Journal: Frontiers in Oncology

Article Title: Cytotoxic Effects of Arsenite in Combination With Gamabufotalin Against Human Glioblastoma Cell Lines

doi: 10.3389/fonc.2021.628914

Figure Lengend Snippet: Prosurvival role of p38 MAPK in the cytotoxicity of U-87 cells treated with the combination of As III and gamabufotalin. (A) Following treatment for 48 h with relatively low concentrations of As III (1, 2 μM) and gamabufotalin (20, 50 nM), alone or in combination, the expression profiles of phospho-p38 (p-p38) and p38 were analyzed using western blotting. A representation image of the expression profile of each protein is shown from three independent experiments. The expression levels were expressed as the ratios between each targeted protein and β-actin protein expression levels, and were compared with those of control group. (B) Following treatment for 48 h with the combined regimen of 2 μM As III + 50 nM gamabufotalin; 3.3 μM As III + 40 nM gamabufotalin, in the presence of absence of 5 μM SB203580, a specific inhibitor for p38 MAPK and its negative control SB202474, cell viability was determined by XTT assay. Relative cell viability was calculated as the ratio of the absorbance at 450 nm of each treatment group against those of the corresponding untreated control group. Data are shown as the means ± SD (n ≥ 3). A p value less than 0.05 was considered as statistically significant ( § p < 0.001; ‡ p < 0.0001 vs. control. $ p < 0.001 vs. As+Gama and As+Gama+SB202474). As, As III ; Gama, gamabufotalin; SB203, SB203580; SB202, SB202474. The images of beta-actin are identical to that in Figure 4B since the same experiment samples were used to analyze.

Article Snippet: Both SB203580, a specific inhibitor of p38 MAPK, and its negative control SB202474 were purchased from Merck KGaA.

Techniques: Expressing, Western Blot, Negative Control, XTT Assay

Journal: Journal of immunotoxicology

Article Title: Acetaminophen and lipopolysaccharide act in synergy for the production of pro-inflammatory cytokines in murine RAW264.7 macrophages.

doi: 10.1080/15476910902938250

Figure Lengend Snippet: Figure 4. Effect of LPS on APAP-induced phosphorylation of MAPK in RAW264.7 cells. (A) RAW264.7 cells were treated with APAP (1 mM) alone, LPS (0.1 ng/ml) alone, or an APAP and LPS combination for up to 8 hr and lysed for assay of MAPK phosphorylation. (B) RAW264.7 cells were treated with APAP (1 mM) alone, LPS (1 ng/ml) alone, or an APAP and LPS combination for up to 8 hr and lysed for assay of MAPK phosphorylation. Whole cell lysates were subjected to Western blotting using antibodies specific for the phosphorylated forms of p38MAPK, ERK1/2, JNK1/2. An anti-p38 MAPK antibody was used as a loading control. Numbers under each image correspond to the ratio (P-protein/p38 MAPK) treated/(P-protein/p38 MAPK) control. Results are representative of two independent experiments.

Article Snippet: Stripping of membranes was performed to analyze total p38 MAPK expression as a loading control with an antibody raised against p38 MAPK (p38 N20, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of immunotoxicology

Article Title: Acetaminophen and lipopolysaccharide act in synergy for the production of pro-inflammatory cytokines in murine RAW264.7 macrophages.

doi: 10.1080/15476910902938250

Figure Lengend Snippet: Figure 5. Effect of the APAP and LPS combination on the NF-B signal- ing pathway in RAW264.7 cells. A. RAW264.7 cells were treated with APAP (1 mM) alone, LPS (0.1 ng/ml) alone, or an APAP and LPS combination for up to 8 hr and lysed for assay of IB degradation by Western blotting. B. RAW264.7 cells were treated with APAP (1 mM) alone, LPS (1 ng/ml) alone, or an APAP and LPS combination for up to 8 hr and lysed for assay of IB degradation. Whole cell lysates were subjected to Western Blotting using antibodies specific for IB. An anti-p38 MAPK antibody was used as a loading control. Numbers under each image correspond to the ratio (IB/p38 MAPK) treated/(IB/p38 MAPK) control. Results are representative of two independent experiments.

Article Snippet: Stripping of membranes was performed to analyze total p38 MAPK expression as a loading control with an antibody raised against p38 MAPK (p38 N20, Santa Cruz Biotechnology).

Techniques: Western Blot, Control

Journal: Journal of immunotoxicology

Article Title: Acetaminophen and lipopolysaccharide act in synergy for the production of pro-inflammatory cytokines in murine RAW264.7 macrophages.

doi: 10.1080/15476910902938250

Figure Lengend Snippet: Figure 6. MAPK inhibitors modulated TNF and IL-1 production induced by APAP, LPS, or APAP and LPS in RAW264.7 cells. After 1 hr-pre- treatment with SB 203580 at 1 µM () or SP600125 at 20 µM () or PD98059 at 30 µM (), APAP (1 mM) or LPS (0.1 ng/ml) or APAP and LPS were added to culture medium for a supplementary 24 hr. Supernatants were ana- lyzed for cytokine production by ELISA. Values (expressed as pg/ml/µg protein) are mean ± SD of three independent experiments.

Article Snippet: Stripping of membranes was performed to analyze total p38 MAPK expression as a loading control with an antibody raised against p38 MAPK (p38 N20, Santa Cruz Biotechnology).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Ecotoxicology and environmental safety

Article Title: 2, 2', 4, 4'-tetrabromodiphenyl ether induces placental toxicity via activation of p38 MAPK signaling pathway in vivo and in vitro.

doi: 10.1016/j.ecoenv.2022.114034

Figure Lengend Snippet: Fig. 6. BDE-47 inhibits the migration and invasion of HTR-8/SVneo cells by activating the p38 MAPK signaling pathway. (A) Representative images of p-p38 and p38 detected by western blot in mouse placenta and quantita tive results (n = 5). (B) Representative images of p-p38 and p38 detected by western blot in HTR-8/SVneo cells and quantitative results (n = 3). (C) Representative images of the inhibi tory effect of SB203580 on p38 MAPK signaling pathway detected by western blot in HTR-8/SVneo cells and quanti tative results (n = 3). (D) Representa tive images of VEGF-A detected by western blot in HTR-8/SVneo cells and quantitative results (n = 4). (E) Representative images of wound heal ing assay and quantitative results (n = 3). Magnification: 100 × , Bar= 200 µm. (F) Representative im ages of Transwell assay and quantita tive results (n = 3). Magnification: 400 × , Bar= 50 µm. (G) Representa tive images of the expressions of invasion-related proteins (MMP9, MMP2, TIMP2, IGF1 and IGFBP3) detected by western blot in HTR-8/ SVneo cells and quantitative results (n = 3). Results are presented as mean ± SD. *P < 0.05, ** P < 0.01 compared to controls; # P < 0.05, ## P < 0.01 compared to 20 μM BDE-47-treated group.

Article Snippet: The p38 MAPK signaling pathway inhibitor SB203580 (Cat. #S1076) was obtained from Selleckchem (Huston, TX, USA).

Techniques: Migration, Western Blot, Transwell Assay

Journal: Ecotoxicology and environmental safety

Article Title: 2, 2', 4, 4'-tetrabromodiphenyl ether induces placental toxicity via activation of p38 MAPK signaling pathway in vivo and in vitro.

doi: 10.1016/j.ecoenv.2022.114034

Figure Lengend Snippet: Fig. 7. BDE-47 inhibits proliferation, increases apoptosis and autophagy in HTR-8/SVneo cells by activating p38 MAPK signaling pathway. (A) Representative images of the expressions of proliferation-related proteins (PCNA and CCND) and quantitative results of western blot analysis. (B) Representative flow histograms depicting cell distribution during cell cycle and quantitative results. (C) Representative flow histograms showing cell apoptosis rates and quantitative results. (D) Representative images of the expressions of apoptosis-related proteins (Bcl2, Bax, Caspase9, cleaved-Caspase9, Caspase3, cleaved-Caspase3 and Cyt c) and quan titative results of western blot analysis. (E) Representative images of the expressions of autophagy-related proteins (p62, Beclin1 and LC3) and quantitative results of western blot analysis. Results are presented as mean ± SD. n = 3; *P < 0.05, ** P < 0.01 compared to controls; # P < 0.05, ## P < 0.01 compared to 20 μM BDE-47- treated group.

Article Snippet: The p38 MAPK signaling pathway inhibitor SB203580 (Cat. #S1076) was obtained from Selleckchem (Huston, TX, USA).

Techniques: Western Blot

Journal: Ecotoxicology and environmental safety

Article Title: 2, 2', 4, 4'-tetrabromodiphenyl ether induces placental toxicity via activation of p38 MAPK signaling pathway in vivo and in vitro.

doi: 10.1016/j.ecoenv.2022.114034

Figure Lengend Snippet: Fig. 8. Outline graph of BDE-47 activation of p38 MAPK signaling pathway leading to placental toxicity.

Article Snippet: The p38 MAPK signaling pathway inhibitor SB203580 (Cat. #S1076) was obtained from Selleckchem (Huston, TX, USA).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Induction of Neurite Outgrowth in PC12 Cells Treated with Temperature-Controlled Repeated Thermal Stimulation

doi: 10.1371/journal.pone.0124024

Figure Lengend Snippet: (A–K) PC12 cells in the differentiation medium were treated with TRTS at 39.5°C (18 h/day), 40 ng/ml BMP4, or left untreated for 7 days in the presence or absence of 2.5 μM U0126, 2.5 μM U0124, 0.5 μM SB203580, or 1.0 μM GW441756. (A) Representative phase-contrast images of PC12 cells on day 7 after treatment with no stimulation; (B) TRTS (18 h/day); (C) TRTS (18 h/day) plus 2.5 μM U0126; (D) TRTS (18 h/day) plus 2.5 μM U0124; (E) TRTS (18 h/day) plus 0.5 μM SB203580; (F) TRTS (18 h/day) plus 1.0 μM GW441756; (G) 40 ng/ml BMP4; (H) 40 ng/ml BMP4 plus 2.5 μM U0126; (I) 40 ng/ml BMP4 plus 2.5 μM U0124; (J) 40 ng/ml BMP4 plus 0.5 μM SB203580; (K) 40 ng/ml BMP4 plus 1.0 μM GW441756. Scale bar, 100 μm. (L, M) PC12 cells were treated with TRTS at 39.5°C (18 h/day), 40 ng/ml BMP4, or left untreated for 7 days in the presence of the indicated concentrations of U0126, U0124, SB203580, or GW441756. The percentage of neurite-bearing cells on day 7 was assayed. The data represent the mean ± SD of three replicates. †† P < 0.01 vs. the day 7 control with no stimulation. ** P < 0.01 vs. TRTS or BMP4 alone on day 7. n.s., not significant. #, not significant vs. the day 7 control with no stimulation. TRTS, temperature-controlled repeated thermal stimulation.

Article Snippet: The MAPK/ERK kinase (MEK)1/2-specific inhibitor U0126 (Calbiochem, San Diego, CA, USA) and a negative control for U0126, U0124 (Merck Millipore, Billerica, MA, USA); p38 MAPK-specific inhibitor SB203580 (Enzo Life Sciences, Farmingdale, NY, USA); the TrkA-specific tyrosine kinase inhibitor GW441756 (Axon Medchem BV, Groningen, The Netherlands) were dissolved in diethyl sulfoxide (DMSO; Wako Pure Chemical Industries, Osaka, Japan).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Transcription of the Transforming Growth Factor ? Activating Integrin ?8 Subunit Is Regulated by SP3, AP-1, and the p38 Pathway *

doi: 10.1074/jbc.M110.113977

Figure Lengend Snippet: p38 regulates ITGB8 expression and αvβ8-mediated TGF-β activation. A, flow cytometry for integrin β8 on adult lung fibroblasts treated with MAPK inhibitors, PD98059 (ERK), SB202190 (p38), and SP600125 (JNK) (± S.E.). MFI, mean fluorescence intensity. B, quantitative RT-PCR results for ITGB8 expression in adult lung fibroblasts treated with SB202190, normalized to GAPDH and β-actin and relative to control (± S.E.). C, immunoblot for phosphorylated HSP 27 and dual-phosphorylated ATF-2 from nuclear extracts from adult lung fibroblasts treated ± SB202190. Immunoblot for the nuclear localized proteins, lamins A and C, was used as a loading control. D, TGF-β activation assays of adult lung fibroblasts treated with anti-β8 blocking antibodies or SB202190 (± S.E.). E, quantitative RT-PCR results for MAPK14 (p38α) and ITGB8 in adult lung fibroblasts transfected with plasmids expressing a p38α dominant-negative isoform (p38αDN) or the empty vector control, pcDNA (± S.E.). The measured transcript is labeled above each respective graph. F, TGF-β activation assays of adult lung fibroblasts transfected with plasmids expressing a p38α dominant-negative isoform (p38αDN) or the empty vector control, pcDNA. Percentage (%) of αvβ8-mediated TGF-β activation shown (± S.E.). * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001.

Article Snippet: The chemical inhibitors of the p38 (SB202190) and extracellular-signaling response kinase (ERK) (PD98059) MAP kinase pathways were obtained from Calbiochem (EMD4 Biosciences, Gibbstown, NJ), whereas the inhibitor of the Jun N-terminal kinase (JNK) pathway (SP600125) was obtained from A.G. Scientific, Inc. (San Diego, CA).

Techniques: Expressing, Activation Assay, Flow Cytometry, Fluorescence, Quantitative RT-PCR, Western Blot, Blocking Assay, Transfection, Dominant Negative Mutation, Plasmid Preparation, Labeling

Journal: PLoS ONE

Article Title: IL-17 Enhances Chemotaxis of Primary Human B Cells during Asthma

doi: 10.1371/journal.pone.0114604

Figure Lengend Snippet: (A) B cells isolated from asthmatic patients were pre-treated with different kinase inhibitors or DMSO carrier for 1 hr before being incubated with Th-17 cytokines in a migration assay. Data is presented as % migration of B cells relative to negative control (medium). (n = 6) (B) Western blots showing p38 MAPK phosphorylation in B cells following Th-17 cytokine stimulation. Asthmatic B cells (2×10 6 ) stimulated with Th-17 cytokines were lysed using 1x RIPA buffer and cell lysates were resolved using western analysis. Blots were probed using anti-phospho-p38 and anti-p38 MAPK antibodies. (C) Ratio of p-p38 over p38 band intensity were determined using densitometer and data were presented as fold increase in p38 phosphorylation ratio following Th-17 cytokine stimulation compared to non-stimulated condition (medium). Data is expressed as means ± SD (n = 5). *P<0.05 is considered significant.

Article Snippet: Specifically, we used the p38 MAPK inhibitor SB203580 (0.1 mM; Axon Medchem, Groningen, The Netherlands), the extracellular signal-regulated kinase (ERK) 1/2 MAPK inhibitor PD184352 (2 mM; United States Biological, Inc, Swampscott, Mass), the NF-kB inhibitor PS1145 (10 mM; Professor Sir Philip Cohen), and the phosphoinositide 3-kinase (PI3K) inhibitor PI103 (5 mM; Cayman Chemical, Ann Arbor, Mich).

Techniques: Isolation, Incubation, Migration, Negative Control, Western Blot, Phospho-proteomics

Journal: Scientific Reports

Article Title: Upregulated long noncoding RNA LOC105375913 induces tubulointerstitial fibrosis in focal segmental glomerulosclerosis

doi: 10.1038/s41598-018-36902-2

Figure Lengend Snippet: Role of p38 MAPK in the expression of LOC105375913 in tubular cells. ( a ) RT-PCR analysis of LOC105375913 in HK-2 cells treated with C3a, SB203580, PD098059 and MK2206 (n = 5); ( b ) RNA pull-down and RT-PCR analysis of the binding between LOC105375913 and miR-27b in HK-2 cells treated with C3a and SB203580 (n = 5); ( c ) Western blot analysis of snail, FN and Col I in HK-2 cells treated with C3a and SB203580 (n = 3); ( d,e ) Western blot analysis of p-p38 in HK-2 cells treated with C3a or plenti-CMV-LOC105375913 plasmid (n = 3); ( f ) RT-PCR analysis of LOC105375913 in HK-2 cells treated with U46619 (n = 5); ( g ) RNA pull-down and RT-PCR analysis of the binding between LOC105375913 and miR-27b in HK-2 cells treated with U46619 (n = 5); ( h ) Western blot analysis of snail, FN and Col I in HK-2 cells treated with U46619 (n = 3). For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for ( a , b and g ) and a two-tailed Student’s t-test was used for ( f ). * P < 0.05 compared with control. # P < 0.05 compared with C3a-treated cells.

Article Snippet: For intervention studies, 1 μM of C3aR antagonist SB290157 (sc-222291, Santa Cruz), 100 μg/ml of eculizumab (Soliris, Alexion Pharmaceuticals), 10 μM of p38 MAPK inhibitor SB203580 (sc-3533, Santa Cruz), 50 μM of ERK inhibitor PD98059 (sc-3532, Santa Cruz), 10 μM of Akt inhibitor MK2206 (sc-364537, Santa Cruz) or 1 μM of U-46619 (sc-201242, Santa Cruz) was given 30 min before treatments.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Western Blot, Plasmid Preparation, Two Tailed Test, Control

Journal: Scientific Reports

Article Title: Upregulated long noncoding RNA LOC105375913 induces tubulointerstitial fibrosis in focal segmental glomerulosclerosis

doi: 10.1038/s41598-018-36902-2

Figure Lengend Snippet: Role of XBP-1s in the expression of LOC105375913 in tubular cells. ( a ) RT-PCR analysis of LOC105375913 in HK-2 cells treated with C3a, XBP-1s siRNA, C/EBPβ siRNA, Elk-1 siRNA, ERα siRNA and GR siRNA (n = 3); ( b ) Level of XBP-1s and p-XBP-1s protein in HK-2 cells treated with C3a and SB203580 (n = 3); ( c ) ChIP analysis of the binding between XBP-1s and LOC105375913 promoter in HK-2 cells treated with C3a (n = 3); ( d ) Schematic of the constructed LOC105375913 promoter-luciferase reporter plasmids; ( e ) Normalized luciferase activity of reporter constructs in HK-2 cells cotransfected with XBP-1s plasmid (n = 5); ( f ) Level of LOC105375913 in HK-2 cells transfected with XBP-1s plasmid (n = 5); ( g ) RNA pull-down and RT-PCR analysis of the binding between LOC105375913 and miR-27b in HK-2 cells transfected with XBP-1s plasmid (n = 5); ( h ) Western blot analysis of snail, FN and Col I in HK-2 cells transfected with XBP-1s plasmid (n = 3); ( i ) RNA pull-down and RT-PCR analysis of the binding between LOC105375913 and miR-27b in HK-2 cells treated with C3a and XBP-1s siRNA (n = 5); ( j ) Western blot analysis of snail, FN and Col I in HK-2 cells treated with C3a and XBP-1s siRNA (n = 3). For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for ( a , e , g and i ), and a two-tailed Student’s t-test was used for ( f ). * P < 0.05 compared with control; # P < 0.05 compared with C3a-treated cells.

Article Snippet: For intervention studies, 1 μM of C3aR antagonist SB290157 (sc-222291, Santa Cruz), 100 μg/ml of eculizumab (Soliris, Alexion Pharmaceuticals), 10 μM of p38 MAPK inhibitor SB203580 (sc-3533, Santa Cruz), 50 μM of ERK inhibitor PD98059 (sc-3532, Santa Cruz), 10 μM of Akt inhibitor MK2206 (sc-364537, Santa Cruz) or 1 μM of U-46619 (sc-201242, Santa Cruz) was given 30 min before treatments.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Construct, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Western Blot, Two Tailed Test, Control