Journal: Biomedicines

Article Title: Extracellular Vesicles from Plasma of Patients with Glioblastoma Promote Invasion of Glioblastoma Cells Even After Tumor Resection

doi: 10.3390/biomedicines12122834

Figure Lengend Snippet: Analysis of influence of EVs on the MAP kinases phosphorylation in astrocytes ( a , b ), GBM 011 cells ( c , d ), and U251 MG cells ( e , f ). The following phosphorylation sites were assayed: pAKT (S473), pERK1/2 (T202/204; T185/Y187), pJNK1/2/3 (T183/Y185), and pp38 (T180/Y182). ( a , c , e ) Cut representative membranes with the bands (marked by frames); ( b , d , f ) quantification of the band intensity reflecting expression of different MAP kinases in the phosphorylated form. Please note that MAP kinases were assayed on the same (pJNK and pp38) or cut (pAKT and pERK) membranes, so they ‘share’ GAPDH. Whole Western blotting membranes are shown in . The quantification data are presented as band intensity (relative to GAPDH) normalized to that of the untreated cells (100%, dashed line) ± SEM (n = 3–8); $ ( p < 0.05) indicates significant difference from the untreated cells according to one-sample Wilcoxon test, followed by post hoc Holm–Sidak’s test; # ( p < 0.05) and ### ( p < 0.001) indicate significant difference between the data groups according to Kruskal–Wallis test followed by post hoc Dunn’s test.

Article Snippet: After that, the membranes were incubated with the rabbit primary antibodies to pAKT (S473) (1:4000, GB150002, ServiceBio, Wuhan, China), pERK1/2 (T202/204; T185/Y187) (1:1000, GB113492, ServiceBio), pJNK1/2/3 (T183/Y185) (1:1000, GB12018, ServiceBio), and pp38 (T180/Y182) (1:1000, GB1133880, ServiceBio) overnight at 4 °C.

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Cells were pretreated with EGF (100ng/ml) and harvested after the indicated times. Lysates were collected, and p38 MAPK phosphorylation levels were analyzed by Western blotting using the antiphospho-p38 antibody. The same membrane was striped and re-probed with a different antibody for measuring total-p38 MAPK protein levels. (B) Caki-1 cells were preincubated with 400pM of the secreted form of the Klotho (KL) protein at the indicated times followed by EGF (100ng/ml) stimulation for additional 5 min. Cell lysates were subjected to Western blot analysis as described in (A). Plots indicate means ± S.E.M of three independent experiments.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: Phospho-proteomics, Western Blot, Membrane

Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Classical in vitro wound healing assay was performed using near-confluent serum-starved Caki-1 cells grown on collagen type 1. Cells were either pretreated with 400pM Klotho (KL) or 1μM p38 MAPK-specific inhibitor SB203580 as positive control, for 60 min followed by EGF (100ng/ml) treatment. Cell culture images shown here were taken at 0 and 24 h. (B) Plots of quantification of the resultant cell motility values were computed by gap surface area measurements for four selected microscopic fields in each assay condition. The degree of migration is expressed as % wound closure compared with zero time point. The results represent means ± S.E.M of three independent experiments. (C) Wound healing assays performed under 3D settings. Images are microphotographs of Caki-1 cells showing hole-closure of “tissue openings” generated with magnetic pattering as described in the materials and method. Cells were pretreated the same way with either Klotho (KL) or SB203580 and stimulated with EGF as described for the classical wound healing assay. (D) Plots of rate closure of holes for Caki-1 cells as a function of EGF exposure in the presence or absence of KL or SB203580.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: In Vitro, Wound Healing Assay, Positive Control, Cell Culture, Migration, Generated

Journal: Basic Research in Cardiology

Article Title: Oxidized low-density lipoprotein (oxLDL) affects load-free cell shortening of cardiomyocytes in a proprotein convertase subtilisin/kexin 9 (PCSK9)-dependent way

doi: 10.1007/s00395-017-0650-1

Figure Lengend Snippet: Participation of MAPK pathways in oxLDL-dependent effects: a cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SP600125 (10 µM). b Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and PD98059 (10 µM). c Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SB202190 (10 µM). In a – c a represents p < 0.095 vs. control. d Representative immunoblot of samples from cardiomyocytes exposed to oxLDL (20 µg/ml) and quantified for p38 MAPK expression and phosphorylation of p38 MAPK. e Quantification of the blot shown in d . f Original western blot showing the oxidative modification of tropomyosin by oxLDL (20 mg/ml) in the left and the control blot under reducing conditions (right). g Quantification of oxidative modification of tropomyosin by oxLDL ( n = 3). Exact p values are given

Article Snippet: Anti-PCSK9 (rabbit polyclonal, ab31762, Abcam, Cambridge, UK), anti p38 MAPK (rabbit polyclonal, #506123, Merck KGaA Darmstadt, Germany), and anti phosphorylated p38 MAPK (rabbit polyclonal, #M0800, Sigma, St. Louis, USA) antibodies were used to detect the expression of PCSK9 protein and phosphorylation of p38 MAPK.

Techniques: Western Blot, Expressing, Modification

Journal: Basic Research in Cardiology

Article Title: Oxidized low-density lipoprotein (oxLDL) affects load-free cell shortening of cardiomyocytes in a proprotein convertase subtilisin/kexin 9 (PCSK9)-dependent way

doi: 10.1007/s00395-017-0650-1

Figure Lengend Snippet: Summary figure of our findings: oxidized LDL (oxLDL) activates the oxLDL receptor. Expression of the receptor by cardiomyocytes was shown in this study and silencing of the receptor by siRNA attenuated all subsequent steps. oxLDL induces oxidative stress as indicated by oxidative modifications of tropomyosin (Trp-ox). Oxidative stress activates p38 MAP kinase as indicated by western blot. Inhibition of p38 MAP kinase activation by SB20190 attenuates this effect. Subsequently, oxLDL causes increased expression of PCSK9 as indicated by western blots and inhibition of transcription by actinomycin D (ActD) or translation by cycloheximide (chx) block all subsequent steps. Silencing of PCSK9 upregulation attenuates future steps

Article Snippet: Anti-PCSK9 (rabbit polyclonal, ab31762, Abcam, Cambridge, UK), anti p38 MAPK (rabbit polyclonal, #506123, Merck KGaA Darmstadt, Germany), and anti phosphorylated p38 MAPK (rabbit polyclonal, #M0800, Sigma, St. Louis, USA) antibodies were used to detect the expression of PCSK9 protein and phosphorylation of p38 MAPK.

Techniques: Expressing, Western Blot, Inhibition, Activation Assay, Blocking Assay

Journal: Oncotarget

Article Title: Estrogen promotes tumor metastasis via estrogen receptor beta-mediated regulation of matrix-metalloproteinase-2 in non-small cell lung cancer

doi: 10.18632/oncotarget.16992

Figure Lengend Snippet: (A) The migration in wound-healing assay of A549 cells after treatment with DMSO or E2 (10 nM), PPT (10 nM), DPN (10 nM), E2+Ful (1 μM) and Ful (1 μM) for 24 h (magnification 40×); (B) The effect on wound closure (percentage) in A549 cells ( *p < 0.05 vs. control group, #p < 0.05 vs. E2 group ); (C) Representative images of Transwell assays to assess A549 cell invasion and migration; (D) The effect of transwell migration assays in A549 cells after treating with DMSO or E2 (10 nM), PPT (10 nM), DPN (10 nM), E2+Ful (1μM) and Ful(1μM) for 24 h; The effect of transwell migration assays in H1793 cells after treatment with the same drugs for 72 h; (E) Representative images of Transwell assays for assessment of H1793 cell invasion and migration; (F) The effect of transwell migration assays in H1793 cells after treatment with DMSO or E2 (10 nM), PPT (10 nM), DPN (10 nM), E2+Ful (1 μM) and Ful (1 μM) for 24 h. The effect of transwell migration assays in H1793 cells after treatment with the same drugs for 72 h; (G-H) Western blot analysis of ERβ, MMP-2, MMP-9, p-p38MAPK, p38MAPK, pAKT and tAKT protein levels at 48 h in A549 cells, respectively. GAPDH was used as a control. All data are shown as the mean ± SD. Results represent three independent experiments. Student's t-test was carried out ( *p < 0.05 vs. control group, #p < 0.05 vs. E2 group ).

Article Snippet: The antibodies used during Western blots included rabbit anti-human ERα from Proteintech, rabbit anti-human ERβ (1:500) from Proteintech, mouse anti-human MMP-1(1:400) from Santa Cruz, mouse anti-human MMP-2(1:400) from Santa Cruz, rabbit anti-human MMP-9 (1:500) from Proteintech, rabbit anti-human p38MAPK(1:500)/p-p38MAPK(1:500) and rabbit anti-human tAkt (1:500)/pAkt(1:500) from Bioworld Technology.

Techniques: Migration, Wound Healing Assay, Western Blot

Journal: Oncotarget

Article Title: Estrogen promotes tumor metastasis via estrogen receptor beta-mediated regulation of matrix-metalloproteinase-2 in non-small cell lung cancer

doi: 10.18632/oncotarget.16992

Figure Lengend Snippet: (A) Western blot analysis of protein expression of ERβ, MMP-2, p-p38MAPK, p38MAPK, pAKT and tAKT in NSCLC cell line A549 when treating with DMSO or E2 (10 nM), E2+LY294002 (10 nM), LY294002 (10 nM), E2+SB203580 (10 nM) and SB203590 (10 nM) for 48 h. (B) the detection of optical density. GAPDH was used as a control. All data are shown as the mean ± SD. Results represent three independent experiments. Student's t-test was carried out ( *p < 0.05 vs. control group, #p < 0.05 vs. E2 group ).

Article Snippet: The antibodies used during Western blots included rabbit anti-human ERα from Proteintech, rabbit anti-human ERβ (1:500) from Proteintech, mouse anti-human MMP-1(1:400) from Santa Cruz, mouse anti-human MMP-2(1:400) from Santa Cruz, rabbit anti-human MMP-9 (1:500) from Proteintech, rabbit anti-human p38MAPK(1:500)/p-p38MAPK(1:500) and rabbit anti-human tAkt (1:500)/pAkt(1:500) from Bioworld Technology.

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Estrogen promotes tumor metastasis via estrogen receptor beta-mediated regulation of matrix-metalloproteinase-2 in non-small cell lung cancer

doi: 10.18632/oncotarget.16992

Figure Lengend Snippet: (A) After bilateral ovariectomy A549 cells (5 × 10 6 /100 μL) suspended in PBS were injected into 4-week-old female BALB/c nude mice via the tail vein, mice were randomly divided into five groups (N = 5/group): E2(0.09 mg/kg), PPT(1.05 mg/kg), DPN(1.89 mg/kg), E2+ Ful(1.46 mg/kg) and blank control. The lungs were removed after 4 weeks subcutaneously under drug treatment. Gross appearance of metastatic lung tumor nodes in different groups is indicated by bright yellow arrows. (B) Representative pathological image of metastatic A549 tumor at magnification 100ificatione pathological image of groups (N = 5/group): The number of metastatic nodules in the lungs (lung nodules number in every mouse of each group), metastatic index in different groups mentioned in ‘‘Materials and methods’’ section for the experimental. (C-E) The mean lung wet weight of each group, The number of metastatic nodules in the lungs (lung nodules number in every mouse of each group), metastatic index in different groups mentioned in ‘‘Materials and methods’’ section for the experimental. (F) Protein expression of ERβ, MMP-2, MMP-9, p-p38MAPK, p38MAPK, pAKT and tAKT in murine lung metastatic nodes was analyzed using Western blot, and (G) the detection of optical density. All data are expressed as the mean ± SD. Student's t-test was carried out ( *p < 0.05 vs. control group, #p < 0.05 vs. E2 group ).

Article Snippet: The antibodies used during Western blots included rabbit anti-human ERα from Proteintech, rabbit anti-human ERβ (1:500) from Proteintech, mouse anti-human MMP-1(1:400) from Santa Cruz, mouse anti-human MMP-2(1:400) from Santa Cruz, rabbit anti-human MMP-9 (1:500) from Proteintech, rabbit anti-human p38MAPK(1:500)/p-p38MAPK(1:500) and rabbit anti-human tAkt (1:500)/pAkt(1:500) from Bioworld Technology.

Techniques: Injection, Expressing, Western Blot