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: Cell metabolism

Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

doi: 10.1016/j.cmet.2020.03.010

Figure Lengend Snippet: (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of AML12 cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.

Article Snippet: Immunoprecipitation, LATS2 Kinase Assay, and GTP-RhoA Assay AML12 cells from 100-mm culture dishes were collected into 0.4 ml 1x ice-cold cell lysis buffer (#9803S, Cell Signaling) and incubated on ice for 5 min. Then cell lysates were centrifuged at 10,000 x g for 10 min at 4°C.

Techniques: Activity Assay, Incubation

Journal: Cell metabolism

Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

doi: 10.1016/j.cmet.2020.03.010

Figure Lengend Snippet: (A) Images and mean fluorescence intensity (MFI) per cell of AML12 cells transduced with cyto-GCaMP6f or ER-GCaMP6f and then incubated for 30 min with vehicle or liposomes (n = 4 biological replicates; means ± SEM; *p < 0.05). Scale bar, 100 μm.

Article Snippet: Immunoprecipitation, LATS2 Kinase Assay, and GTP-RhoA Assay AML12 cells from 100-mm culture dishes were collected into 0.4 ml 1x ice-cold cell lysis buffer (#9803S, Cell Signaling) and incubated on ice for 5 min. Then cell lysates were centrifuged at 10,000 x g for 10 min at 4°C.

Techniques: Fluorescence, Transduction, Incubation

Journal: Cell metabolism

Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

doi: 10.1016/j.cmet.2020.03.010

Figure Lengend Snippet: (A) TAZ immunoblot of siScr- or siGnas-transfected AML12 cells that were incubated for 16 h with liposomes and then 8 h with Lipo-Chol).

Article Snippet: Immunoprecipitation, LATS2 Kinase Assay, and GTP-RhoA Assay AML12 cells from 100-mm culture dishes were collected into 0.4 ml 1x ice-cold cell lysis buffer (#9803S, Cell Signaling) and incubated on ice for 5 min. Then cell lysates were centrifuged at 10,000 x g for 10 min at 4°C.

Techniques: Western Blot, Transfection, Incubation

Journal: Cell metabolism

Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

doi: 10.1016/j.cmet.2020.03.010

Figure Lengend Snippet: (A) RhoA activity of AML12 cells that were incubated for 24 h with vehicle or liposomes or for 2 h with liposomes and then 3 h with Lipo-Chol ± 10 μM ALOD4 (n = 6 biological replicates; means ± SEM; ***p < 0.001 vs. both Veh groups).

Article Snippet: Immunoprecipitation, LATS2 Kinase Assay, and GTP-RhoA Assay AML12 cells from 100-mm culture dishes were collected into 0.4 ml 1x ice-cold cell lysis buffer (#9803S, Cell Signaling) and incubated on ice for 5 min. Then cell lysates were centrifuged at 10,000 x g for 10 min at 4°C.

Techniques: Activity Assay, Incubation