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: 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

Journal: Frontiers in Genetics

Article Title: Long Noncoding Ribonucleic Acid SNHG18 Promotes Glioma Cell Motility via Disruption of α-Enolase Nucleocytoplasmic Transport

doi: 10.3389/fgene.2019.01140

Figure Lengend Snippet: Top 10 proteins identified using in mass spectrometry analysis among those proteins pulled down using SNHG18.

Article Snippet: For the RIP assay, the anti-ENO1 antibody was provided by Abcam (# ab155102).

Techniques: Mass Spectrometry

Journal: Frontiers in Genetics

Article Title: Long Noncoding Ribonucleic Acid SNHG18 Promotes Glioma Cell Motility via Disruption of α-Enolase Nucleocytoplasmic Transport

doi: 10.3389/fgene.2019.01140

Figure Lengend Snippet: (A) The results of RNA pulldown using SNHG18 , its antisense RNA, and a negative control RNA, as analyzed using western blotting for α-enolase (ENO1). The input was the total protein used for RNA pulldown (mean ± SD, n = 3, *P < 0.05, Student's t-test). (B) The results of RIP assays assessed using real-time reverse transcription polymerase chain reaction (RT-PCR) for SNHG18 . Quantitative RT-PCR (mean ± SD, n = 3, *P < 0.05, Student's t-test) (C) and western blotting (D) analysis of ENO1 expression in M059K cells silenced for SNHG18 , in M059J cells overexpressing SNHG18 , and in their control groups (mean ± SD, n = 3; *P < 0.05, Student's t-test). (E) M059K cells silenced for SNHG18 stained for nuclei [2-(4-amidinophenyl)-1H-indole-6-carboxamidine, blue fluorescence], and ENO1 (anti-ENO1 monoclonal antibody followed by fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G, green fluorescence) (scale bar = 10 μm). (F) ENO1 expression in nuclear or cytoplasm extracts by western blotting.

Article Snippet: For the RIP assay, the anti-ENO1 antibody was provided by Abcam (# ab155102).

Techniques: Negative Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Staining, Fluorescence

Journal: Frontiers in Genetics

Article Title: Long Noncoding Ribonucleic Acid SNHG18 Promotes Glioma Cell Motility via Disruption of α-Enolase Nucleocytoplasmic Transport

doi: 10.3389/fgene.2019.01140

Figure Lengend Snippet: (A) Western blotting detection of the level of α-enolase (ENO1) in U251- SNHG18 cells transfected with an short interfering RNA (siRNA) targeting ENO1 (siENO1). (B) Quantitative analysis of Transwell invasion and wound-healing assays in SNHG18 overexpressed U251 cells treated with siENO1 and their control groups (mean ± SD, n = 3, *P < 0.05, Student's t-test). (C) Transwell invasion (Panel a-d) and wound-healing assays (Panel e-h), and immunofluorescence (Panel i-l) were performed in SNHG18 overexpressed U251 cells treated with siENO1 and their control groups. Panel i-l: Tetramethyl rhodamine iso-thiocyanate-conjugated phalloidin was used to detect the distribution of F-actin (red). The nuclei are stained blue with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine. Arrows show clustering of F-actin signals (Panel a-h: scale bar = 200 μm, Panel i-l: scale bar = 10 μm). (D) Western blotting to detect the levels of β-catenin, SNAIL, Vimentin, N-cadherin, SLUG, E-cadherin, MMP-2, MMP-9, and ENO1-total and ENO1-cyto (ENO1 in total cellular or cytoplasm) in U251- SNHG18 cells transfected with the siRNA targeting ENO1 (siENO1).

Article Snippet: For the RIP assay, the anti-ENO1 antibody was provided by Abcam (# ab155102).

Techniques: Western Blot, Transfection, Small Interfering RNA, Immunofluorescence, Staining