and SK-Hep-1-SR cells were treated with regorafenib (0 and 5 μM) and β-HB (0, 2.5, 5, and 10 mM) for 48 h, and cell viability was then analyzed using an MTT assay. (C) Western blotting was conducted to analyze the protein expression of phosphorylated (p)- and total (t)-MAPK-related signaling cascades; α-tubulin was used as an internal control. (D) Western blot images from Fig. 4 C were quantified using ImageJ software. (E) Representative contour plots of apoptosis were detected by flow cytometry stained with Annexin V-FITC and PI. (F) Apoptosis rates were assessed in dose-dependent regorafenib with or without β-HB treatment in Huh7-SR and Sk-Hep-1-SR with Annexin V/PI staining. * p < 0.05; * * p < 0.01; * ** p < 0.001 vs. 0 mM β-HB cells. Data are presented as mean ± SD.

Article Snippet: The following antibodies were used in the experiments: HMGCS2 (1:1000, #ab137043, Abcam, Cambridge, UK), hexokinase I (1:1000, #2024, Cell Signaling, Danvers, MA, USA), hexokinase II (1:1000, #2867, Cell Signaling), phosphofructokinase (1:1000, #8164, Cell Signaling), LDHA (1:1000, #3582, Cell Signaling), PDH (1:1000, #3205, Cell Signaling), IDH (1:1000, #8137, Cell Signaling), phosphorylated (p)- and total (t)-B-raf (1:1000, #2696 and #9433, Cell Signaling), p- and t-MAPK kinase (MEK; 1:1000, #2338 and #9122, Cell Signaling), ERK (1:1000, #9101 and #4695, Cell Signaling), β-catenin (1:1000, #8480, Cell Signaling), ZO-1 (1:1000, #8193, Cell Signaling), Vimentin (1:1000, #5741, Cell Signaling), N-cadherin (1:1000, #13116, Cell Signaling) and antiα-tubulin (1:5000 dilution, #T9026, Sigma-Aldrich).

Techniques: MTT Assay, Western Blot, Expressing, Control, Software, Flow Cytometry, Staining

Journal: FEBS Open Bio

Article Title: Caspase 3‐specific cleavage of MEK1 suppresses ERK signaling and sensitizes cells to stress‐induced apoptosis

doi: 10.1002/2211-5463.13574

Figure Lengend Snippet: MEK1 cleavage by caspase‐3 abolishes its kinase activity and promotes apoptosis. (A) In vitro kinase assay of full‐length or caspase‐3‐mediated cleaved forms of GST‐MEK1. GST‐MEK1 was first incubated with or without purified, recombinant caspase‐3‐GST, and was then mixed and further incubated with a MEK1 substrate, kinase‐defective GST‐ERK2(K/N), for the indicated times. Phosphorylated ERK2(K/N) was detected by immunoblotting (top). Total GST‐ERK2(K/N) and GST‐MEK1 (full‐length or cleaved forms) were also probed with an anti‐GST antibody (bottom). (B) Expression levels of endogenous MEK1 and exogenous Flag‐MEK1 (WT or D282N) in wild‐type MEF, MEK1 −/− MEF, MEF‐MEK1, and MEF‐MEK1(D283N) cells were analyzed by immunoblotting. (C) In vitro kinase assay of recombinant GST‐MEK1 or GST‐MEK1(D282N). The assay was performed as in (A). (D, E) MEF‐MEK1 or MEF‐MEK1(D282N) cells were exposed to osmotic stress (0.6 m sorbitol) for the indicated times. The phosphorylation status of ERK, and the cleavage of MEK1 and PARP were analyzed by immunoblotting. In (E), the intensity of the P‐ERK bands from three independent experiments was quantified (right graph). Data are mean ± SEM. P ‐values were assessed using a two‐tailed Student's t ‐test. N.S., not significant. (F) MEK1 −/− MEF, MEF‐MEK1, and MEF‐MEK1(D282N) cells were treated with osmotic stress (0.6 m sorbitol for 3 h), stained with annexin V‐FITC, and visualized by fluorescence microscopy (upper panels). The scale bar represents 50 μm. The percentage of annexin V‐positive apoptotic cells was quantified (right graph). Data are mean ± SEM from three independent experiments. More than 100 cells were counted per sample. P ‐values were assessed using one‐way ANOVA followed by Tukey's multiple comparisons test. N.S., not significant. (G) The cells were exposed to osmotic stress (0.6 m sorbitol for 4 h) in the presence or absence of a MEK inhibitor (10 μ m trametinib). The phosphorylation status of ERK, and the cleavage of MEK1 and PARP were analyzed by immunoblotting. (H) The indicated cells were stimulated with etoposide (60 μ m for 24 h), sorbitol (0.6 m for 12 h), or anti‐Fas antibody (200 ng·mL −1 for 10 h) as indicated. Where indicated, the cells were pretreated with (+) or without (−) the pan‐caspase inhibitor Z‐VAD‐FMK. The phosphorylation states of ERK and RSK, and the cleavage of MEK1 were monitored by immunoblotting. (B, D, E, G, H) Actin served as a loading control.

Article Snippet: Where indicated, cells were pretreated with a pan‐caspase inhibitor [100 μ m Z‐VAD‐FMK (Santa Cruz, Dallas, TX, USA)], a caspase‐8 inhibitor [20 μ m Z‐IETD‐FMK (R&D Systems, Minneapolis, MN, USA, FMK007)], or a caspase‐9 inhibitor [20 μ m Z‐LEHD‐FMK (R&D Systems, FMK008)] for 2 h, or with a MEK inhibitor [10 μ m Trametinib (LC Laboratories, T‐8123)] for 30 min before stimulation.

Techniques: Activity Assay, In Vitro, Kinase Assay, Incubation, Purification, Recombinant, Western Blot, Expressing, Two Tailed Test, Staining, Fluorescence, Microscopy