Journal: Oxidative Medicine and Cellular Longevity

Article Title: Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α 7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm

doi: 10.1155/2022/9964689

Figure Lengend Snippet: Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on RhoA-GTP, RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.

Article Snippet: Western blot analyses were performed according to standard protocol [ ] using the following antibodies against: α 7-nAChR (ab216485; 1 : 1000), p38 (ab31828; 1 : 1000), p-p38 (phospho T180+Y182) (ab4822; 1 : 1000), IL-6 (ab9324; 1 : 1000), inducible NO synthase (ab3523; 1 : 1000), and GAPDH (ab9484; 1 : 10,000), purchased from Abcam (Abcam plc., Cambridge, UK), and RhoA (#2117; 1 : 1000), RhoA-GTP (#8820; 1 : 1000), ROCK1 (#4035; 1 : 1000), ROCK2 (#9029; 1 : 1000), t-MBS (#2634; 1 : 1000), and p-MBS (#3040; 1 : 1000) from Cell Signaling Technology (Cell Signaling, Danvers, MA, USA) in Supplementary Table .

Techniques: Activation Assay, Western Blot, Derivative Assay, Expressing, Activity Assay, Fluorescence, shRNA

Journal: Nature Communications

Article Title: Construction of a synthetic methodology-based library and its application in identifying a GIT/PIX protein–protein interaction inhibitor

doi: 10.1038/s41467-022-34598-7

Figure Lengend Snippet: a Transwell assays were employed to detect the anti-invasion abilities of 14-5-18 (10 and 50 μM) in the MGC803 (left) and MKN45 (right) cell lines for 24 h. The right columns show quantification, n = 3 biologically independent samples. Scale bar = 100 µm. b Expression levels of total Rac1/Cdc42 and the activated forms GTP-Rac1/Cdc42 were detected after 14-5-18 was added to MKN45 and MGC803 at concentrations of 5, 20 and 50 μM for 24 h, n = 3 biologically independent samples. c In vivo imaging of nude mice after intravenous injection of luciferase-expressing MGC803 cells. Compound 14-5-18 were administrated at the indicated dosages by gavage once a day for 18 days ( n = 6 biologically independent animals), and solvent DMSO (2%) + Tween 20 (5%) in water was used as control. Otherwise noted, DMSO was used as solvent control. Data are presented as mean values ± SD, and error bars indicate SD. Statistical analysis: One-way ANOVA, Dunnett’s multiple-comparisons test ( a–c , each group was compared with the control group). Source data are provided as a Source Data file.

Article Snippet: Active Rac1/Cdc42 Detection assays were performed following the protocols of Active Rho/Cdc42 Detection Kit (Cell Signaling Technology, USA).

Techniques: Expressing, In Vivo Imaging, Injection, Luciferase