Journal: Molecular medicine reports

Article Title: CREB1 and Smad3 mediate TGF‑β3‑induced Smad7 expression in rat hepatic stellate cells.

doi: 10.3892/mmr.2017.7654

Figure Lengend Snippet: Figure 3. TGF‑β3 activates CREB1 via p38 to induce Smad7 expression. (A) HSCs were treated with the c‑Jun N‑terminal kinase inhibitor SP600125 (20 µM), the p38 inhibitor SB203580 (20 µM), the mitogen‑activated protein kinase kinase inhibitor PD98059 (20 µM), or the protein kinase A inhibitor H89 (5 µM) for 30 min, then treated with or without exogenous TGF‑β3 (10 ng/ml) for 2 h. Smad7 mRNA expression levels were assessed using reverse transcrip tion‑quantitative polymerase chain reaction. Control is defined as 1 (n=6). Data are expressed as the mean ± standard deviation. *P<0.05 compared to cells treated with exogenous TGF‑β3 in the control group. (B) HSCs were treated with the p38 inhibitor SB203580 (20 µM) for 30 min and then with or without exogenous TGF‑β3 (10 ng/ml) for 2 h. Protein expression levels of CREB1, p‑CREB1 and Smad7 were assessed using western blot analysis. Three independent experiments were performed and a representative blot is presented. TGF, transforming growth factor; CREB, cAMP responsive element binding protein; HSC, hepatic stellate cell; p‑, phosphorylated.

Article Snippet: SP600125, a c-Jun N-terminal kinase (JNK) inhibitor; SB203580, a p38 inhibitor; PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor; and H89, a protein kinase A (PKA) inhibitor, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Polymerase Chain Reaction, Control, Standard Deviation, Western Blot, Binding Assay

Journal: International Journal of Endocrinology

Article Title: Effect of Gelam Honey on the Oxidative Stress-Induced Signaling Pathways in Pancreatic Hamster Cells

doi: 10.1155/2013/367312

Figure Lengend Snippet: Effect of quercetin and Gelam honey extract on pAkt (Ser473) expression.Quantitative analysis and representative western blot analysis of pAkt (Ser473) in HIT-T15 cells pretreated with quercetin and honey extract in cells cultured in 20 mM ((a), (c), (e)) and 50 mM ((b), (d), (f)) glucose. A sustained increase in the level of pAkt (ser473) was observed after pretreatment with quercetin and honey extract. Akt inhibitor VIII prevented the expression Akt ser473 phosphorylation induced by quercetin and honey extract. The results were normalized with β actin antibody. Data were presented as the mean ± standard deviation. (e) * P < 0.05; # P < 0.005 quercetin and honey extract treated compared to the 20 mM glucose alone. (f) * P < 0.05; # P < 0.005 quercetin and honey extract treated compared to the 50 mM glucose alone.

Article Snippet: To investigate inhibitory effects on Akt signaling pathway, cells were incubated with 5 μ M Akt inhibitor VIII (Santa Cruz Biotechnology sc-203173) for one hour before pretreatment with quercetin and honey extract.

Techniques: Expressing, Western Blot, Cell Culture, Phospho-proteomics, Standard Deviation

Journal: International Journal of Endocrinology

Article Title: Effect of Gelam Honey on the Oxidative Stress-Induced Signaling Pathways in Pancreatic Hamster Cells

doi: 10.1155/2013/367312

Figure Lengend Snippet: The effect of flavonoids and Gelam honey extract on insulin content. (a) Effect of pretreatment with quercetin and Gelam honey extract and the addition of Akt inhibitor VIII on the insulin content in cells cultured in 20 mM glucose. There was a significant increase in insulin content (* P < 0.05) when the cells were pretreated with quercetin and honey. There was a significant decrease in insulin content (* P < 0.05) when the cells were treated with Akt inhibitor VIII, before pretreating with quercetin and Gelam honey extract. (b) Effect of pretreatment with quercetin and Gelam honey and the addition of Akt inhibitor VIII on the insulin content in cells cultured in 50 mM glucose. There was a significant increase in insulin content (* P < 0.05, # P < 0.005) when the cells were pretreated with quercetin and honey. There was a significant decrease in insulin content (* P < 0.05, # P < 0.005) when the cells were treated with Akt inhibitor VIII, before pretreating with quercetin and Gelam honey extract.

Article Snippet: To investigate inhibitory effects on Akt signaling pathway, cells were incubated with 5 μ M Akt inhibitor VIII (Santa Cruz Biotechnology sc-203173) for one hour before pretreatment with quercetin and honey extract.

Techniques: Cell Culture

Journal: Experimental & Molecular Medicine

Article Title: The acute phase reactant orosomucoid-2 directly promotes rheumatoid inflammation

doi: 10.1038/s12276-024-01188-0

Figure Lengend Snippet: a Effect of NF-κB and p38 MAP kinase inhibitors on ORM2-stimulated IL6 and CXCL8 expression. RA-FLSs were pretreated with PDTC (10 μM), BAY 117082 (40 μM), or SB203580 (10 μM) for 1 h and then stimulated with recombinant ORM2 (1 μg/mL) for 6 h. IL6 and CXCL8 mRNA levels were assessed by qRT‒PCR. The data are presented as the mean ± SEM of more than three independent experiments. ** P < 0.01 and **** P < 0.0001 versus ORM2 according to the Kruskal–Wallis test ( IL6 : P < 0.0001, CXCL8 : P < 0.0001) with post hoc pairwise comparisons test using the Mann–Whitney U test. b Immunocytochemistry analysis of NF-κB p65 in RA-FLSs. Cells were activated with ORM2 (1 μg/mL) or LPS (100 ng/mL) for the indicated times. Representative confocal images of p65 translocation to the nucleus are presented. The extent of nuclear translocation (%) was manually counted and is presented in the bar graph. The data are presented as the mean ± SEM of more than three independent experiments. ** P < 0.01 and *** P < 0.001 versus no ORM2 according to two-way ANOVA ( P < 0.0001) with Sadik’s multiple comparisons. Scale bar: 20 μm. c Western blot analysis of IκB-α, NF-κB phospho-p65 ( p -p65), and NF-κB p65 in RA-FLSs stimulated with ORM2 for the indicated times (minutes [m]). d , e Decrease in ORM2-induced IL6 and CXCL8 mRNA levels induced by knockdown of NF-κB p65 . RA-FLSs were transfected with NF-κB p65 siRNAs (si-p65, 50 nM) or control siRNAs (si Con, 50 nM) for 24 h. NF-κB p65 expression was determined by qRT‒PCR (left in d ) and Western blot analysis (right in d ). IL6 and CXCL8 expression levels were determined by qRT‒PCR ( e ). f Total p38 and phospho-p38 ( p -p38) expression levels in RA-FLSs determined by Western blotting after stimulation with ORM2 for the indicated times (minutes). g Downregulation of p38 expression after 24 h of transfection with p38 siRNAs (si p38 , 50 nM), as determined by qRT‒PCR (left) and Western blot analysis (right). h qRT‒PCR analysis of IL6 and CXCL8 expression. i NF-κB p65 and p38 expression in double-knockdown cells was analyzed via qRT‒PCR. After p38 transcripts were knocked down for 24 h, RA-FLSs ( n = 6) were transfected again with si- p65 for an additional 24 h. j qRT‒PCR analysis of IL6 and CXCL8 expression. The qRT‒PCR data in ( e ), ( h ), and ( j ) were obtained for the siRNA-transfected RA-FLSs ( n = 5) 6 h after stimulation with ORM2 (1 μg/mL). The Western blot data in ( c ), ( d ), ( f ), and ( g ) are representative of three independent experiments. The bar graphs represent the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 according to the Mann‒Whitney U test for ( d ) and ( g ); one-way ANOVA ( IL6 in ( e ): P = 0.0003; IL6 in ( h ): P = 0.0004) with Tukey’s multiple comparisons test; Kruskal‒Wallis test ( CXCL8 in ( e ): P < 0.0001; CXCL8 in ( h ): P < 0.0001; p38 in ( i ): P = 0.0002; and IL6 in ( j ): P = 0.001) with post hoc pairwise comparisons test using a Mann‒Whitney U test; and Brown-Forsythe and Welch ANOVA ( P < 0.0001) with Dunnett T3 multiple-comparison test for CXCL8 in ( j ).

Article Snippet: Scrambled control siRNA and NF-κB p65, p38 MAP kinase, and GYPC siRNAs were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Recombinant, MANN-WHITNEY, Immunocytochemistry, Translocation Assay, Western Blot, Knockdown, Transfection, Control, Comparison

Journal: Chemico-biological interactions

Article Title: A mechanism of perhexiline’s cytotoxicity in hepatic cells involves endoplasmic reticulum stress and p38 signaling pathway

doi: 10.1016/j.cbi.2020.109353

Figure Lengend Snippet: Inhibition of p38 signaling pathway attenuates the cytotoxicity of perhexiline. HepG2 cells were pre-treated with 10 μM SB239063 before exposed to 25 μM perhexiline. (A) Cytotoxicity of perhexiline was assessed using LDH release. (B) The effectiveness of SB239063 was assessed using Western blotting. (C) Caspase 3/7 activity was assessed after 2 h perhexiline treatment. The results shown are mean ± S.D. from 3 independent experiments. *p < 0.05 compared to DMSO control.

Article Snippet: SB239063 (p38 inhibitor) was from LC laboratories (Woburn, MA).

Techniques: Inhibition, Western Blot, Activity Assay

Journal: Redox Biology

Article Title: The CH24H metabolite, 24HC, blocks viral entry by disrupting intracellular cholesterol homeostasis

doi: 10.1016/j.redox.2023.102769

Figure Lengend Snippet: CH24H is enriched in the mouse cortex and is upregulated via MAPK pathways upon VSV infection in vitro . A. Basal levels of CH24H mRNA in different tissues of C57/BL6 mice (n = 5) were analyzed by qPCR. B. The basal level of CH24H protein in different areas of the brain was analyzed by Western blotting (WB), and β-actin was included as a reference gene. C. C57BL/6 mice (n = 5) were infected intranasally (i.n.) with VSV at 10 5 FFU or mock infected with DMEM (Mock). At 6 days post-infection (d.p.i.), mouse brains were harvested and qPCR was performed to detect CH24H mRNA levels. D. Mouse cortical neurons were isolated and infected with VSV at MOI 0.01, cells were harvested at 24 h post-infection (h.p.i.), and CH24H mRNA and protein levels were measured by qPCR (n = 3) and WB. E, F. N2a cells were infected with VSV at MOI 0.01 and cells were harvested at the indicated time points. CH24H mRNA and protein levels (E), and IL-6 mRNA levels (F) were measured by qPCR (n = 3) and WB. G, H. N2a cells were incubated with LPS (100 ng/ml) for the indicated periods. CH24H mRNA and protein levels (G) and IL-6 mRNA levels (H) were measured by qPCR (n = 3) and WB. I, J. N2a cells were incubated with IFNβ at different concentrations (50 ng/ml and 100 ng/ml) for 24 h (h), and CH24H (I) and CH25H (J) mRNA levels were measured by qPCR (n = 3). K. N2a cells were mock infected or infected with VSV at an MOI of 0.01, at different time points after VSV infection, the culture medium was replaced in the presence of specific inhibitors (p38 inhibitor, 1 μM, 24 h; NF-κB inhibitor, 5 μM，18 h; JNK inhibitor, 0.5 μM, 12 h; ERK inhibitor, 5 μM, 18 h) or DMSO. CH24H mRNA levels were measured by qPCR (n = 3). L. N2a cells were infected with VSV at an MOI of 0.01, at different time points after VSV infection, the culture medium was replaced in the presence of specific inhibitors (p38 inhibitor, 1 μM, 24 h; NF-κB inhibitor, 5 μM，18 h; JNK inhibitor, 0.5 μM, 12 h; ERK inhibitor, 5 μM, 18 h) or DMSO. CH24H protein levels were measured by WB. Statistical analysis of comparisons between groups was performed by Student's t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant). Bar graph shows mean ± SD. The Western blot data are representative of at least two independent experiments.

Article Snippet: The JNK inhibitor (#GC13841), the NF-κB inhibitor (#GC11751), the p38 inhibitor (#GC18602), the ERK inhibitor (#GC43624), 25-Hydroxycholesterol (25HC, #GC33860), 24-Hydroxycholesterol (24HC, #GC33673), Filipin III (#GC12048), Nile Red (#GC15539), and MβCD (#GC32697) were all purchased from Glpbio (GLPBIO, Montclair, USA).

Techniques: Infection, In Vitro, Western Blot, Isolation, Incubation

Journal: Redox Biology

Article Title: The CH24H metabolite, 24HC, blocks viral entry by disrupting intracellular cholesterol homeostasis

doi: 10.1016/j.redox.2023.102769

Figure Lengend Snippet: The transcription factor c-Jun element regulates the activity of the CH24H promoter. A. N2a cells were transfected with CH24H promoter-reporter plasmids (TSS-500, TSS-1000, TSS-1500, TSS-2000, and TSS-2500) for 24 h. Then the cells were either infected with VSV at an MOI of 0.01 or uninfected, and the luciferase activities were measured after another 18 h of infection (n = 3). B. N2a cells were transfected with the CH24H promoter-reporter plasmid: TSS-500. At 24 h post-transfection, cells were infected with VSV at an MOI of 0.01. At different time points after VSV infection, the culture medium was replaced in the presence of different signaling pathway-specific inhibitors (p38 inhibitor, 1 μM, 24 h; NF-κB inhibitor, 5 μM, 18 h; JNK inhibitor, 0.5 μM, 12 h; ERK inhibitor, 5 μM, 18 h) or DMSO. After 18 h of infection, the luciferase activity of the CH24H promoter was measured (n = 3). C. Schematic representation of point mutations in the wild-type promoter-reporter (TSS-500). N2a cells were transfected with the CH24H promoter reporter (TSS-500 and its mutant: TSS-500-mut). At 24 h post-transfection, cells were either mock-infected or infected with VSV at MOI 0.01, and luciferase activity was measured after 18 h of infection (n = 3). D. N2a cells were co-transfected with CH24H promoter-reporter (TSS-500) plasmid and pCAGSS-HA-c-Jun expressing plasmid (named HA-c-Fos) or pCAGGS vector for 24 h, and then CH24H promoter luciferase activity was measured (n = 3). E. N2a cells were mock-infected (top) or infected with VSV-GFP (bottom) at an MOI of 0.01. At 18 h post-infection, the cells were fixed, and c-Jun proteins (left), GFP (middle), and cell nuclei (right) were observed by confocal microscopy. Scale bar = 10 μm. F. N2a cells were transfected with HA-c-Jun for 24 h and then infected with VSV at an MOI of 0.01 for 18 h. Fixed chromatin from N2a cells was prepared and immunoprecipitated with anti-HA antibodies. ChIP primers were designed to amplify the region containing the c-Jun binding site in the CH24H promoter. PCR products were separated by acrylamide gel electrophoresis. Statistical analysis of comparisons between groups was performed by Student's t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Bar graph shows mean ± SD.

Article Snippet: The JNK inhibitor (#GC13841), the NF-κB inhibitor (#GC11751), the p38 inhibitor (#GC18602), the ERK inhibitor (#GC43624), 25-Hydroxycholesterol (25HC, #GC33860), 24-Hydroxycholesterol (24HC, #GC33673), Filipin III (#GC12048), Nile Red (#GC15539), and MβCD (#GC32697) were all purchased from Glpbio (GLPBIO, Montclair, USA).

Techniques: Activity Assay, Transfection, Infection, Luciferase, Plasmid Preparation, Mutagenesis, Expressing, Confocal Microscopy, Immunoprecipitation, Binding Assay, Acrylamide Gel Assay, Electrophoresis