Journal: Theranostics

Article Title: Dioscin Exerts Protective Effects Against Crystalline Silica-induced Pulmonary Fibrosis in Mice

doi: 10.7150/thno.20270

Figure Lengend Snippet: Dioscin inhibits macrophages and fibroblasts from secreting pro-inflammatory cytokines. (A) Western blot analyses of ASK1 phosphorylation and downstream MAPK proteins (p38 and JNK/SAPK) in LPS-primed RAW 264.7 macrophages treated with CS (50 μg/cm 2 ) together with different does of dioscin for 6 h. Anisomycin was used to reverse dioscin's effect (2 ng/mL). Quantification of each protein level relative to β-Actin is shown below each band . (B-D) The levels of phospho-ASK1, phospho-p38 and phosphor-JNK were normalized to those of β-Actin (n=3). (E-H) ELISA analyses of pro-inflammation cytokines in the culture medium of different treated macrophages at 6 h. (E) MCP-1, (F) IL-1β, (G) IL-6, (H)TNF-α (n=3). Data are the mean of three independent experiments (n=3). (I) Western blot analyses of ASK1 phosphorylation and downstream MAPK proteins (p38 and JNK/SAPK) in TNF-α- (25 ng/mL) treated NIH-3T3 fibroblasts together with different does of dioscin for 30 min. Quantification of each protein level relative to β-Actin is shown below each band . (J-L) The levels of phospho-ASK1, phospho-p38 and phosphor-JNK were normalized to those of β-Actin (n=3). (M) qPCR analysis of Il-6 mRNA levels in TNF-α- (25 ng/mL) treated fibroblasts together with different concentrations of dioscin for 12 h (n=3). *, P<0.05; **, P<0.01. Error bars indicate the mean ± SD. The experiment was performed twice with similar results.

Article Snippet: Anisomycin (2 ng/mL, Cell Signaling Technology), activator of JNK and p38 MAPKs, was used to test the biological effect of dioscin on the MAPKs.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Dioscin Exerts Protective Effects Against Crystalline Silica-induced Pulmonary Fibrosis in Mice

doi: 10.7150/thno.20270

Figure Lengend Snippet: Dioscin regulated the TGF-β1-Samd3 signaling pathway and regulatory T cell functions. (A, B) Western blot analysis and quantification of TGF-β1 in lungs of dioscin-treated mice at 56 d (n=3). β-Actin was used as the loading control. Quantification of TGF-β1 levels relative to β-Actin is shown below the band. (C) qPCR analysis of Tgf-β1 in the lung tissues (n=6-8 per group). *, P<0.05; **, P<0.01. Error bars indicate the mean ± SD. The experiment was performed in triplicate. (D, E) Western blot analysis and quantification of Smad3 signaling in the lungs of treated mice at 56 d (n=3). β-Actin was used as the loading control. Quantification of p-Smad3 levels relative to β-Actin is shown below the band. (F, G) Western blot analysis of p-Smad3 and Smad3 in NIH-3T3 fibroblast, 30 min after TGF-β1 (5 ng/mL) and dioscin treatment. Quantification of p-Smad3 levels relative to β-Actin is shown below the band. (H) Schematic design of transwell experiment: RAW 264.7 macrophages were primed by LPS (25 ng/mL) for 3 h, and then treated with CS (50 μg/cm 2 ) together with dioscin or anisomycin (2 ng/mL) for 24 h in the lower plate, while NIH-3T3 fibroblasts were attached in the upper chamber. After that, the upper chamber was inserted back to the lower plate, and macrophages and fibroblasts were co-cultured for another 24 h. Then, the mRNA expressions of Col1a1 (I) and α-SMA (J) in NIH-3T3 fibroblast were determined by qPCR (n=3). (K, L) Percentage of CD4+CD25+FoxP3+ cells (regulatory T cells) in hilar lymph nodes was assayed by FACS analysis (n=5). *, P<0.05; **, P<0.01. Error bars indicate the mean ± SD.

Article Snippet: Anisomycin (2 ng/mL, Cell Signaling Technology), activator of JNK and p38 MAPKs, was used to test the biological effect of dioscin on the MAPKs.

Techniques: Western Blot, Cell Culture

Journal: BMC Cell Biology

Article Title: A role for p38 MAPK in the regulation of ciliary motion in a eukaryote

doi: 10.1186/1471-2121-12-6

Figure Lengend Snippet: Sequence alignment and domain analysis of S. mansoni p38 MAPK . The S. mansoni putative p38 MAPK sequence (XP_002571000) was aligned with those for S. japonicum MAPK14a, Homo sapiens (p38 MAPKα, also known as MAPK14 isoform 1), Drosophila melanogaster (p38a MAPK and p38b MAPK), Caenorhabditis elegans (p38 MAPK family member, PMK1) and Danio rerio (MAPK 14a); accession numbers are shown. Multiple alignments were achieved using Geneious Pro 4.85 with Blosum62 cost matrix and default settings; shading of residues: black = 100% similar, dark grey = 80-100% similar, light grey = 60-80% similar and white < 60% similar. The ATP binding site, kinase interacting motif (KIM) docking site, and activation loop are indicated by coloured arrows. Within the activation loop, the conserved Thr-Gly-Tyr (TGY) phosphorylation motif is highlighted by the yellow box, with the phosphorylated residues (Thr and Tyr) central to kinase activation indicated with green asterisks; the substrate binding site is shown by the orange box. Sequence highlighted by the blue box is that used to generate the monoclonal anti-phospho p38 MAPK antibodies. The residues within the ATP binding site that are known to confer specificity and sensitivity of SB 230580 towards p38 MAPK are highlighted by the red box with the residue (Thr) most responsible for p38 MAPK inhibition indicated by the red asterisk; red crosses denote other known interaction sites between SB 203580 and p38 MAPK (see text for further details).

Article Snippet: The effect of the p38 MAPK activator anisomycin on p38 MAPK phosphorylation (activation) in S. mansoni was assessed by western blotting using anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies (Cell Signalling Technology, New England Biolabs, Hitchin, UK) that recognize only the phosphorylated (activated) form of the enzyme.

Techniques: Sequencing, Binding Assay, Activation Assay, Inhibition

Journal: BMC Cell Biology

Article Title: A role for p38 MAPK in the regulation of ciliary motion in a eukaryote

doi: 10.1186/1471-2121-12-6

Figure Lengend Snippet: Biochemical characterization of S. mansoni p38 MAPK . (A) Immunodetection of phosphorylated S. mansoni p38 MAPK. Protein from astrocytoma (U251 MG) cells (lane 1), 900 freshly-hatched swimming miracidia (lane 2), or an adult worm pair (lane 3) was processed for western blotting using anti-phospho p38 MAPK antibodies. (B) Activated S. mansoni p38 MAPK phosphorylates activating transcription factor-2 (ATF-2), and SB 203580 inhibits p38 MAPK activity. p38 MAPK from adult worm pairs was immunoprecipitated using immobilized anti-phospho p38 MAPK antibodies and the immunoprecipitated protein used in an in vitro kinase assay to phosphorylate ATF-2 either in the presence of SB 203580 (1-5 μM) or DMSO (D+), or with neither (D-). Phosphorylation of ATF-2 by immunoprecipitated p38 MAPK was assessed by western blotting using anti-phospho ATF-2 antibodies. (C) Anisomycin activates p38 MAPK in S. mansoni miracidia. Freshly-hatched miracidia were exposed to anisomycin (20 μM) for various durations and phosphorylation of p38 MAPK in miracidia detected by western blotting with anti-phospho p38 MAPK antibodies; blots were also probed with anti-actin antibodies to demonstrate equal protein loading between lanes. Results shown in (A-C) represent those obtained in at least two independent experiments.

Article Snippet: The effect of the p38 MAPK activator anisomycin on p38 MAPK phosphorylation (activation) in S. mansoni was assessed by western blotting using anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies (Cell Signalling Technology, New England Biolabs, Hitchin, UK) that recognize only the phosphorylated (activated) form of the enzyme.

Techniques: Immunodetection, Western Blot, Activity Assay, Immunoprecipitation, In Vitro, Kinase Assay

Journal: BMC Cell Biology

Article Title: A role for p38 MAPK in the regulation of ciliary motion in a eukaryote

doi: 10.1186/1471-2121-12-6

Figure Lengend Snippet: Activated p38 MAPK localizes to cilia in S. mansoni miracidia . (A) Scanning electron micrograph of the anterior of a freshly-hatched miracidium showing numerous cilia (c), and sensory endings (se) associated with the semi-spherical terebratorium (tb). (B - J) Immunolocalization of activated p38 MAPK (green) in S. mansoni miracidia following fixing and staining of parasites with anti-phospho p38 MAPK primary antibodies. (B) Freshly-hatched miracidium incubated without primary antibodies but with secondary antibodies alone (negative control). (C) Freshly-hatched swimming miracidium displaying weak patchy staining only localized adjacent to the tegument (tg) in areas corresponding to the cilia. (D) Serial optical z-section of a miracidium treated with 20 μM anisomycin for 30 min showing p38 MAPK activity in the regions occupied by cilia. (E and F) High power, optically zoomed, z-sections through discrete regions of cilia/tegument to capture the miracidium surface in one plane with (E) showing p38 MAPK localized to the shafts of the cilia (arrowed) and regions proximal to the tegument, and (F) showing co-localization with cilia (red) detected using anti-acetylated tubulin antibodies. (G) Miracidium treated with 20 μM anisomycin for 30 min for direct comparison with (C). (H) Autofluorescence (red) of S. mansoni egg containing (I, and J overlay) miracidium (m) with p38 MAPK activity in regions occupied by cilia. (K) Scanning electron micrograph of S. mansoni egg revealing common position of rupture during hatching, and correlating to rupture in H-J. Z-axis projections are shown in maximum pixel brightness mode. Bars: A, E and F = 2 μm; B-D, and G = 15 μm; H-K = 25 μm.

Article Snippet: The effect of the p38 MAPK activator anisomycin on p38 MAPK phosphorylation (activation) in S. mansoni was assessed by western blotting using anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies (Cell Signalling Technology, New England Biolabs, Hitchin, UK) that recognize only the phosphorylated (activated) form of the enzyme.

Techniques: Staining, Incubation, Negative Control, Activity Assay

Journal: BMC Cell Biology

Article Title: A role for p38 MAPK in the regulation of ciliary motion in a eukaryote

doi: 10.1186/1471-2121-12-6

Figure Lengend Snippet: p38 MAPK activation and inhibition affects S. mansoni miracidia swim velocity . Freshly-hatched swimming miracidia were collected and incubated with (A) SB 203580 (1 μM) or (B) anisomycin (20 μM) in spring water, or spring water alone, and the effects on miracidia swim behaviour over 60 min captured by digital video microscopy. Calculation of swim velocities (mm/s) for 30 miracidia for each treatment and each time point was achieved using ImageJ and mean swim speeds (± SEM) were calculated. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: The effect of the p38 MAPK activator anisomycin on p38 MAPK phosphorylation (activation) in S. mansoni was assessed by western blotting using anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies (Cell Signalling Technology, New England Biolabs, Hitchin, UK) that recognize only the phosphorylated (activated) form of the enzyme.

Techniques: Activation Assay, Inhibition, Incubation, Microscopy

Journal: BMC Cell Biology

Article Title: A role for p38 MAPK in the regulation of ciliary motion in a eukaryote

doi: 10.1186/1471-2121-12-6

Figure Lengend Snippet: p38 MAPK activation accelerates the liberation of ciliated plates in transforming S. mansoni larvae . Cultures of larvae were exposed to (A) SB 203580 or (B) anisomycin, during transformation in vitro and the proportion of fully-ciliated parasites determined by microscopic examination over 50 h, compared to CBSS controls. Values shown are means (± SEM, n = 90 for each time point and each treatment from three independent experiments). *P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: The effect of the p38 MAPK activator anisomycin on p38 MAPK phosphorylation (activation) in S. mansoni was assessed by western blotting using anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies (Cell Signalling Technology, New England Biolabs, Hitchin, UK) that recognize only the phosphorylated (activated) form of the enzyme.

Techniques: Activation Assay, Transformation Assay, In Vitro

Journal: BioMed Research International

Article Title: Effect of TRPV4-p38 MAPK Pathway on Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion

doi: 10.1155/2016/6978923

Figure Lengend Snippet: The effects of the reagents on CCD-induced mechanical allodynia. (a–d) The PWMTs of CCD rats (4 days after operation) 1, 2, 4, and 8 h after RR, 4 α -PDD, SB203580, and anisomycin intrathecal injections ( n = 6; the data are expressed as means ± SEMs); ∗ P < 0.05 and ∗∗ P < 0.01 compared ipsilaterally with the saline group; one-way ANOVA followed by Tukey's post hoc test.

Article Snippet: Four days after CCD surgery, the TRPV4 inhibitor Ruthenium Red (RR, Sigma, Germany), the TRPV4 agonist 4 α -PDD (CST, USA), the p38 inhibitor SB203580 (CST, USA), or the p38 agonist anisomycin (CST, USA) was given to the experimental groups at the recommended concentrations via intrathecal injection.

Techniques:

Journal: BioMed Research International

Article Title: Effect of TRPV4-p38 MAPK Pathway on Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion

doi: 10.1155/2016/6978923

Figure Lengend Snippet: Distribution changes of the p38-positive neurons in DRG tissue. (a–f) p38 immunohistochemical staining of the DRG neurons in the control, CCD, CCD+RR 10 nmol/L, CCD+4 α -PDD 10 nmol/L, CCD+SB203580 20 μ mol/L, and CCD+anisomycin 25 μ g/mL groups. Scale bars: 100 μ m. (g) The analysis of the p38-positive neurons (shown as means ± SEMs); ∗ P < 0.05 and ∗∗ P < 0.01 compared with controls; ## P < 0.01 compared with the CCD group.

Article Snippet: Four days after CCD surgery, the TRPV4 inhibitor Ruthenium Red (RR, Sigma, Germany), the TRPV4 agonist 4 α -PDD (CST, USA), the p38 inhibitor SB203580 (CST, USA), or the p38 agonist anisomycin (CST, USA) was given to the experimental groups at the recommended concentrations via intrathecal injection.

Techniques: Immunohistochemical staining, Staining

Journal: BioMed Research International

Article Title: Effect of TRPV4-p38 MAPK Pathway on Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion

doi: 10.1155/2016/6978923

Figure Lengend Snippet: The effect of reagents on the protein expression of TRPV4, p38, and P-p38. (a–d) The expression of TRPV4, p38, and P-p38 proteins after RR, 4 α -PDD, SB203580, and anisomycin intrathecal injections given to rats 4 days after CCD surgery. The bars represent means ± SEMs based on six experiments; ∗ P < 0.05 and ∗∗ P < 0.01, TRPV4 compared with controls. # P < 0.05 and ## P < 0.01, p38 compared with controls. & P < 0.05 and && P < 0.01, P-p38 compared with controls.

Article Snippet: Four days after CCD surgery, the TRPV4 inhibitor Ruthenium Red (RR, Sigma, Germany), the TRPV4 agonist 4 α -PDD (CST, USA), the p38 inhibitor SB203580 (CST, USA), or the p38 agonist anisomycin (CST, USA) was given to the experimental groups at the recommended concentrations via intrathecal injection.

Techniques: Expressing

Journal: BioMed Research International

Article Title: Effect of TRPV4-p38 MAPK Pathway on Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion

doi: 10.1155/2016/6978923

Figure Lengend Snippet: Altered distribution of TRPV4-positive neurons in DRG tissue. (a–f) TRPV4 immunohistochemical staining of the DRG neurons in the control, CCD, CCD+RR 10 nmol/L, CCD+4 α -PDD 10 nmol/L, CCD+SB203580 20 μ mol/L, and CCD+anisomycin 25 μ g/mL groups, respectively. Scale bars: 100 μ m. (g) The analysis of the TRPV4-positive neurons (shown as means ± SEMs); ∗∗ P < 0.01 compared with controls; ## P < 0.01 compared with the CCD group.

Article Snippet: Four days after CCD surgery, the TRPV4 inhibitor Ruthenium Red (RR, Sigma, Germany), the TRPV4 agonist 4 α -PDD (CST, USA), the p38 inhibitor SB203580 (CST, USA), or the p38 agonist anisomycin (CST, USA) was given to the experimental groups at the recommended concentrations via intrathecal injection.

Techniques: Immunohistochemical staining, Staining

Journal: BioMed Research International

Article Title: Effect of TRPV4-p38 MAPK Pathway on Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion

doi: 10.1155/2016/6978923

Figure Lengend Snippet: Ectopic discharges after CCD surgery and reagent injection. (a–f) represent discharges of the control, CCD, CCD+RR 10 nmol/L, CCD+4 α -PDD 10 nmol/L, CCD+SB203580 20 μ mol/L, and CCD+anisomycin 25 μ g/mL groups. Cut from BL-420E+ biological and functional experimental system. (g) and (h) show the amplitudes and frequencies for the different groups (data are expressed as means ± SEM); ∗∗ P < 0.01, compared with the CCD group (7-8 rats in each group).

Article Snippet: Four days after CCD surgery, the TRPV4 inhibitor Ruthenium Red (RR, Sigma, Germany), the TRPV4 agonist 4 α -PDD (CST, USA), the p38 inhibitor SB203580 (CST, USA), or the p38 agonist anisomycin (CST, USA) was given to the experimental groups at the recommended concentrations via intrathecal injection.

Techniques: Injection, Functional Assay

Journal: Frontiers in Nutrition

Article Title: Maternal Organic Selenium Supplementation Relieves Intestinal Endoplasmic Reticulum Stress in Piglets by Enhancing the Expression of Glutathione Peroxidase 4 and Selenoprotein S

doi: 10.3389/fnut.2022.900421

Figure Lengend Snippet: Effect of HMSeBA supplementation on the expression of selenoproteins and the antioxidative capacity in IPEC-J2 cells. IPEC-J2 cells were treated with 1.2 μM HMSeBA for 24 h. (A) Relative mRNA levels of selenoproteins. n = 3 for each group. (B) Relative protein abundance of GPX4, SELENOS and SELENOP. n = 4 for each group. (C) The activity of antioxidant related enzymes. n = 6 for each group. GSH-Px, glutathione peroxidase; T-SOD, total superoxide dismutase; CAT, catalase; T-AOC, total antioxidant capacity. Data are expressed as mean ± SE. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The PVDF membrane was then blocked with 1% BSA containing TBST, followed by incubation with primary antibodies at 4°C for more than 8 h. The primary antibodies were as follows: SELS (21 KDa, 15591-1-AP, Proteintech), GPX4 (22 KDa, 52455S, Cell Signaling Technology), SEPP1 (57/45 KDa, sc-376858, Santa Cruz Biotechnology), GRP78 (78 KDa, ab21685, Abcam), p-IRE1α (110 KDa, ab48187, Abcam), IRE1α (110 KDa, ab37073, Abcam), p-PERK (170 KDa, 3179S, Cell Signaling Technology), PERK (170 KDa, 3192S, Cell Signaling Technology), CHOP (27 KDa, 381679, Zen BioScience) and β-actin (45 kDa, #4967, Cell Signaling Technology).

Techniques: Expressing, Activity Assay

Journal: Frontiers in Nutrition

Article Title: Maternal Organic Selenium Supplementation Relieves Intestinal Endoplasmic Reticulum Stress in Piglets by Enhancing the Expression of Glutathione Peroxidase 4 and Selenoprotein S

doi: 10.3389/fnut.2022.900421

Figure Lengend Snippet: Roles of GPX4 and SELENOS in regulating ER stress induced by H 2 O 2 in IPEC-J2 cells. (A) IPEC-J2 cells were infected with AdshGPX4, then were treated with H 2 O 2 plus HMSeBA. Protein levels of ER stress related markers were detected. (B) IPEC-J2 cells were infected with AdshSELS, then were treated with H 2 O 2 plus HMSeBA. Protein levels of ER stress related markers were detected. (C) IPEC-J2 cells were infected with AdshGPX4 or/and AdshSELS, then were treated with H 2 O 2 . Protein levels of ER stress related markers were detected n = 4 or 3 for western blot assay. Data are expressed as mean ± SE. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The PVDF membrane was then blocked with 1% BSA containing TBST, followed by incubation with primary antibodies at 4°C for more than 8 h. The primary antibodies were as follows: SELS (21 KDa, 15591-1-AP, Proteintech), GPX4 (22 KDa, 52455S, Cell Signaling Technology), SEPP1 (57/45 KDa, sc-376858, Santa Cruz Biotechnology), GRP78 (78 KDa, ab21685, Abcam), p-IRE1α (110 KDa, ab48187, Abcam), IRE1α (110 KDa, ab37073, Abcam), p-PERK (170 KDa, 3179S, Cell Signaling Technology), PERK (170 KDa, 3192S, Cell Signaling Technology), CHOP (27 KDa, 381679, Zen BioScience) and β-actin (45 kDa, #4967, Cell Signaling Technology).

Techniques: Infection, Western Blot

Journal: Frontiers in Nutrition

Article Title: Maternal Organic Selenium Supplementation Relieves Intestinal Endoplasmic Reticulum Stress in Piglets by Enhancing the Expression of Glutathione Peroxidase 4 and Selenoprotein S

doi: 10.3389/fnut.2022.900421

Figure Lengend Snippet: General view of the effect of HMSeBA on intestinal ER stress. A model for HMSeBA supplementation regulates ER stress induced by LPS or H 2 O 2 in vivo and vitro . Only core components of the pathway are shown. LPS or H 2 O 2 administration induce ER stress through activating the GRP78-IRE1α-CHOP signaling pathway. While HMSeBA treatment improves the expression of GPX4 and SELS, thereby suppresses the ER stress signal, ameliorates the redox status. Maternal HMSeBA is involved in regulating the ER stress signal and health of intestine.

Article Snippet: The PVDF membrane was then blocked with 1% BSA containing TBST, followed by incubation with primary antibodies at 4°C for more than 8 h. The primary antibodies were as follows: SELS (21 KDa, 15591-1-AP, Proteintech), GPX4 (22 KDa, 52455S, Cell Signaling Technology), SEPP1 (57/45 KDa, sc-376858, Santa Cruz Biotechnology), GRP78 (78 KDa, ab21685, Abcam), p-IRE1α (110 KDa, ab48187, Abcam), IRE1α (110 KDa, ab37073, Abcam), p-PERK (170 KDa, 3179S, Cell Signaling Technology), PERK (170 KDa, 3192S, Cell Signaling Technology), CHOP (27 KDa, 381679, Zen BioScience) and β-actin (45 kDa, #4967, Cell Signaling Technology).

Techniques: In Vivo, Expressing