Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 and NF- κ B signalling pathways are deregulated in human CC, with ERK5 expression correlating with increased NF- κ B activation. ( a ) Representative immunoblots of steady-state protein expression levels of MEK5, ERK5, NF- κ B, I κ B and β -actin in normal colon, colon adenomas and pMMR and dMMR colon carcinomas; ( b ) MEK5 and ERK5 steady-state protein levels; ( c ) NF- κ B, I κ B and NF- κ B/I κ B ratio; ( d ) correlations between ERK5 steady-state protein levels and NF- κ B, I κ B or NF- κ B/I κ B ratio; ( e ) representative immunoblots of steady-state levels of p-NF- κ B, NF- κ B, p-I κ B, I κ B and β -actin in normal colon, colon adenoma, and pMMR and dMMR colon carcinomas; and ( f ) representative immunohistochemistry for ERK5, p-NF- κ B and NF- κ B in human colon cancer samples. Immunoblot statistical significance was determined using the non-parametric statistical analysis Kruskal–Wallis test with Dunn's post test for selected comparisons and results are expressed as mean±S.E.M. for samples in each category; correlation statistical significance was determined using the non-parametric stastistical analysis Spearman test. pMMR, proficient mismatch repair system; dMMR, deficient mismatch repair system. Scale bar=100 μ m. * P <0.05, † P <0.01 and ‡ P <0.001 from normal colon

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Expressing, Activation Assay, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: Correlation between MEK5, ERK5, NF- κ B and I κ B steady-state protein expression, NF- κ B activation and clinicopathological characteristics

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Expressing, Activation Assay

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation accelerates cell cycle progression in SW620 cells. ( a ) Representative immunoblot of ERK5 protein levels in SW620 cell lines with differential ERK5 activation, stable constitutive MEK5 activation ( CA-MEK5 ), dominant-negative MEK5 ( DN-MEK5 ), empty control ( Empty ) and wild-type SW620 cell line (WT). The developed cell model consistently showed that DN-MEK5 led to constitutive inhibition of ERK5 activation, CA-MEK5 led to constitutive ERK5 activation and Empty control cells displayed basal ERK5 activation. ( b ) FACS cell cycle analysis of CA-MEK5, DN-MEK5 and Empty SW620 cells, following release from dual-thymidine block and exposed to ( c ) XMD8-92 or ( d ) BAY11-7085. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P <0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Western Blot, Dominant Negative Mutation, Control, Inhibition, Cell Cycle Assay, Blocking Assay

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation accelerated cell cycle is associated with NF- κ B activation via reduction of I κ B steady-state levels. Immunoblot analysis of steady-state protein levels of ( a ) p-ERK5, ERK5 and p-ERK5/ERK5, and of ( b ) p-NF- κ B/NF- κ B, NF- κ B/I κ B ratios, p-I κ B and I κ B. ( c ) Representative immunoblots for p-ERK5, ERK5, p-NF- κ B, NF- κ B, p-I κ B, I κ B and β -actin in Empty, DN-MEK5 and CA-MEK5 SW620 cells when treated for 24 h with XMD8-92 and BAY11-7085, or untreated (no addition). Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P <0.05, † P <0.01 and ‡ P <0.001 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation increases cell migration in vitro . Cell migration was assessed by ( a ) wound-healing assay at 24 and 48 h after ‘wound' formation and ( b ) transwell migration assay, with cells allowed to migrate for 9 h after cell platting. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P <0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Migration, In Vitro, Wound Healing Assay, Transwell Migration Assay

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation increased cell migration is associated with increased vimentin expression and NF- κ B activation. Immunoblot analysis of steady-state protein levels of ( a ) vimentin and ( b ) of p-NF- κ B/NF- κ B, NF- κ B/I κ B ratios, p-I κ B and I κ B. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. of at least three independent experiments. * P <0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Migration, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation increases NF- κ B nuclear translocation and transcriptional activity via I κ B phosphorylation and degradation. Immunoblot analysis of steady-state protein levels of (a) p-I κ B, I κ B and p-I κ B/I κ B ratios, and ( b ) nuclear and cytosolic NF- κ B; ( c ) NF- κ B transcriptional activity, evaluated by dual luciferase assay with reporter plasmids. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P< 0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Translocation Assay, Activity Assay, Phospho-proteomics, Western Blot, Luciferase

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: In vivo , MEK5/ERK5 activation is associated with local invasion and regional lymphnode metastasis. As shown in the graphical abstract, ( a ) the injected tumours may grow locally in the caecum/colon infiltrating mucosa, sub-mucosa, muscularis propria and eventually sub-serosa. In addition, tumour growth may be focal (restricted to one foci, at the injection site) or multifocal (numerous foci spread throughout the caecum and colon, not restricted to the injection site). Furthermore, in addition to local invasion by tumour cells, the metastatic cascade encompasses intravasation into lymph vessels, extravasation out of the circulation, and survival and growth at secondary site. We injected 5 × 10 5 SW620 DN-MEK5 or CA-MEK5 cells into the wall of the caecum, in BALB/c scid mice, and mice were killed 30 or 60 days post injection. ( a ) Histopathological characteristics of DN-MEK5 and CA-MEK5 tumours regarding local invasion, extravasation and distant metastasis (to regional lymphnodes), and ( b) representative microphotographs of the multifocallity of CA-MEK5 tumours, compared with the focal lesions seen in DN-MEK5 tumours (white arrows, upper panel), of the lymph vessel invasion (black arrowhead, middle panel) and of the lymphnode metastasis (lower panel). *Tumour cells

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: In Vivo, Activation Assay, Injection

Journal: Experimental eye research

Article Title: Signaling pathways activated by resolvin E1 to stimulate mucin secretion and increase intracellular Ca 2+ in cultured rat conjunctival goblet cells

doi: 10.1016/j.exer.2018.04.015

Figure Lengend Snippet: Cultured goblet cells were preincubated with the Ca2+/CaMK inhibitor KN93 and its negative control KN92, both at 10−7 M for 30 min. [Ca2+]i over time is shown in A. Change in peak [Ca2+]i is shown in B. Data are mean ± SEM from 4 independent experiments. * indicates significant difference from basal.

Article Snippet: Ro-318220, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 and {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}} U73343 , KN92 and KN93 were purchased from Tocris Bioscience (Ellisville, MO).

Techniques: Cell Culture, Negative Control

Journal: Experimental eye research

Article Title: Signaling pathways activated by resolvin E1 to stimulate mucin secretion and increase intracellular Ca 2+ in cultured rat conjunctival goblet cells

doi: 10.1016/j.exer.2018.04.015

Figure Lengend Snippet: Cultured goblet cells were incubated with U73122, U73343, 2-APB, Ro 31–8220, 1-but, t-but, aris acid, KN93, or KN92 before stimulation with RvE1 (10−9 M). Glycoconjugate secretion was measured. Data are mean ± SEM from 4 independent experiments and shown as fold above basal, which set to 1. * indicates significant difference from basal; # indicates significance from RvE1 alone.

Article Snippet: Ro-318220, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 and {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}} U73343 , KN92 and KN93 were purchased from Tocris Bioscience (Ellisville, MO).

Techniques: Cell Culture, Incubation