Journal: Neurochemistry international

Article Title: Enolase inhibition alters metabolic hormones and inflammatory factors to promote neuroprotection in spinal cord injury

doi: 10.1016/j.neuint.2020.104788

Figure Lengend Snippet: Effects of ENOblock treatment on several key metabolic factors after SCI in rats. SCI rats were treated with vehicle alone or ENOblock (100 μg/kg given 15min, 24h, and 3d post-injury via intravenous tail vein injection), and blood samples were obtained at sacrifice, 7d post-injury. Sham operated rats (T10 laminectomy) were used as controls. Samples were analyzed by using Eve Tech’s Mouse, Rat Metabolic Array (MRDMET) Discovery Assay. (A-B) Pancreatic polypeptide (PP) and peptide tyrosine tyrosine (PYY). PP concentrations in plasma samples were not significantly changed by ENOblock treatment. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 130.3 and 131.6 pg/ml respectively; the distributions in the two groups did not differ significantly (Mann–Whitney U = 14, n1 = n2 = 6,). PYY levels were significantly increased in ENO-treated rats as compared to vehicle treated rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 63.81 and 137.8 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U = 0, n1 = n2 = 6, **p < 0.01 two-tailed). (C-D) Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotrophic polypeptide (GIP). Plasma levels of GLP-1 in ENO-treated SCI rat samples were significantly increased as compared to vehicle treated rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 500.1 and 678.3 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U = 0, n1 = n2 = 6, **p < 0.01 two-tailed). ENOblock treatment increased GIP levels after SCI in rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 3416 and 3858 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U =2, n1 = n2 = 6, **p < 0.01 two-tailed). (E-F) Glucagon and insulin. ENOblock treatment increased glucagon levels significantly as compared with vehicle treated SCI rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 374.3 and 737.3 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U = 0, n1 = n2 = 6, **p < 0.01 two-tailed). (B) ENOblock treatment significantly increased insulin levels as compared with vehicle treated SCI rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 893.2 and 1264 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U = 0, n1 = n2 = 6, **p < 0.01 two-tailed).

Article Snippet: ENOblock (AP-111-a4, Adooq Bioscience, 100μg/kg) was administered systemically via intravenous tail vein injection at four time points: 15min, 24h, and 3d post-injury.

Techniques: Injection, MANN-WHITNEY, Two Tailed Test

Journal: Neurochemistry international

Article Title: Enolase inhibition alters metabolic hormones and inflammatory factors to promote neuroprotection in spinal cord injury

doi: 10.1016/j.neuint.2020.104788

Figure Lengend Snippet: Effects of ENOblock treatment on several key metabolic factors after SCI in rats. SCI rats were treated with vehicle alone or ENOblock (100 μg/kg given 15min, 24h, and 3d post-injury via intravenous tail vein injection), and blood samples were obtained at sacrifice, 7d post-injury. Sham operated rats (T10 laminectomy) were used as controls. Samples were analyzed by using Eve Tech’s Mouse, Rat Metabolic Array (MRDMET) Discovery Assay. (A-B) Pancreatic polypeptide (PP) and peptide tyrosine tyrosine (PYY). PP concentrations in plasma samples were not significantly changed by ENOblock treatment. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 130.3 and 131.6 pg/ml respectively; the distributions in the two groups did not differ significantly (Mann–Whitney U = 14, n1 = n2 = 6,). PYY levels were significantly increased in ENO-treated rats as compared to vehicle treated rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 63.81 and 137.8 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U = 0, n1 = n2 = 6, **p < 0.01 two-tailed). (C-D) Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotrophic polypeptide (GIP). Plasma levels of GLP-1 in ENO-treated SCI rat samples were significantly increased as compared to vehicle treated rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 500.1 and 678.3 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U = 0, n1 = n2 = 6, **p < 0.01 two-tailed). ENOblock treatment increased GIP levels after SCI in rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 3416 and 3858 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U =2, n1 = n2 = 6, **p < 0.01 two-tailed). (E-F) Glucagon and insulin. ENOblock treatment increased glucagon levels significantly as compared with vehicle treated SCI rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 374.3 and 737.3 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U = 0, n1 = n2 = 6, **p < 0.01 two-tailed). (B) ENOblock treatment significantly increased insulin levels as compared with vehicle treated SCI rats. Median concentrations in SCI-vehicle and SCI-ENOblock treated groups were 893.2 and 1264 pg/ml respectively; the distributions in the two groups differed significantly (Mann–Whitney U = 0, n1 = n2 = 6, **p < 0.01 two-tailed).

Article Snippet: ENOblock (AP-111-a4, Adooq Bioscience, 100μg/kg) was administered systemically via intravenous tail vein injection at four time points: 15min, 24h, and 3d post-injury.

Techniques: Injection, Clinical Proteomics, MANN-WHITNEY, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Keratin 17 covalently binds to alpha-enolase and exacerbates proliferation of keratinocytes in psoriasis

doi: 10.7150/ijbs.83141

Figure Lengend Snippet: Loss of ENO1 inhibits glycolysis and proliferation of psoriatic KCs. ( A ) Relative ENO1 activity in keratinocytes (KCs) treated with the ENO1 inhibitor ENOBlock ( n = 3). ( B ) The extracellular acid ratio (ECAR), ( C ) intracellular ATP levels, ( D ) extracellular lactate production and ( E ) the uptake of glucose were analyzed in KCs treated with the ENO1 inhibitor ENOBlock. ( n = 3). ( F ) Cell proliferation was analyzed by Cell Counting Kit-8 (CCK8) assay, ( G ) EdU assay, the percentage of EdU positive cells and ( H ) PCNA protein levels in KCs treated with the ENO1 inhibitor ENOBlock. ( I ) Ear phenotype and H&E staining of lesional skin sections. Scale bar, 10 μm. ( J ) Epidermal thickness and ( K ) immunofluorescence staining of Ki67 in lesional skin from ENO1 inhibitor ENOBlock-treated IMQ-induced psoriasis-like mice; DMSO was used as a control ( n = 3-5). Ki67 (red) and Hoechst (blue). Scale bar, 50 μm. Data are presented as the mean ± SEM (n = 3-5 ) . * p <0.05, ** p <0.01, *** p <0.001. p values were calculated by unpaired Student's t test. All experiments were repeated at least three times.

Article Snippet: To evaluate the effect of ENO1 inhibition or phosphorylation of K17-Ser 44 inhibition on the development of psoriasis, 2.5 nmol/kg ENO1 siRNA (Ribobio, Guangzhou, China), 10 mg/kg ENOBlock (Ribobio, Guangzhou, China), and 0.5 mg/kg BI-D1870 (Ribobio, Guangzhou, China) were individually mixed with vehicle and applied topically to the right ear of mice after 3-4 h of challenge with IMQ every other day for 5 days.

Techniques: Activity Assay, Cell Counting, CCK-8 Assay, EdU Assay, Staining, Immunofluorescence, Control

Journal: International Journal of Biological Sciences

Article Title: Keratin 17 covalently binds to alpha-enolase and exacerbates proliferation of keratinocytes in psoriasis

doi: 10.7150/ijbs.83141

Figure Lengend Snippet: ENO1 promotes cell glycolysis and proliferation by regulating the phosphorylation of K17. ( A ) Protein expression of phosphorylated K17, K17 and ENO1 in keratinocytes (KCs) treated with Pso Mix, K17-overexpressing KCs and K17 siRNA-transfected KCs. ( B ) Phosphorylation of K17 in KCs treated with Pso Mix and ENO1 siRNA-transfected or ENOBlock-treated KCs. ( C ) RSK1 kinase activity in Pso Mix and ENO1 siRNA-transfected, ENOBlock-treated and BI-D1870-treated KCs. ( D ) The extracellular acid ratio (ECAR) of K17 S44A mutant-transfected KCs. ( E ) Cell proliferation was analyzed using the CCK8 assay, ( F ) EdU assay and the percentage of EdU positive cells in mutant K17 S44A-transfected KCs. ( G ) Immunofluorescence staining for the punctate form of EGFP-K17 in BI-D1870-treated EGFP-K17-transfected KCs ( n = 3). K17 (green) and Hoechst (blue). Scale bar, 10 μm. ( H ) The ECAR of Pso Mix and BI-D1870-treated KCs. ( I ) Cell proliferation was analyzed by a CCK8 assay in BI-D1870-treated KCs. Data are presented as the mean ± SEM (n = 3-5 ) . * p <0.05, ** p <0.01, *** p <0.001. p values were calculated by unpaired Student's t test . All experiments were repeated at least three times.

Article Snippet: To evaluate the effect of ENO1 inhibition or phosphorylation of K17-Ser 44 inhibition on the development of psoriasis, 2.5 nmol/kg ENO1 siRNA (Ribobio, Guangzhou, China), 10 mg/kg ENOBlock (Ribobio, Guangzhou, China), and 0.5 mg/kg BI-D1870 (Ribobio, Guangzhou, China) were individually mixed with vehicle and applied topically to the right ear of mice after 3-4 h of challenge with IMQ every other day for 5 days.

Techniques: Phospho-proteomics, Expressing, Transfection, Activity Assay, Mutagenesis, CCK-8 Assay, EdU Assay, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: Keratin 17 covalently binds to alpha-enolase and exacerbates proliferation of keratinocytes in psoriasis

doi: 10.7150/ijbs.83141

Figure Lengend Snippet: Phosphorylation of K17-Ser 44 promotes its translocation to the nucleus. ( A ) Immunofluorescence staining for K17 in the nucleus of keratinocytes (KCs) of lesional skin from psoriasis patients ( n = 3). K17 (green) and Hoechst (blue). Scale bar, 10 μm. ( B ) Immunofluorescence staining of K17 distributed in the nuclei of KCs transfected with EGFP-K17 and treated with Pso Mix and the intensity of nuclear fluorescence per unit area. Scale bar, 10 μm. ( C ) K17 and phosphorylated K17-Ser 44 protein levels in the nucleus and cytoplasm of K17-overexpressing and Pso Mix-treated KCs. ( D ) Immunofluorescence staining for the punctate form of EGFP-K17 in ENO1 siRNA-transfected, ENOBlock- or BI-D1870-treated EGFP-K17-transfected KCs ( n = 3). K17 (green), ENO1 (red) and Hoechst (blue). White arrows: the punctate and diffuse forms of EGFP-K17. Scale bar, 10 μm. ( E ) K17 and phosphorylated K17-Ser 44 protein levels in the nucleus and cytoplasm of Pso Mix and ENO1 siRNA-transfected or ENOBlock-treated KCs. ( F ) K17 and phosphorylation of K17-Ser 44 protein levels in the nucleus and cytoplasm of Pso Mix and RSK kinase inhibitor BI-D1870-treated KCs. Data are presented as the mean ± SEM (n = 3-5) . ChIP assays of K17 binding to the LDHA promoter in ( G ) K17-overexpressing KCs and ( H ) BI-D1870-treated K17-overexpressing KCs. ( I ) Luciferase reporter assays in HEK293T cells to test the LDHA WT and mutants. * p <0.05, ** p <0.01, *** p <0.001, ns, not significant. p values were calculated by unpaired Student's t test . All experiments were repeated at least three times.

Article Snippet: To evaluate the effect of ENO1 inhibition or phosphorylation of K17-Ser 44 inhibition on the development of psoriasis, 2.5 nmol/kg ENO1 siRNA (Ribobio, Guangzhou, China), 10 mg/kg ENOBlock (Ribobio, Guangzhou, China), and 0.5 mg/kg BI-D1870 (Ribobio, Guangzhou, China) were individually mixed with vehicle and applied topically to the right ear of mice after 3-4 h of challenge with IMQ every other day for 5 days.

Techniques: Phospho-proteomics, Translocation Assay, Immunofluorescence, Staining, Transfection, Fluorescence, Binding Assay, Luciferase