Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: α-Enolase reduces cerebrovascular Aβ deposits by protecting Aβ amyloid formation

doi: 10.1007/s00018-022-04493-x

Figure Lengend Snippet: Reduced CAA pathology and improved cognitive impairment in APP23 mice by ENO1. a Schedule of administration of ENO1 (or vehicle control) and behavioral testing for 18-month-old male APP23 and wild-type mice. ENO1 (1 mM, 100 μl) or vehicle control (100 μl) was infused into the brain continuously for 7 days. b Representative immunoblot of 18-month-old male APP23 mouse brain lysates treated with ENO1 (right) and vehicle control (left), analyzed by using anti-human Aβ antibody (6E10), with quantification of relative intensity of monomeric Aβ. c Representative dot blots of 18-month-old male APP23 mouse brain lysates treated with ENO1 (right) and vehicle control (left), analyzed using anti-Aβ fibril antibody, with quantification of relative intensity of Aβ fibrils. d–g Quantification of soluble and insoluble Aβ40 and Aβ42 (n = 3 per group). h Representative immunoblots of lysates from the hippocampus (left) and cerebral cortex (right) showing APP expression, with quantification of the relative intensities of APP (n = 3 per group). i, j Y-maze test results. Average alternation (%) (i) and total number of entries into each arm (j) for each group (vehicle-infused wild-type mice, n = 6; ENO1-infused wild-type mice, n = 5; vehicle-infused APP23 mice, n = 7; ENO1-infused APP23 mice, n = 7, heat-inactivated ENO1-infused APP23 mice, n = 5). k Representative histopathological images of cerebrovascular Aβ deposits (upper) and parenchymal Aβ plaques (lower) in vehicle-infused, ENO1-infused, and heat-inactivated ENO1-infused APP23 mice. Scale bars = 500 μm (upper), 100 μm (lower). l, m Quantitative analyses of the numbers of vessels with Aβ deposits (l), as well as the percent area of Aβ plaques (m) (vehicle-infused APP23, n = 4; ENO1-infused APP23, n = 4; heat-inactivated ENO1-infused APP23, n = 5). Monomeric Aβ, Aβ fibrils, and APP protein levels were normalized to β-actin protein levels. Data are shown as mean ± SEM. *P < 0.05, ***P < 0.001. NS not significant

Article Snippet: We then used Western blotting to quantify nitrated ENO1 levels using an anti-ENO1 antibody (1:1000 dilution; Proteintech Group) and an anti-3-nitrotyrosine antibody (1:1000 dilution; Abcam).

Techniques: Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: α-Enolase reduces cerebrovascular Aβ deposits by protecting Aβ amyloid formation

doi: 10.1007/s00018-022-04493-x

Figure Lengend Snippet: Interaction of Aβ and ENO1 in vitro. a, b ThT fluorescence of Aβ—Aβ40, 50 μM (a); Aβ42, 10 μM (b)—incubated with ENO1 at a final concentration of 1 μM. c, d Representative TEM images of Aβ40 incubated with ENO1 (d) and Aβ42 incubated with ENO1 (d). Scale bars = 2 μm. e, f Representative immunoblots of Aβ40 incubated with ENO1 (e) and Aβ42 incubated with ENO1 (f), analyzed using anti-human Aβ antibody (6E10), with quantification of the relative intensities of Aβ fibrils. g, h ThT fluorescence of Aβ fibrils—Aβ40 fibrils, 50 μM (g); Aβ42 fibrils, 10 μM (h)—incubated with ENO1 at a final concentration of 1 μM. i, j Representative TEM images of Aβ40 fibrils incubated with ENO1 (i) and Aβ42 fibrils incubated with ENO1 (j). Scale bars = 2 μm. k, l Representative immunoblots of Aβ40 fibrils incubated with ENO1 (k) and Aβ42 fibrils incubated with ENO1 (l), analyzed using anti-human Aβ antibody (6E10), with quantification of the relative intensities of Aβ fibrils. Data are shown as mean ± SEM of three independent experiments. ***P < 0.001. NS not significant

Article Snippet: We then used Western blotting to quantify nitrated ENO1 levels using an anti-ENO1 antibody (1:1000 dilution; Proteintech Group) and an anti-3-nitrotyrosine antibody (1:1000 dilution; Abcam).

Techniques: In Vitro, Fluorescence, Incubation, Concentration Assay, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: α-Enolase reduces cerebrovascular Aβ deposits by protecting Aβ amyloid formation

doi: 10.1007/s00018-022-04493-x

Figure Lengend Snippet: Reversal of the inhibitory effects of ENO1 on Aβ fibril formation by the ENO1 inhibitor NaF. a ThT fluorescence kinetics of Aβ40 (50 μM) incubated with ENO1 (1 μM) and NaF at the indicated concentrations (10 mM, 1 mM, and 100 μM) for 7 days. b Representative TEM images of Aβ40 incubated with ENO1 and NaF. Scale bars = 2 μm. c Representative immunoblot of Aβ40 incubated with ENO1 and NaF, analyzed using anti-human Aβ antibody (6E10), with quantification of the relative intensities of Aβ fibrils. d ThT fluorescence kinetics of Aβ42 (10 μM) incubated with ENO1 (1 μM) and NaF at the indicated concentrations for 7 days. e Representative TEM images of Aβ42 incubated with ENO1 and NaF. Scale bars = 2 μm. f Representative immunoblot of Aβ42 incubated with ENO1 and NaF, analyzed using anti-human Aβ antibody (6E10), with quantification of the relative densities of Aβ fibrils. Data are shown as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. NS not significant

Article Snippet: We then used Western blotting to quantify nitrated ENO1 levels using an anti-ENO1 antibody (1:1000 dilution; Proteintech Group) and an anti-3-nitrotyrosine antibody (1:1000 dilution; Abcam).

Techniques: Fluorescence, Incubation, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: α-Enolase reduces cerebrovascular Aβ deposits by protecting Aβ amyloid formation

doi: 10.1007/s00018-022-04493-x

Figure Lengend Snippet: Induction of ENO1 expression by Aβ. a, b ENO1 mRNA levels in cerebrovascular smooth muscle cells treated with freshly prepared Aβ40 (a) or Aβ42 (b) at a final concentration of 10 μM for 24 h, or treated with PBS (control). ENO1 mRNA levels were normalized to β-actin mRNA levels. c, d Representative immunoblots and quantification of the relative ENO1 protein levels of cultured cell lysates treated with freshly prepared Aβ40 (c) or Aβ42 (d) at a final concentration of 10 μM for 24 h, or treated with PBS (control). e, f Representative immunoblots and quantification of the relative ENO1 protein levels in culture medium treated with freshly prepared Aβ40 (e) or Aβ42 (f). g, h ENO1 mRNA levels in cerebrovascular smooth muscle cells treated with Aβ40 fibrils (g) or Aβ42 fibrils (h) at a final concentration of 10 μM for 24 h, or treated with PBS (control). ENO1 mRNA levels were normalized to β-actin mRNA levels. i, j Representative immunoblots and quantification of the relative ENO1 protein levels of cultured cell lysates treated with Aβ40 fibrils (i) or Aβ42 fibrils (j) at a final concentration of 10 μM for 24 h, or treated with PBS (control). k, l Representative immunoblots and quantification of the relative ENO1 protein levels in culture medium treated with Aβ40 fibrils (k) or Aβ42 fibrils (l). m ENO1 mRNA levels in lysates of cultured murine cerebrovascular smooth muscle cells treated with H2O2 for 24 h. n, o Representative immunofluorescence images—ENO1 (green) and Aβ (red)—of cerebrovascular Aβ deposits (n) and parenchymal Aβ deposits (o) in 18-month-old APP23 mice. p, q ENO1 mRNA levels in the hippocampus, cerebral cortex, and cerebral blood vessels from 4-month-old wild type (n = 6) and APP23 (n = 4) mice (p), and from 18-month-old wild type (n = 7) and APP23 (n = 5) mice (q). ENO1 mRNA and protein levels were normalized to β-actin mRNA and protein levels. Data are shown as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. NS not significant. Scale bars = 50 μm

Article Snippet: We then used Western blotting to quantify nitrated ENO1 levels using an anti-ENO1 antibody (1:1000 dilution; Proteintech Group) and an anti-3-nitrotyrosine antibody (1:1000 dilution; Abcam).

Techniques: Expressing, Concentration Assay, Western Blot, Cell Culture, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: α-Enolase reduces cerebrovascular Aβ deposits by protecting Aβ amyloid formation

doi: 10.1007/s00018-022-04493-x

Figure Lengend Snippet: ENO1-induced reduced Aβ cytotoxicity in cerebrovascular smooth muscle cells. a Significantly reduced Aβ40-induced caspase-3/7 activity after administration of ENO1 together with Aβ40 for 24 h. b Representative images from immunofluorescence studies with anti-human Aβ antibody (1:100 dilution, 6E10) in cultured cells treated with Aβ40 (10 μM) for 24 h in the absence (left) or presence (right) of ENO1 (1 μM). Scale bars = 100 μm. c Quantification of the percentages of Aβ40-positive areas obtained by averaging 10 random fields of cultured cells in the absence or presence of ENO1. d Enhancement of Aβ40-induced caspase-3/7 activity by ENO1 knockdown. Data are shown as mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001. NS not significant

Article Snippet: We then used Western blotting to quantify nitrated ENO1 levels using an anti-ENO1 antibody (1:1000 dilution; Proteintech Group) and an anti-3-nitrotyrosine antibody (1:1000 dilution; Abcam).

Techniques: Activity Assay, Immunofluorescence, Cell Culture

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: α-Enolase reduces cerebrovascular Aβ deposits by protecting Aβ amyloid formation

doi: 10.1007/s00018-022-04493-x

Figure Lengend Snippet: Reversal of the inhibitory effects of ENO1 on Aβ fibril formation by Pefabloc, a serine protease inhibitor. a ThT fluorescence kinetics of Aβ40 (50 μM) incubated with ENO1 (1 μM) and Pefabloc (pefa) for 7 days. b Representative TEM images of Aβ40 incubated with ENO1 and Pefabloc. Scale bars = 2 μm. c Representative immunoblot of Aβ40 incubated with ENO1 and Pefabloc, analyzed by using anti-human Aβ antibody (6E10), with quantification of the relative intensities of Aβ fibrils. d Total amount of uncleaved Aβ (left), and total amount of cleaved Aβ (right), with quantification of the relative densities of Aβ. e–h ThT fluorescence kinetics of Aβ40 (50 μM) incubated with ENO1 (1 μM) and protease inhibitors (described below) for 7 days. i-l Representative TEM images of Aβ40 incubated with ENO1 and protease inhibitors. Scale bars = 2 μm. m-p Representative immunoblots of Aβ40 incubated with ENO1 and protease inhibitors, analyzed using anti-human Aβ antibody (6E10), with quantification of the relative intensities of Aβ fibrils. Protease inhibitors were used at the indicated concentrations: Pefabloc SC, a serine protease inhibitor, 2.5 mM, 1 mM; pepstatin A, an aspartic protease inhibitor, 10 μM, 1 μM; E-64, a cysteine protease inhibitor, 1 mM, 100 μM; phosphoramidon, a metalloprotease inhibitor, 1 mM, 100 μM; and EDTA, a metalloprotease inhibitor, 5 mM, 1 mM. Data are shown as mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001. NS not significant

Article Snippet: We then used Western blotting to quantify nitrated ENO1 levels using an anti-ENO1 antibody (1:1000 dilution; Proteintech Group) and an anti-3-nitrotyrosine antibody (1:1000 dilution; Abcam).

Techniques: Protease Inhibitor, Fluorescence, Incubation, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: α-Enolase reduces cerebrovascular Aβ deposits by protecting Aβ amyloid formation

doi: 10.1007/s00018-022-04493-x

Figure Lengend Snippet: Nitrative modification of ENO1 in vivo and in vitro. a Representative immunoblots of immunoprecipitated ENO1 from the hippocampus, cerebral cortex, and cerebral blood vessels obtained from 18-month-old wild-type and APP23 mice; probes were anti-3-nitrotyrosine and anti-ENO1 antibodies. Quantified nitrated ENO1 levels in the hippocampus, cerebral cortex, and cerebral blood vessels appear below the immunoblots. Data were normalized to ENO1 protein levels. b Representative immunoblots of ENO1, analyzed using anti-3-nitrotyrosine and anti-ENO1 antibodies, with quantification of the relative intensity of nitrated ENO1. Data were normalized to ENO1 protein levels. c Quantified ENO1 enzymatic activity of non-nitrated ENO1 and nitrated ENO1. d, e Representative immunofluorescence images—ENO1 (green) and 3-nitrotyrosine (red)—of cerebrovascular Aβ deposits (d) and parenchymal Aβ deposits (e) in 18-month-old APP23 mice. Data are shown as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. NS not significant. Scale bars = 50 μm

Article Snippet: We then used Western blotting to quantify nitrated ENO1 levels using an anti-ENO1 antibody (1:1000 dilution; Proteintech Group) and an anti-3-nitrotyrosine antibody (1:1000 dilution; Abcam).

Techniques: Modification, In Vivo, In Vitro, Western Blot, Immunoprecipitation, Activity Assay, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: α-Enolase reduces cerebrovascular Aβ deposits by protecting Aβ amyloid formation

doi: 10.1007/s00018-022-04493-x

Figure Lengend Snippet: Elimination by heat-inactivated ENO1 of the inhibitory effects of ENO1 on Aβ40 fibril formation. a Quantified enzymatic activities of ENO1. b ThT fluorescence kinetics of Aβ40 (50 μM) incubated with heat-treated ENO1 (60 ℃ or 90 ℃ for 5 min) at a final concentration of 1 μM for 7 days. c Representative TEM images of Aβ40 incubated alone, with ENO1, and with heat-treated ENO1. Scale bars = 2 μm. d Representative immunoblot of Aβ40 incubated alone, with ENO1, and with heat-treated ENO1, analyzed using anti-human Aβ antibody (6E10), with quantification of the relative intensity of Aβ40 fibrils. e The bar graph shows ThT fluorescence values on Day 3; Aβ40 fibrils (50 μM) incubated with heat-treated ENO1 (90 ℃ for 5 min) at a final concentration of 1 μM for 3 days. f Representative TEM images Aβ40 fibrils incubated alone, or incubated with heat-treated ENO1 (90 ℃ for 5 min). Scale bars = 2 μm. g Representative immunoblot of Aβ40 fibrils incubated alone, or incubated with heat-treated ENO1 (90 ℃ for 5 min), analyzed using anti-human Aβ antibody (6E10), with quantification of relative intensity of Aβ40 fibrils. h ThT fluorescence kinetics of Aβ42 (10 μM) incubated with heat-treated ENO1 at a final concentration of 1 μM for 7 days. i Representative TEM images of Aβ42 incubated alone, with ENO1, and with heat-treated ENO1 (60 ℃ or 90 ℃ for 5 min). Scale bars = 2 μm. j Representative immunoblot of Aβ42 incubated with heat-treated ENO1, analyzed using anti-human Aβ antibody (6E10), with quantification of the relative densities of Aβ42 fibrils. Data are shown as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. NS not significant

Article Snippet: We then used Western blotting to quantify nitrated ENO1 levels using an anti-ENO1 antibody (1:1000 dilution; Proteintech Group) and an anti-3-nitrotyrosine antibody (1:1000 dilution; Abcam).

Techniques: Fluorescence, Incubation, Concentration Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: A The expression level of ENO1 in LO2exo, HepG2exo, MHCC97Lexo, and HCCLM3exo determined by proteomics analysis. B Western blot analysis of ENO1 levels in LO2, HepG2, MHCC97L, and HCCLM3 cells. A representative of three independent experiments is shown. C Differential expression of ENO1 in 94 pairs of HCC and adjacent non-tumor tissues. D IHC staining of ENO1 expression in normal liver tissues, cirrhosis tissues, paired of HCC and non-tumor tissues and metastasis tissues. Representative photographs of ENO1 staining in different tissues are shown. Scale bar represent 800 μm and 100 μm. E Kaplan–Meier analysis of overall survival of 94 HCC patients with different ENO1 expression levels. F Kaplan–Meier analysis of overall survival of 365 HCC patients in the TCGA cohort. G Forest plot showing the association between ENO1 expression and HCC survival as revealed by univariate and multivariate analyses (HR, hazard ratio; CI, confidence interval). All data are represented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 according to two-tailed Student’s t -test.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: Correlations between ENO1 expression and clinicopathological parameters in patients with HCC.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: A CCK-8 assays, B colony formation assays, C wound healing assays, D transwell migration and invasion assays of HCCLM3 cells after ENO1 knockdown. E Western blot analysis of E-cadherin, N-cadherin and Vimentin levels in HCCLM3 cells after ENO1 knockdown. f CCK-8 assays, G colony formation assays, H transwell migration and invasion assays of MHCC97L and HepG2 cells after ENO1 overexpression. I Western blot analysis of E-cadherin, N-cadherin and Vimentin levels in MHCC97L and HepG2 cells after ENO1 overexpression. A representative of three independent experiments is shown. All data are represented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 according to two-tailed Student’s t -test.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: CCK-8 Assay, Migration, Western Blot, Over Expression, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: A Western blot analysis of ENO1 level in exosomes derived from HCCLM3-NC, HCCLM3-shENO1, MHCC97L-NC, MHCC97L-ENO1, HepG2-NC, and HepG2-ENO1 cells, and HA protein expression in MHCC97L-ENO1exos and HepG2-ENO1exos. B NTA analysis of the impact of altered ENO1 expression in HCC cells on the number of exosomes released. C Fluorescence microscopy analysis of PKH67-labeled exosomes with high ENO1 expression incorporation (green) by HCCLM3-shENO1, MHCC97L-NC, and HepG2-NC cells with relatively low ENO1 expression. Exosomes were isolated from HCCLM3-NC, MHCC97L-ENO1 and HepG2-ENO1 cells. Scale bar represent 10 μm. D Cell CCK-8 assays, E transwell migration and invasion assays of HCCLM3-shENO1, MHCC97L-NC, and HepG2-NC cells educated with HCCLM3-NCexos, MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 μg/ml). F Western blot analysis of ENO1, HA, E-cadherin, N-cadherin and Vimentin levels in HCCLM3-shENO1, MHCC97L-NC, and HepG2-NC cells educated with HCCLM3-NCexos, MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 μg/ml). G Evaluation of HCC metastasis by tumor size and number of nude mice with lung metastasis in mice injected in the tail vein with HCCLM3-shENO1 and MHCC97L-NC cells following education with HCCLM3-NCexos, MHCC97L-ENO1exos or self-secreted exosomes. For the in vivo experiments, four nude mice per group were used. A representative of three independent experiments is shown. All data are represented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 according to two-tailed Student’s t -test.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Western Blot, Derivative Assay, Expressing, Fluorescence, Microscopy, Labeling, Isolation, CCK-8 Assay, Migration, Injection, In Vivo, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: A Immunofluorescence quantification of integrin α6 and β4 expression in arbitrary units (a.u.) in MHCC97L-NC and HepG2-NC cells educated with MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 μg/ml). Scale bar represent 25 μm. B Western blot analysis of the impact of exosome-derived ENO1 on the expression of integrin α6β4 and the activation of integrin-mediated FAK/Src-p38MAPK pathway in MHCC97L-NC and HepG2-NC cells educated with MHCC97L-ENO1exos, HepG2-ENO1exos or self-secreted exosomes (50 μg/ml). A representative of three independent experiments is shown. All data are represented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 according to two-tailed Student’s t -test.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Immunofluorescence, Expressing, Western Blot, Derivative Assay, Activation Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Exosome-derived ENO1 regulates integrin α6β4 expression and promotes hepatocellular carcinoma growth and metastasis

doi: 10.1038/s41419-020-03179-1

Figure Lengend Snippet: Exosome-derived ENO1 facilitates the expression of integrin α6β4 and the activation of the FAK/Src pathway, supporting downstream signaling via p38MAPK in HCC cells.

Article Snippet: Then, primary antibodies against E-cadherin (1:5000, Proteintech, 20874-1-AP), N-cadherin (1:2000, Proteintech, 22018-1-AP), Vimentin (1:2000, Proteintech, 10366-1-AP), MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), GM130 (1:1000, Abcam, ab52649), GRP94 (1:1000, Abcam, ab238126), ALIX (1:10,000, Abcam, ab186429), TSG101 (1:1000, Abcam, ab125011), CD63 (1:1000, Abcam, ab134045), CD81 (1:1000, Abcam, ab109201), ENO1 (1:1000, Abcam, ab155102), HA (1:4000, Abcam, ab9110), integrin α1 (1:500, Proteintech, 22146-1-AP), integrin α2 (1:10,000, Abcam, ab133557), integrin α3 (1:500, Proteintech, 66070-1-Ig), integrin α5 (1:1000, Abcam, ab150361), integrin α6 (1:2000, Abcam, ab181551), integrin αV (1:5,000, Abcam, ab179475), integrin β1 (1:1000, Abcam, ab52971), integrin β3 (1:1000, Abcam, ab119992), integrin β4 (1:1000, Abcam, ab182120), integrin β6 (1:10,000, Abcam, ab187155), FAK (1:2000, Abcam, ab40794), FAK (Y397) (1:1000, Abcam, ab81298), FAK (Y576 + Y577) (1:50,000, Abcam, ab76244), FAK (Y861) (1:10,000, Abcam, ab81293), FAK (Y925) (1:1000, Abcam, ab230813), Src (1:10,000, Abcam, ab109381), Src (Y418) (1:1000, Abcam, ab40660), Src (Y419) (1:5000, Abcam, ab185617), Src (Y529) (1:5000, Abcam, ab32078), p38MAPK (1:1000, Abcam, ab170099), p38MAPK (T180 + Y182) (1:1000, Abcam, ab195049), GAPDH (1:10,000, Abbkine, A01020, USA), and β-tubulin (1:10,000, Abbkine, A01030) were diluted in primary antibody diluent (KeyGEN BioTECH) and incubated with membranes overnight at 4 °C.

Techniques: Derivative Assay, Expressing, Activation Assay

Journal: PLoS ONE

Article Title: Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

doi: 10.1371/journal.pone.0122994

Figure Lengend Snippet: Protein Identification Summary.

Article Snippet: Passive Lysis Buffer (Promega), PVDF membrane (Millipore), Luminata forte Western HRP substrate (Millipore), anti alpha-tubulin monoclonal antibody (Invitrogen Life Technologies), DAPI (Invitrogen Life Technologies), Primary antibodies- anti-Cyclophilin A antibody (ab58144), anti-eIF5A antibody [EP527Y] (ab32407), anti-ENO1 antibody (ab85086), anti-OTUB1 antibody (ab98280), anti-RhoGDI antibody [1F2] (ab118159), and anti-VDAC1/Porin antibody—N-terminal (ab135585) were purchased from abcam, UK.

Techniques: Sequencing, Binding Assay, Activity Assay, Translocation Assay, Transduction, Activation Assay, Conjugation Assay

Journal: PLoS ONE

Article Title: Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

doi: 10.1371/journal.pone.0122994

Figure Lengend Snippet: Western blotting of representative proteins revealed similar quantitative profiles as suggested by 2DGE analysis. Contrasting effect of Nef variants observed upon expression of the six proteins- Cyclophilin A, EIF5A-1, Rho GDI, VDAC1, OTUB1, and ENO1. alpha tubulin was used as a loading control. Data are presented as the mean ± SD of three independent experiment.

Article Snippet: Passive Lysis Buffer (Promega), PVDF membrane (Millipore), Luminata forte Western HRP substrate (Millipore), anti alpha-tubulin monoclonal antibody (Invitrogen Life Technologies), DAPI (Invitrogen Life Technologies), Primary antibodies- anti-Cyclophilin A antibody (ab58144), anti-eIF5A antibody [EP527Y] (ab32407), anti-ENO1 antibody (ab85086), anti-OTUB1 antibody (ab98280), anti-RhoGDI antibody [1F2] (ab118159), and anti-VDAC1/Porin antibody—N-terminal (ab135585) were purchased from abcam, UK.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

doi: 10.1371/journal.pone.0122994

Figure Lengend Snippet: Immunofluorescence study to compare cell surface expression of six proteins in SupT1 cells (1 μm section) was captured by Confocal microscopy at 40 X and 63 X magnification. Figure shows immuostained SupT1 cells for expression of Vector/Nef (green), respective proteins (red) with their nucleus stained with DAPI (blue). Fluorescence intensity was measured for 5–8 Nef/vector transfected cells from 6 different fields of each sample and mean was calculated. Data is presented as values from two different experiments. displays ENO1 and VDAC1 (A-C) and EIF5A, OTUB1, CYPA and RhoGDI (D-H).

Article Snippet: Passive Lysis Buffer (Promega), PVDF membrane (Millipore), Luminata forte Western HRP substrate (Millipore), anti alpha-tubulin monoclonal antibody (Invitrogen Life Technologies), DAPI (Invitrogen Life Technologies), Primary antibodies- anti-Cyclophilin A antibody (ab58144), anti-eIF5A antibody [EP527Y] (ab32407), anti-ENO1 antibody (ab85086), anti-OTUB1 antibody (ab98280), anti-RhoGDI antibody [1F2] (ab118159), and anti-VDAC1/Porin antibody—N-terminal (ab135585) were purchased from abcam, UK.

Techniques: Immunofluorescence, Expressing, Confocal Microscopy, Plasmid Preparation, Staining, Fluorescence, Transfection

Journal: PLoS ONE

Article Title: Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

doi: 10.1371/journal.pone.0122994

Figure Lengend Snippet: (A)Western blots showing reverse effect of Nef RP01 mutant upon downmodulation of α-enolase (ENO1) and VDAC1 as caused by Nef RP01. Alpha-tubulin was used as a loading control. (B)Graphs representing the difference in effect of Nef RP14 Nef RP01 and Nef RP01 mutant upon expression of α-enolase (ENO1) and VDAC1. Data are presented as the mean ± SD of two independent experiment.

Article Snippet: Passive Lysis Buffer (Promega), PVDF membrane (Millipore), Luminata forte Western HRP substrate (Millipore), anti alpha-tubulin monoclonal antibody (Invitrogen Life Technologies), DAPI (Invitrogen Life Technologies), Primary antibodies- anti-Cyclophilin A antibody (ab58144), anti-eIF5A antibody [EP527Y] (ab32407), anti-ENO1 antibody (ab85086), anti-OTUB1 antibody (ab98280), anti-RhoGDI antibody [1F2] (ab118159), and anti-VDAC1/Porin antibody—N-terminal (ab135585) were purchased from abcam, UK.

Techniques: Western Blot, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

doi: 10.1371/journal.pone.0122994

Figure Lengend Snippet: Predicted network map for Cyclophilin A, EIF5A-1, Rho GDI, VDAC1, OTUB1, and ENO1 with their top partners and association with HIV-1 Nef was created in STRING. Proteins in boxes were underexpressed proteins identified in our work. Rest of proteins were host proteins from the database. Protein labeled in Yellow is Nef, with known interaction with Cyclophilin A, RAC1, CDC42 and BCL2L1 (connected with red line) as given in HIV-1, Human Protein Interaction Database.

Article Snippet: Passive Lysis Buffer (Promega), PVDF membrane (Millipore), Luminata forte Western HRP substrate (Millipore), anti alpha-tubulin monoclonal antibody (Invitrogen Life Technologies), DAPI (Invitrogen Life Technologies), Primary antibodies- anti-Cyclophilin A antibody (ab58144), anti-eIF5A antibody [EP527Y] (ab32407), anti-ENO1 antibody (ab85086), anti-OTUB1 antibody (ab98280), anti-RhoGDI antibody [1F2] (ab118159), and anti-VDAC1/Porin antibody—N-terminal (ab135585) were purchased from abcam, UK.

Techniques: Labeling

Journal: BMC Veterinary Research

Article Title: Overexpression of α-enolase correlates with poor survival in canine mammary carcinoma

doi: 10.1186/1746-6148-7-62

Figure Lengend Snippet: Immunohistochemical semiquantitation of ENO1 expression with the Quick score in canine mammary tumor

Article Snippet: The lysates were resolved in a 10% SDS-containing polyacrylamide gel, blotted on a nitrocellulose membrane, and probed with anti-ENO1 antibody (clone 8G8, Abnova Co., Taipei, Taiwan) in 1:2000 dilution, or with pre-immunized mouse total IgG.

Techniques: Immunohistochemical staining, Expressing

Journal: BMC Veterinary Research

Article Title: Overexpression of α-enolase correlates with poor survival in canine mammary carcinoma

doi: 10.1186/1746-6148-7-62

Figure Lengend Snippet: Overexpression of ENO1 in canine mammary tumor

Article Snippet: The lysates were resolved in a 10% SDS-containing polyacrylamide gel, blotted on a nitrocellulose membrane, and probed with anti-ENO1 antibody (clone 8G8, Abnova Co., Taipei, Taiwan) in 1:2000 dilution, or with pre-immunized mouse total IgG.

Techniques: Over Expression, Expressing

Journal: BMC Veterinary Research

Article Title: Overexpression of α-enolase correlates with poor survival in canine mammary carcinoma

doi: 10.1186/1746-6148-7-62

Figure Lengend Snippet: Kaplan-Meier curves for cause-specific survival of canine mammary carcinoma patients with and without ENO1 overexpression . Sixteen of the fifty cases lacked survival data and were excluded from the analysis.

Article Snippet: The lysates were resolved in a 10% SDS-containing polyacrylamide gel, blotted on a nitrocellulose membrane, and probed with anti-ENO1 antibody (clone 8G8, Abnova Co., Taipei, Taiwan) in 1:2000 dilution, or with pre-immunized mouse total IgG.

Techniques: Over Expression

Journal: BMC Veterinary Research

Article Title: Overexpression of α-enolase correlates with poor survival in canine mammary carcinoma

doi: 10.1186/1746-6148-7-62

Figure Lengend Snippet: Overexpression of ENO1 and age in the Cox regression model for predicting Cause-specific survival

Article Snippet: The lysates were resolved in a 10% SDS-containing polyacrylamide gel, blotted on a nitrocellulose membrane, and probed with anti-ENO1 antibody (clone 8G8, Abnova Co., Taipei, Taiwan) in 1:2000 dilution, or with pre-immunized mouse total IgG.

Techniques: Over Expression