Journal: BMC cell biology

Article Title: Phosphorylation of p68 RNA helicase by p38 MAP kinase contributes to colon cancer cells apoptosis induced by oxaliplatin.

doi: 10.1186/1471-2121-13-27

Figure Lengend Snippet: Figure 2 MAPKPhosphorylation of p68 by p38 MAPK. (A) Threonine phosphorylations of p68 in HCT116 cells that are treated with 20 μM of oxaliplatin for different times are analyzed by immunobloting the p68 that are immunoiprecipitated (IP:p68) from cell lysates using antibody against phorsphor-threonine (IB:14B3). Phosphorylation of p38 MAPK under the same treatment is analyzed by immunoblot of cell lysates using antibody against the phosphorylated p38. Immunoblot of p68 (IB:p68) in the immunoprecipitates indicate the amounts of p68 that are precipitated. Immunoblot of p38 in the cell lysate (IB:p38) indicate the cellular levels of p38, as a loading control. (B) Co-immunoprecipitation of p38 and p68 in the cell extracts of HCT116 cells with/without oxaliplatin treatment (Oxa, +/−20 μM) was analyzed by immunoblot of p68 immunoprecipitates (IP:p68) using antibody against p38 (IB:p38). Immunoblot of p68 (IB:p68) in the immunoprecipitates indicate the amounts of p68 that are precipitated. IP:IgG is the immunoprecipitation using rabbit IgG, serving as a negative control IP. (C) Phosphorylation of recombinant His-p68 or BSA, as a control, by recombinant p38 in the presence of [γ-32P]-ATP is revealed by autoradiography. The amounts of proteins used in the phosphorylation reactions are shown by coomasie blue stains (CBS). (D) Phosphorylation of p68 by exogenous expression of Flag-tagged p38 MAPK, wild type and constitutively active mutant D176A-F327L, in HCT116 cells are analyzed by immunoblotting the p68 that are immunoiprecipitated (IP:p68) from cell lysates using antibody against phorsphor-threonine (IB:14B3). Immunoblot of p68 (IB:p68) in immunoprecipitates indicate the amounts of p68 that are precipitated. Immunoblot of Flag-tag (IB:FLAG) indicate the exogenous p38 levels. Immunoblot of GAPDH (IB:GAPDH) is a loading control.

Article Snippet: P38α cDNA (Origene) was subcloned into p3XFLAGmyc-CMVTM-24 Expression Vector (Sigma).

Techniques: Western Blot, Phospho-proteomics, Control, Immunoprecipitation, Negative Control, Recombinant, Autoradiography, Expressing, Mutagenesis, FLAG-tag

Journal: BMC cell biology

Article Title: Phosphorylation of p68 RNA helicase by p38 MAP kinase contributes to colon cancer cells apoptosis induced by oxaliplatin.

doi: 10.1186/1471-2121-13-27

Figure Lengend Snippet: Figure 3 Phosphorylation site(s) of p68 by p38 MAPK. (A) Prediction of potential p38 MAPK phosphorylation site(s) in the p68 reading frame and compared to the consensus p38 MAPK phosphorylation sites of several authentic p38 MAPK substrates by a web-based phosphorylation site prediction program NetPhos 2.0, (B) Phosphorylation of recombinant His-p68 and mutants with single site mutation (Upper) and double site mutations (Lower) by recombinant p38 in the presence of [γ-32P]-ATP is revealed by autoradiography. The amounts of proteins used in the phosphorylation reactions are shown by coomasie blue stains (CBS). (C) Phosphorylation of exogenously expressed HA-p68s, wild type (WT) and mutants (Single site mutation, Upper, and Double site mutations, Lower), in HCT116 cells with/without oxaliplatin treatment (Oxa, +/−) are analyzed by immunoblotting the p68 that is immunoiprecipitated (IP:p68) from cell lysates using antibody against phorsphor-theronine (IB:14B3). Immunoblot of p68 (IB:p68) in immunoprecipitates indicate the amounts of p68 that are precipitated.

Article Snippet: P38α cDNA (Origene) was subcloned into p3XFLAGmyc-CMVTM-24 Expression Vector (Sigma).

Techniques: Phospho-proteomics, Recombinant, Mutagenesis, Autoradiography, Western Blot

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: tRNA synthetase inhibition causes persistent ribosome stalls. ( A ) U-2 OS cells expressing GFP-G3BP1 were treated with 20 µM halofuginone (HF) or 0.2% DMSO carrier. Images were collected every 2 h for 16 h and representative images are shown ( n = 3 independent experiments). ( B ) Cells were treated with DMSO (−) or 20 µM HF for 4 or 16 h, and P-eIF2α and total eIF2α were detected by Western blotting. Representative blots with total protein are shown at the left , and the average ± SEM of n = 3 independent experiments is shown at the right . Molecular weights (in kilodaltons) are shown. ( C ) Cells were treated with 20 µM HF for 1 h (black line), 4 h (purple line), or 16 h (green line), and polysome profiling was performed. A representative profile is shown at the left , and the average ± SEM of the polysome:monosome ratio from n = 3 independent replicates is shown at the right . Pink, gray, and green dots represent results from each replicate. ( D ) Cells were treated with either 0.2% DMSO carrier or 20 µM HF in the presence or absence of 250 µM sodium arsenite (As) or 2 µM rocaglamide A (RocA), and GFP-G3BP1 was imaged every 0.5 h for 3 h. Representative images are shown at the left with the average ± SEM. The percentage of cells with SGs from n = 3 independent experiments is shown at the right ; ≥288 cells were counted per treatment across all replicates. ( E ) Cells were treated with 0.2% DMSO (black line), 2 µM RocA (green line), or 2 µM RocA plus 20 µM HF (purple line) for 3 h followed by polysome profiling. Representative profiles are shown at the left , and the average ± SEM from n = 3 independent experiments is shown at the right , with pink, gray, and green dots representing each replicate. ( F ) Cells were untreated (DMSO carrier) or treated for 24, 4, or 1 h with 20 µM anisomycin (ANI) or 20 µM halofuginone, and Western blotting for p-p38 and p-JNK was done, with the total protein shown below . A representative blot of n = 2 independent experiments is shown. Scale bars, 10 µm. Significance was assessed with ordinary one-way ANOVAs and Tukey's multiple comparisons tests. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005.

Article Snippet: p-p38 (Thr180/Tyr182) , Proteintech , 28796 , 1:1000 (Western).

Techniques: Inhibition, Expressing, Western Blot

Journal: Cancer Research

Article Title: Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

doi: 10.1158/0008-5472.can-08-3797

Figure Lengend Snippet: Figure 5. Cytoskeleton and VEGF-dependent signaling in cultured endothelial cells isolated from Shb knockout or wild-type liver. A, the cytoskeleton of isolated endothelial cells maintained in tissue culture for 5 to 7 d was visualized by staining with rhodamine-phalloidin, showing a less regular shape with numerous extensions in Shb null cells. B, corresponding wild-type control. No clear cytoskeletal difference between wild-type or knockout cells was noted after stimulation with VEGF-A (C, Shb knockout; D, Shb wild type). Addition of VEGF to the control cells (D) produced changes that made the cells resemble the Shb null cells in the absence of VEGF-A. Horizontal scale bar is shown. Equal amounts of protein from the experimental groups (+/, 20 ng/mL VEGF-A for 2 min) were subjected to Western blot analysis for the phosphorylated proteins indicated. Shb blot shows unstimulated cells only. The blots were subjected to densitometric analysis for phosphorylated FAK, total FAK, phosphorylated p38, total p38, pMLC, phosphorylated ERK, and total ERK in three separate experiments. Quantitation of the relative phosphorylation of FAK, MLC, p38, and ERK is also given. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01 (paired Students’ t test).

Article Snippet: The samples were electrophoresed on SDS-polyacrylamide gels, protein was transferred on to Hybond-P filters (GE Healthcare), and these were then probed for pY-1175 VEGFR-2, total VEGFR-2, pY-397 FAK, total FAK, phosphorylated extracellular signal-regulated kinase (ERK), total ERK, and phosphorylated Akt, total Akt, phosphorylated myosin light chain (pMLC), pY-658 VE-cadherin, phosphorylated p38, and total p38 (all antibodies were from Cell Signaling, except for total VEGFR-2 from R&D Systems, pY-397 FAK from Biosource, pY-VE-cadherin from ProSci, and pMLC from Santa Cruz Biotechnology) before incubation with secondary antibodies and enhanced chemiluminescence.

Techniques: Cell Culture, Isolation, Knock-Out, Staining, Control, Produced, Western Blot, Quantitation Assay, Phospho-proteomics

Journal: Molecular medicine reports

Article Title: Fluoxetine protects against methamphetamine‑induced lung inflammation by suppressing oxidative stress through the SERT/p38 MAPK/Nrf2 pathway in rats.

doi: 10.3892/mmr.2016.6072

Figure Lengend Snippet: Figure 4. SERT protein expression and P‑38 MAPK phosphorylation in rat lungs in the different groups. (A) SERT protein expression and (B) the rela tive p‑p38 MAPK expression in lungs were determined and quantified. The data are expressed as the mean ± standard deviation (n=6; *P<0.05, **P<0.01 vs. the control group; +P<0.05 vs. the MA group). SERT, serotonin trans porter; MAPK, mitogen activated protein kinases; MA, methamphetamine; F2, fluoxetine at 2 mg/kg; F10, fluoxetine at 10 mg/kg.

Article Snippet: The membranes were probed with primary mouse monoclonal anti-β‐actin (1:2,000; cat. no. sc‐130300; Santa Cruz Biotechnology, Inc., Dallas, USA) and α‐tubulin 1:2,000; cat. no. 66031; ProteinTech Group, Inc., Chicago, IL, USA) and rabbit polyclonal anti‐SERT (1:200; cat. no. bs 1893R), IL‐6 (1:200; cat. no. bs 0379R), tumor necrosis factor‐α (TNF-α; 1:200; cat. no. bs 0078R), human heme oxygenase‐1 (HO‐1; 1:400; cat. no. bs 2075R) (Biosynthesis Biotechnology Co. Ltd.), anti‐Nrf2 (1:600; cat. no. 16396‐1‐AP; ProteinTech Group, Inc.), p38 (1:600; cat. no. ZS‐7149), and phosphorylated (p)‐p38 (1:600; cat. no. ZS‐101759; Zhongshan Golden Bridge Biotechnology Co., Ltd.) overnight at 4 ̊C.

Techniques: Expressing, Phospho-proteomics, Standard Deviation, Control

Journal: Nature Communications

Article Title: Inhibition of iRhom1 by CD44-targeting nanocarrier for improved cancer immunochemotherapy

doi: 10.1038/s41467-023-44572-6

Figure Lengend Snippet: a Correlation of iRhom1 gene expression levels with survivals in BRCA-TNBC or COAD patients receiving chemotherapy. For BRCA-TNBC-Chemo: n(Low expression) = 52, n (High expression) = 33; For COAD-Chemo: n (Low expression)=89, n (High expression) = 56. b CTRP-based prediction of the correlation between iRhom1 gene expression levels and the responses to different types of anticancer drugs. HDACi: histone deacetylase inhibitor, MAi: microtube assembly inhibitor, Topoi: topoisomerase inhibitor, PLK1i: polo-like kinase 1 inhibitor, NAMPTi: nicotinamide phosphoribosyltransferase inhibitor. c The impact of siRNA-mediated iRhom1 knockdown on the cytotoxicity of DOX or CPT-SAHA in several cancer cell lines. n = 6 independent samples. d Changes in protein levels of iRhom1 following treatment with DOX or CPT-SAHA. n = 4 independent experiments and data were quantified by densitometry. e RNA-seq analysis of gene enrichment in various signaling pathways in iRhom1 KO CT26 cells compared to WT cells. f Changes in the protein levels of MAPK14-HSP27 axis in iRhom1 KO cells compared to WT cells. n = 5 independent experiments and data were quantified by densitometry. g Changes in the protein levels of MAPK14-HSP27 axis in iRhom1 OE cells compared to WT cells. n = 5 independent experiments and data were quantified by densitometry. h Changes in the protein levels of MAPK14-HSP27 axis after treatment with various concentrations of CPT-SAHA in iRhom1 KO cells and WT cells. n = 3 independent experiments and data were quantified by densitometry. i MTT cytotoxicity assay of treatments with various concentrations of CPT-SAHA in CT26 WT or CT26 iRhom1 OE cells, with or without knockdown of MAPK14 respectively. n = 6 independent samples. j Synergy between Ivermectin (IVM) and chemotherapy drug (CPT-SAHA or DOX). k Immunoprecipitation of HA-iRhom1 with anti-HA beads led to pulldown of p-38α (MAPK14) in HA-iRhom1 expressing 293 T cells and reciprocal immunoprecipitation of FLAG-MAPK14 with anti-FLAG beads led to pulldown of iRhom1 in FLAG-MAPK14 expressing 293 T cells. Data are presented as mean ± s.e.m. in ( c , i ) and mean ± s.d. in ( d , f , g ). Statistical analysis was performed by log rank test for comparison in ( a ), two-tailed Student’s t-test for comparison in ( c , f , g ), and one-way ANOVA with Tukey’s post hoc test for comparison in ( d and i ). Data are representative of two independent experiments in ( e ), i – k three independent experiments in ( c , h ), four independent experiments in ( d ), and five independent experiments in ( f , g ). Source data are provided as a file for ( c , d , f , g , h , i , j , k ).

Article Snippet: Flag tagged MAPK14 Mouse ORF Clone (Cat.MR227429) was purchased from Origene (MD, USA).

Techniques: Gene Expression, Expressing, Histone Deacetylase Assay, Knockdown, RNA Sequencing, Protein-Protein interactions, Cytotoxicity Assay, Immunoprecipitation, Comparison, Two Tailed Test

Journal: Molecular medicine reports

Article Title: Helicobacter pylori infection enhances heparanase leading to cell proliferation via mitogen‑activated protein kinase signalling in human gastric cancer cells.

doi: 10.3892/mmr.2018.9558

Figure Lengend Snippet: Figure 2. H. pylori infection mediates the HPA protein expression increase in MKN‑45 cells via the MAPK signalling pathway. (A) Western blotting was used to detect the expression levels of p‑p38 MAPK, p38 MAPK, p‑p65 NF‑κB and p65 NF‑κB following different co‑culture durations. (B) p‑p38 MAPK protein expression. (C) p‑p65 NF‑κB protein expression levels were quantitatively analysed using densitometry. (D) Western blots and (E) quantitatively analysed expression of NF‑κB in MKN‑45 cells pre‑treated with SB203580 prior to co‑culture with H. pylori. *P<0.05 and **P<0.01, as indicated. MAPK, mitogen‑activated protein kinase; NF‑κB, nuclear factor‑κB; p‑, phosphorylated; H. pylori or Hp, Helicobacter pylori.

Article Snippet: Following the blocking step, the membranes were incubated with the following primary antibodies: anti-HPA1 (1:1,000; cat. no. ab128931), anti-phosphorylated (p)-p38 MAPK (1:1,000; cat. no. ab195049), anti-p38 MAPK (1:1,000; cat. no. ab170099), anti-p-p65 NF-κB (1:1,000; cat. no. ab76302), anti-p65 NF-κB (1:1,000; no. ab32536; all Abcam, Cambridge, UK) and anti-β-actin (1:2,000; cat. no. TA-09; OriGene Technologies, Inc., Beijing, China).

Techniques: Infection, Expressing, Western Blot

Journal: PLoS ONE

Article Title: AMP activated kinase negatively regulates hepatic Fetuin-A via p38 MAPK-C/EBPβ/E3 Ubiquitin Ligase Signaling pathway

doi: 10.1371/journal.pone.0266472

Figure Lengend Snippet: [A] HepG2 cells were incubated with either low- or high-glucose in the absence or presence of AICAR for 12hr and used for Western blot analysis for ERK1/2 phosphorylation expression (n = 3). [B] HepG2 cells were incubated with different concentration of AICAR for 12 h and cell lysates were analyzed by Western blotting for ERK1/2, p38MAPK and JNK phosphorylation (n = 3). [C] Cells were treated with p38 MAPK inhibitor [SB202190, n = 4] before treatment of AICAR for 12 hr. Cell lysate or media were analyzed by Western blotting for indicated proteins and [D] Fet-A levels, as a ratio to GAPDH were determined. [E] Cells were treated with AMPK inhibitor [Comp C, n = 4] before treatment of AICAR for 12hr. Cell lysates were analyzed by Western blotting for phosphorylated p38 MAPK (pP38MAPK) and [F] pP38MAPK levels, as a ratio to GAPDH were determined. [G] Knockdown of p38 MAPK was performed using MAPK14 [p38 MAPK] small interfering RNA [siRNA] in HepG2 cells. Following AICAR treatment for 12 h, cell lysates were analyzed by Western blotting for expression of p38 MAPK, phosphorylated p38 MAPK, Fet-A, and pAMPK. [H] Efficiency of p38MAPK siRNA in HepG2 cells were determined by immunoblotting transfected cells for p38MAPK and levels were expressed as a ratio to GAPDH. [I] Effect of AICAR on Fet-A expression in scrambled or p38MAPK siRNA transfected cells were determined by expressing Fet-A levels, as a ratio to GAPDH (n = 4). [J] Effect of protein synthesis inhibitors, cycloheximide and puromycin, were compared with anisomycin, also a protein synthesis inhibitor, for effects on Fet-A and phosphorylated p38 MAPK expression (n = 3). Data are shown as Means ± SEM. P values were determined accordingly by either by unpaired two-tailed test (E) or one-way ANOVA followed by Tukey’s multiple comparison tests (C-F). * Indicates p < 0.05.

Article Snippet: To silence the p38 MAPK gene expression, Mapk14 mouse siRNA oligo duplex (Locus ID 26416) was used (OriGene).

Techniques: Incubation, Western Blot, Phospho-proteomics, Expressing, Concentration Assay, Knockdown, Small Interfering RNA, Transfection, Two Tailed Test, Comparison

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: (a) TGFβ stimulates p38α activity, inhibited by SB2036580 or β-LGND. Bar graph is the mean±SD from 3 exps combined. *p < 0.05 vs. control, +p < 0.05 for TGFβ vs same + SB2036580 or β-LGND. (b) TGFβ inhibits KLF15 mRNA and protein in cardiomyocytes, blocked by the p38 antagonist SB2036580 (0.1μM) (c) TAK1 activating phosphorylation is stimulated by AngII or TGFβ, inhibited by β-LGND. *p<0.05 vs. control, + p<0.05 for TGFβ or AngII vs same plus β-LGND, n=3 exps. (d) TAK1 siRNA diminishes TGFβ or AngII-stimulated p38α activity. The latter was seen as phosphorylation at tyrosine182. *p<0.05 vs control, +p<0.05 for TGFβ or AngII vs same + β-LGND, n=3 exps. TAK1 siRNA validation is also shown. (e) Flow cytometry analysis of β-LGND inhibition of phospho-kinases due to cAMP/PKA. *p<0.05 for control vs. AngII-stimulated phospho-TAK1, phospho-p38α, or KLF15 proteins. +p<0.05 for AngII vs AngII + β-LGND, ++p<0.05 for AngII + β-LGND vs same + either H-89 (PKA inhibitor) or RP-8-Br-cAMP (cAMP inhibitor), n=3 exps.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Activity Assay, Control, Phospho-proteomics, Biomarker Discovery, Flow Cytometry, Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: AngII acting through TGFβ stimulates a TAK1-p38α kinase axis that inhibits KLF15 expression and nuclear localization of the protein. This contributes to increased gene expression and cardiomyocyte hypertrophy. ERβ acting through protein kinase A opposes TAK1-p38α activation. This restores KLF15 abundance and nuclear localization, contributing in part to inhibition of AngII-induced gene expression and cardiomyocyte hypertrophy.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Expressing, Gene Expression, Activation Assay, Inhibition

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 8. Effects of GADD45a on the olaquindox-induced MAPKs pathways (A) HepG2 and HepG2-iGADD45a cells were exposed to 0, 200, 400 and 800 µg/mL olaquindox for 24 h. Expression of p-JNK/JNK, p-p38/p38, p-ERK/ERK was detected by western blotting. GAPDH was used for loading control. The densitometric analysis results of p-JNK/JNK, p-p38/p38 and p-ERK/ERK were shown on (B–D). All data and results were represented as means ± SD from three or more independent experiments. (* p < 0.05, ** p < 0.01, compared with the control; # p < 0.05, ## p < 0.01, compared to HepG2 groups).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Expressing, Western Blot, Control

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 9. Effects of JNK/p38 pathways on olaquindox-induced cell death. HepG2 cells were pretreated with SP600125 or SB203580 for 1 h, followed to replace with or without olaquindox at the final concentration of 400 µg/mL for additional 24 h. (A) Cell viability was measured by MTT method; (B) Morphologic observation (400×); (C) Cell apoptosis was measured by staining with Hoechst 33342 (400×); (D) Quantification of apoptosis in HepG2 cells. Bright blue cells were counted as apoptotic cells from five independent microscopic fields. Results were presented as mean ± SD, from three independent experiments. (* p < 0.05, compared to the olaquindox alone group).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Concentration Assay, Staining

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 10. JNK/p38 pathways played protect role in olaquindox-induced DNA damage. (A) HepG2 cells were treated with olaquindox (400 µg/mL) for 4 h after preincubation with SP600125 or SB203580 for 1 h; (B) % tail DNA; (C) tail length; (D) tail moment; (E) HepG2 cells were treated with olaquindox (400 µg/mL) for 24 h after preincubation with SP600125 or SB203580 for 1 h. Cells were observed under a Leica inverted fluorescence microscope (400×). 1000 binucleated cells were recorded from each experiment and three independent experiments were carried out. All results were presented as mean ± SD. (** p < 0.01, compared with the control group; # p < 0.05, compared to olaquindox group).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Microscopy, Control

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 11. JNK/p38 pathways inhibited olaquindox-induced S-phase arrest. (A) HepG2 cells were treated with 400 µg/mL of olaquindox for 24 h after preincubation with SP600125 or SB203580 for 1 h; (B) Effect of HepG2 cells after preincubation with SP600125 or SB203580 on cell cycle distribution; (C) Effect of HepG2 cells treated with 400 µg/mL of olaquindox for 24 h after preincubation with SP600125 or SB203580 for 1 h on cell cycle distribution. Samples were measured by a flow cytometer. A minimum of 20,000 cells were recorded from each experiment. All results were presented as mean ± SD and three independent experiments were carried out. (** p < 0.01, compared with the control; # p < 0.05, compared to HepG2 groups).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Cytometry, Control

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 12. The relationship between GADD45a and JNK/p38 pathways in olaquindox response in the HepG2 cells. (A,B) Effects of SP600125 and SB203580 on the protein level of GADD45a in HepG2 cells. Cells were pre-treated with these inhibitors for 1 h before olaquindox treatment; (C) Effects of SP600125 and SB203580 on the cell viability both in HepG2 and HepG2-iGADD45a cells. All data and results were represented as means ± SD from three or more independent experiments. (* p < 0.05, ** p < 0.01, compared with the control; # p < 0.05, compared to HepG2 groups).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Control

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 13. A schematic diagram of the protective effect of GADD45a on olaquindox induced DNA damage and cell cycle arrest in HepG2 cells. Olaquindox could induce the exceeding reactive oxygen species (ROS) in the metabolic process, subsequently induced the DNA damage and activated GADD45a and JNK/p38 pathways. The activation of JNK/p38 may play a protective role in olaquindox induced cell death, DNA damage and cell cycle arrest. GADD45a could effective inhibit olaquindox induced DNA damage and S-phase arrest in HepG2 cell and JNK/p38 pathways may partly contribute to GADD45a regulated olaquindox-induced DNA damage and S-phase arrest.

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Activation Assay

Journal: Molecular medicine reports

Article Title: Fluoxetine protects against methamphetamine‑induced lung inflammation by suppressing oxidative stress through the SERT/p38 MAPK/Nrf2 pathway in rats.

doi: 10.3892/mmr.2016.6072

Figure Lengend Snippet: Figure 4. SERT protein expression and P‑38 MAPK phosphorylation in rat lungs in the different groups. (A) SERT protein expression and (B) the rela tive p‑p38 MAPK expression in lungs were determined and quantified. The data are expressed as the mean ± standard deviation (n=6; *P<0.05, **P<0.01 vs. the control group; +P<0.05 vs. the MA group). SERT, serotonin trans porter; MAPK, mitogen activated protein kinases; MA, methamphetamine; F2, fluoxetine at 2 mg/kg; F10, fluoxetine at 10 mg/kg.

Article Snippet: The membranes were probed with primary mouse monoclonal anti-β‐actin (1:2,000; cat. no. sc‐130300; Santa Cruz Biotechnology, Inc., Dallas, USA) and α‐tubulin 1:2,000; cat. no. 66031; ProteinTech Group, Inc., Chicago, IL, USA) and rabbit polyclonal anti‐SERT (1:200; cat. no. bs 1893R), IL‐6 (1:200; cat. no. bs 0379R), tumor necrosis factor‐α (TNF-α; 1:200; cat. no. bs 0078R), human heme oxygenase‐1 (HO‐1; 1:400; cat. no. bs 2075R) (Biosynthesis Biotechnology Co. Ltd.), anti‐Nrf2 (1:600; cat. no. 16396‐1‐AP; ProteinTech Group, Inc.), p38 (1:600; cat. no. ZS‐7149), and phosphorylated (p)‐p38 (1:600; cat. no. ZS‐101759; Zhongshan Golden Bridge Biotechnology Co., Ltd.) overnight at 4 ̊C.

Techniques: Expressing, Phospho-proteomics, Standard Deviation, Control