Journal: Experimental and Therapeutic Medicine

Article Title: Proliferation, apoptosis and invasion of human lung cancer cells are associated with NFATc1

doi: 10.3892/etm.2022.11748

Figure Lengend Snippet: Knockdown of NFATc1 expression induces cell apoptosis whilst inhibiting the MAPK and epithelial-to-mesenchymal transition signaling pathways in lung cancer cells. (A) Protein expression levels of Bax, cleaved caspase-3, CDK4 and c-Myc in both cell lines were examined by western blot analysis. GAPDH was used as the loading control. (B) Protein expression levels of E-cadherin and N-cadherin were examined by western blotting. GAPDH was used as the loading control. (C) Protein expression levels of ERK and p38, along with their corresponding phosphorylation levels were detected by western blotting. GAPDH was used as the loading control. * P<0.05, ** P<0.01 and *** P<0.001 vs. shCtrl. Ctrl, control; NFATc1, nuclear factor of activated T cells c1; p-, phosphorylated; sh, short hairpin RNA.

Article Snippet: The blots were blocked in 5% skimmed milk for 1 h at room temperature, followed by incubation with primary antibodies against ERK (cat. no. 9107; 1:2,000; Cell Signaling Technology, Inc.), E-cadherin (cat. no. 14472; 1:500; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK (cat. no. 4376; 1:1,000; Cell Signaling Technology, Inc.), N-cadherin (cat. no. ab18203; 1:500; Abcam), Bax (cat. no. ab32503; 1:2,000; Abcam), p38 (cat. no. 8690; 1:3,000; Cell Signaling Technology, Inc.), cleaved caspase-3 (cat. no. 9664; 1:500; Cell Signaling Technology, Inc.), p-p38 (cat. no. 4631; 1:500; Cell Signaling Technology, Inc.), c-Myc (cat. no. ab32072; 1:1,000; Abcam), CDK4 (cat. no. ab137675; 1:1,000; Abcam), NFAT2 (cat. no. ab2796; 1:1,000; Abcam) and GAPDH (cat. no. ab37168; 1:5,000; Abcam) at 4 ̊C with gentle shaking overnight.

Techniques: Knockdown, Expressing, Protein-Protein interactions, Western Blot, Control, Phospho-proteomics, shRNA

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hibiscus sabdariffa L.: A potent natural neuroprotective agent for the prevention of streptozotocin-induced Alzheimer's disease in mice.

doi: 10.1016/j.biopha.2020.110303

Figure Lengend Snippet: Fig. 6. Effects of Hibiscus extracts and celecoxib on the activity of p38MAPK (A), and COX-2 (B), and on concentration of mPGES-1 (C) and PGE2 (D). p38MAPK and COX-2 levels in celecoxib (30 mg/kg), white and red Hibiscus (200 mg/kg each) groups were decreased compared to STZ (3 mg/kg, ICV) group and increased compared to the normal group. mPGES-1 and PGE2levels were normalized in the three treated groups. Panel 4 represents immunoblot analysis shown in Fig. 6.A. Panel 5 represents immunoblot analysis shown in Fig. 6.B. Data are expressed as means ± S.D. The significance of the dif- ference between means was tested by ANOVA followed by Tukey Kramer multiple compar- isons test. * P < 0.05 vs normal; @ P < 0.05 vs STZ-treated group; # P < 0.05 vs celecoxib. n = 6 mice, DF = 29. For p38MAPK: F = 354.1, R2 = 0.9827. For COX-2: F = 350.5, R2 = 0.9825. For m-PGES-1: F = 430.4, R2 = 0.9857. For PGE2: F = 354.3, R2 = 0.9827.

Article Snippet: The antibodies used were rabbit BACE1 monoclonal [EPR19523] (abcam, ab183612), mouse Cox-2 (H-3) monoclonal (Santa cruze Inc, sc-376861), rabbit PSENEN polyclonal (Boster Biological Technology, A04504) and rabbit p38 MAPK polyclonal (Biorbyt Ltd., orb127559).

Techniques: Activity Assay, Concentration Assay, Western Blot

Journal: Disease Markers

Article Title: Aldolase A Promotes Colorectal Cancer Progression through Targeting COPS6 and Regulating MAPK Signaling Pathway

doi: 10.1155/2023/1702125

Figure Lengend Snippet: Graphical abstract. By binding to and targeting COPS6, aberrantly expressed ALDOA promoted the EMT process and activated the ERK1/2 and P38 signaling pathways, ultimately accelerating CRC cell proliferation and metastasis.

Article Snippet: The following antibodies were used: ALDOA (mouse monoclonal; cat. no. sc-390733; 1 : 1000 dilution; Santa Cruz, CA, USA), HRP-conjugated DYKDDDDK Tag (monoclonal; cat. no. HRP-66008; 1 : 5000 dilution; Proteintech Group), ALDOB (rabbit polyclonal; cat. no. 18065-1-AP; 1 : 1000 dilution; Proteintech Group), ALDOC (rabbit polyclonal; cat. no. 14884-1-AP; 1 : 1000 dilution; Proteintech Group), E-cadherin (rabbit monoclonal; cat. no. 3195; 1 : 1000 dilution; Cell Signaling Technology), N-cadherin (rabbit monoclonal; cat. no. 13116; 1 : 1000 dilution; Cell Signaling Technology), vimentin (rabbit monoclonal; cat. no. 5741; 1 : 1000 dilution; Cell Signaling Technology), p38 (rabbit monoclonal; cat. no. 8690; 1 : 1000 dilution; Cell Signaling Technology), p-p38 (rabbit monoclonal; cat. no. 8632; 1 : 1000 dilution; Cell Signaling Technology), ERK1/2 (rabbit monoclonal; cat. no. 4695; 1 : 1000 dilution; Cell Signaling Technology), p-ERK1/2 (rabbit monoclonal; cat. no. 4376; 1 : 1000 dilution; Cell Signaling Technology), ACTB (rabbit monoclonal; cat. no. AC038; 1 : 10000 dilution; ABclonal Technology, Wuhan, China), GAPDH (rabbit monoclonal; cat. no. 60004-1-Ig; 1 : 10000 dilution; Proteintech Group), lamin B1 (rabbit polyclonal; cat. no. 12987-1-AP; 1 : 2000 dilution; Proteintech Group), PKM (rabbit polyclonal; cat. no. 10078-2-AP; 1 : 1000 dilution; Proteintech Group), HSP90AB (rabbit polyclonal; cat. no. RK05737; 1 : 1000 dilution; ABclonal Technology), CSN6 (mouse monoclonal; cat. no. sc-393023; 1 : 1000 dilution; Santa Cruz, CA, USA), caspase-3 (rabbit monoclonal; cat. no. 9662; 1 : 1000 dilution; Cell Signaling Technology), and cleaved caspase-3 (rabbit monoclonal; cat. no. 9661; 1 : 1000 dilution; Cell Signaling Technology).

Techniques: Binding Assay, Protein-Protein interactions

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: ( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p < 0.05 compared to the negative control group.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Inhibition, Western Blot, Phospho-proteomics, In Vitro, Migration, Negative Control, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon up-regulates p38β expression in hepatic cells. Pokemon was delivered by expression plasmid pcDNA3.1(−)-Pokemon with pcDNA3.1(−) as a negative control. Pokemon silencing was triggered by si-RNA. ( a ) Targeted expression or silencing of Pokemon in HepG2 cells. Cells were collected at 60 h after transfection or silencing (Left panel). And the quantification of western blot data was displayed on the Right panel; ( b ) Real-time quantitative polymerase chain reaction (qPCR) at 48 h after transfection in HepG2 cells. Upper panel: Ectopic expression of Pokemon; lower panel: Silencing of Pokemon; ( c ) Ectopic expression of Pokemon in HL7702 cells. Upper panel: Western blot in which cells were lysed and total proteins were collected at 36, 48, 60 and 72 h, respectively. Lower panel: Real-time qPCR at 48 and 60 h after transfection; ( d ) Silencing of Pokemon in BEL7404 cells. Upper panel: Western blot; lower panel: Real-time qPCR. * p < 0.05 compared to the negative control. N.C. means negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Negative Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon stimulates p38β promoter activity. ( a ) ChIP assays in HepG2 and BEL7404 cells. Protein-DNA complexes are immunoprecipitated either with anti-Pokemon antibody or anti-IgG as negative control, followed by PCR with primers specific to p38β promoter sequence and agarose-gel electrophoresis for visualization. Total lysates were used as the input samples and positive control; ( b ) Dual luciferase reporter assay. Luciferase activities were normalized to Renilla activity. Y axis stands for the relative fold changes of activity as the pcDNA3.1(−)-Pokemon plasmid increases. * p < 0.05 compared to the negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Activity Assay, Immunoprecipitation, Negative Control, Sequencing, Agarose Gel Electrophoresis, Positive Control, Luciferase, Reporter Assay, Plasmid Preparation

Journal: Drug Design, Development and Therapy

Article Title:

Resveratrol downregulates TNF-α-induced monocyte chemoattractant protein-1 in primary rat pulmonary artery endothelial cells by P38 mitogen-activated protein kinase signaling

doi: 10.2147/dddt.s184785

Figure Lengend Snippet: Figure 8 MCP-1 mRNA expression mediated by the p38 MAPK-dependent signaling pathway in pulmonary artery cells. The levels of MCP-1 mRNA expression were measured using real-time PCR assay at 8 hrs time point in pulmonary artery cells (mean ± SD, n=6 each group). ★p<0.05: RPAs-TNF-α vs RPAs-control, RPAs-TNF-α + resveratrol, RPAs-TNF-α + SB203580 and RPAs-TNF-α + resveratrol + SB203580groups. #p<0.05: RPAs-TNF-α + resveratrol and RPAs-TNF-α + SB203580 vs RPAs-control and RPAs-TNF-α + resveratrol + SB203580; **p<0.05: RPAs-TNF-α + resveratrol + SB203580 vs RPAs-control. Abbreviation: RPAs, rat pulmonary artery endothelial cells.

Article Snippet: Reagents A polyclonal rabbit anti-rat MCP-1 antibody of Abcam (http://www.abcam.com/), rabbit polyclonal antibodies for total and phospho-p38 MAPK [Cell Signaling Technology (http://www.cellsignal.com/)] and SB203580 and resveratrol of [Sigma–Aldrich (http://www.sigmaaldrich.com)] were purchased from the above companies.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Cell

Article Title: Ribosome collisions trigger general stress responses to regulate cell fate

doi: 10.1016/j.cell.2020.06.006

Figure Lengend Snippet: (A) Immunoblots for phosphorylation of eIF2α and mTOR in MCF10A cells treated with ANS (0.1–100 mg/L, 0.5 h). Total eIF2α and β-actin as loading controls. (B) eIF2α phosphorylation induced by intermediate doses of ANS (0.5 mg/L, 0.5 h) in MCF10A cells pretreated with GCN2 inhibitor (A-92), PERK inhibitor (GSK 2606414), or p38 inhibitor (BIRB 796). (C) eIF2α phosphorylation induced by ANS in WT or ZAK KO MCF10A cells. (D) eIF2α phosphorylation in WT, ZAK KO, or ZAK KO MCF10A complemented with ZAKα, ZAKα -K45M, or ZAKβ under ANS treatment (0.5 mg/L, 0.5 h). (E) Polysome profiles from DSP-crosslinked WT and ZAK KO MCF10A cells. Fractions were analyzed by immunoblotting with indicated antibodies.

Article Snippet: Antibodies for phospho-p38 (Thr180/Tyr182, 9211), p38 (9212), phospho-JNK (Thr183/Tyr185, 4668), β-actin (51255), phospho-mTOR (Ser2448, 2971) and eIF2α (9722S) were from Cell Signaling Technology.

Techniques: Western Blot, Phospho-proteomics

Journal: Oncogene

Article Title: Transcriptional factor HBP1 targets P16(INK4A), upregulating its expression and consequently is involved in Ras-induced premature senescence.

doi: 10.1038/onc.2010.252

Figure Lengend Snippet: Figure 1 p16INK4A and HBP1 participate in Ras-induced premature senescence. (a) p16INK4A protein level is increased in Ras- transfected WI-38 cells. Levels of HBP1, p16INK4A, p-p38, p38 and FLAG-Ras were determined by western blot in Ras-transfected WI-38 cells with or without p16INK4A knockdown. Ras increases p-p38, HBP1 and p16INK4A protein levels in WI-38 cells. (b) p16INK4A is required for Ras-induced premature senescence. WI-38 cells (PD10) were transfected with pSR (as control), Ras or Ras with p16shRNA. Cells were then stained for SA-b-gal at day 14 after transfection. Significant SA-b-gal staining is apparent in the Ras- transfected cells, whereas few cells are stained in Ras-transfected with p16INK4A knockdown cells. (c) The percentage of cells positive for SA-b-gal in WI-38 cells transfected with pSR (as control), Ras or Ras with p16shRNA. Values are means±standard deviations for triplicates. At least 300 cells were counted for each sample. (d) HBP1 and p16INK4A proteins increase during replicative senescence. Levels of HBP1 and p16INK4A in young (PD10) and senescent (PD50) cells are shown. The level of p38 MAPK protein is used as a loading control. (e) HBP1 protein level is increased in Ras-transfected WI-38 cells. Levels of HBP1, p16INK4A, p38 and FLAG-Ras were determined by western blot in Ras-transfected WI-38 cells with or without HBP1 knockdown. Ras increases HBP1 and p16INK4A protein levels in WI-38 cells. p16INK4A protein level decreases in HBP1 knockdown cells. (f) The percentage of cells positive for SA-b-gal in WI-38 cells transfected with pSR (as control), Ras or Ras with HBP1shRNA. Values are means±standard deviations for triplicates. At least 300 cells were counted for each sample.

Article Snippet: The primary antibodies used were for HBP1 (N-20X; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p16INK4A (C-20; Santa Cruz Biotechnology), p38 (A-12; Santa Cruz Biotechnology), phospho-p38 (no. 9211; Cell Signaling, Danvers, MA, USA) and HA.11 antibody (Covance, San Diego, CA, USA).

Techniques: Transfection, Western Blot, Knockdown, Control, Staining

Journal: Oncogene

Article Title: Transcriptional factor HBP1 targets P16(INK4A), upregulating its expression and consequently is involved in Ras-induced premature senescence.

doi: 10.1038/onc.2010.252

Figure Lengend Snippet: Figure 2 HBP1 regulates p16INK4A expression. (a) HBP1 increases p16INK4A mRNA level. p16INK4A mRNA level was measured by RT–PCR in WI-38 cells (PD15) transfected with pBabe-HBP1 or pBabe (as control). (b) p16INK4A knockdown by introduction of shRNA. Levels of p16INK4A and p38 MAPK were determined by western blot in WI-38 cells (PD15) transfected with HBP1 or control vector with or without introduction of p16shRNA. (c) p16INK4A knockdown prevents HBP1-induced SA-b-gal expression. The percentages of cells positive for SA-b-gal in WI-38 cells transfected with p16shRNA in the presence of constitutively HBP1 were measured. Values are means±standard deviations for triplicates. At least 300 cells were counted for each sample. (d) HBP1 knockdown decreases p16INK4A mRNA level. HBP1 and p16INK4A mRNA levels were measured by RT–PCR with WI-38 cells (PD40) transfected with pSuper.retro (as control) or pHBP1shRNA. (e) HBP1 knockdown decreases p16INK4A protein level. HBP1 and p16INK4A protein levels were measured by western blot in WI-38 cells (PD40) transfected with pSuper.retro or pHBP1shRNA. (f) HBP1 knockdown prevents HBP1-induced S-phase inhibition. WI-38 cells (PD40) transfected with pSuper.retro or pHBP1shRNA were labeled with BrdU, and BrdU-positive nuclei were stained at day 14 after transfection. The percentages of cell nuclei positive for BrdU are plotted in the graph. Values are means±standard deviations for three independent experiments. At least 300 cells were counted for each sample.

Article Snippet: The primary antibodies used were for HBP1 (N-20X; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p16INK4A (C-20; Santa Cruz Biotechnology), p38 (A-12; Santa Cruz Biotechnology), phospho-p38 (no. 9211; Cell Signaling, Danvers, MA, USA) and HA.11 antibody (Covance, San Diego, CA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Knockdown, shRNA, Western Blot, Plasmid Preparation, Inhibition, Labeling, Staining