Journal: Neuromolecular Medicine

Article Title: Neuronal Subtype-Specific Expression of γ-Enolase: Its Role in Neuronal Differentiation

doi: 10.1007/s12017-025-08902-9

Figure Lengend Snippet: Expression patterns of α-enolase and γ-enolase in SH-SY5Y cells differentiated into specific neuronal subtypes. ( A , B ) ELISA results of α-enolase and γ-enolase expression in ( A ) dopaminergic-like neuronal cells differentiated with retinoic acid (RA) and phorbol 12-myristate 13-acetate (PMA) and cholinergic-like neuronal cells differentiated with RA and brain-derived neurotrophic factor (BDNF) and ( B ) adrenergic-like cells differentiated with dibutyryl cyclic AMP (dbcAMP). Data are shown as mean ± 95% CI from N = 2 independent experiments, each performed in duplicate. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: ( A ) α-enolase, p = 0.108; γ-enolase - Control vs. RA, p = 0.582, Control vs. RA + PMA, p = 0.061; Control vs. RA + BDNF, p = 0.001; RA vs. RA + PMA, p = 0.205; RA vs. RA + BDNF, p = 0.001; RA + PMA vs. RA + BDNF, p = 0.001; ( B ) α-enolase - Control vs. dbcAMP, p = 0.005; γ-enolase – Control vs. dbcAMP, p = 0.005. ( C , D ) Representative western blots (top) and quantification (bottom) of the expression of α-enolase and γ-enolase and the active-to-total γ-enolase ratio in ( C ) dopaminergic-, cholinergic-, and ( D ) adrenergic-like neuronal cells. Protein levels are normalized to GAPDH. Data are shown as mean ± 95% CI from N = 2–7 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: ( C ) α-enolase, p = 0.660; γ-enolase (total form), p = 0.224; γ-enolase (active form) - Control vs. RA, p = 0.070, Control vs. RA + PMA, p = 0.029; Control vs. RA + BDNF, p = 0.001; RA vs. RA + PMA, p = 0.900; RA vs. RA + BDNF, p = 0.002; RA + PMA vs. RA + BDNF, p = 0.005; γ-enolase (active/total form) - Control vs. RA, p = 0.726, Control vs. RA + PMA, p = 0.381; Control vs. RA + BDNF, p = 0.019; RA vs. RA + PMA, p = 0.900; RA vs. RA + BDNF, p = 0.158; RA + PMA vs. RA + BDNF, p = 0.425; ( D ) α-enolase - Control vs. dbcAMP, p = 0.378; γ-enolase (total from) - Control vs. dbcAMP, p = 0.130; γ-enolase (active from) - Control vs. dbcAMP, p = 0.150; γ-enolase (active/total from) - Control vs. dbcAMP, p = 0.123. Data were obtained after 7 days of differentiation into dopaminergic- and cholinergic-like neuronal cells and after 4 days of differentiation into adrenergic-like cells, and are expressed relative to the control

Article Snippet: Two specific mouse monoclonal antibodies were used: one raised against amino acids 416–433 of γ-enolase, which detects the C-terminal region and therefore recognizes only the full-length variant (1:250; sc-21738, Santa Cruz Biotechnology, Dallas, TX, USA), and another raised against amino acids 271–285, which detects both the full-length and C-terminally truncated (ΔC) variants, representing total γ-enolase expression (1:350; sc-21737, Santa Cruz Biotechnology).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Western Blot

Journal: Neuromolecular Medicine

Article Title: Neuronal Subtype-Specific Expression of γ-Enolase: Its Role in Neuronal Differentiation

doi: 10.1007/s12017-025-08902-9

Figure Lengend Snippet: Morphological changes and expression of β-tubulin after γ-enolase upregulation in differentiated SH-SY5Y neuronal subtypes. ( A ) Representative phase-contrast images of SH-SY5Y cells transfected with the ΔC γ-enolase pcDNA3-GFP and total γ-enolase pcDNA3-GFP plasmids. Black arrows indicate cell extensions. Scale bars: 400 μm. ( B ) Neurite lengths were measured in pixels using ImageJ when the extensions were longer than the cell diameter. Data are shown as mean ± 95% CI from N = 2 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: Control - Wild type vs. Total γ-enolase pcDNA3-GFP, p = 0.051; Wild type vs. ΔC γ-enolase pcDNA3-GFP, p = 0.069; Wild type vs. pcDNA3-GFP, p = 0.181; Total γ-enolase pcDNA3-GFP vs. ΔC γ-enolase pcDNA3-GFP, p = 0.900; Total γ-enolase pcDNA3-GFP vs. pcDNA3-GFP, p = 0.541; ΔC γ-enolase pcDNA3-GFP, vs. pcDNA3-GFP, p = 0.713; RA – Wild type vs. Total γ-enolase pcDNA3-GFP, p = 0.098; Wild type vs. ΔC γ-enolase pcDNA3-GFP, p = 0.390; Wild type vs. pcDNA3-GFP, p = 0.844; Total γ-enolase pcDNA3-GFP vs. ΔC γ-enolase pcDNA3-GFP, p = 0.529; Total γ-enolase pcDNA3-GFP vs. pcDNA3-GFP, p = 0.203; ΔC γ-enolase pcDNA3-GFP, vs. pcDNA3-GFP, p = 0.744; RA + PMA – Wild type vs. Total γ-enolase pcDNA3-GFP, p = 0.064; Wild type vs. ΔC γ-enolase pcDNA3-GFP, p = 0.900; Wild type vs. pcDNA3-GFP, p = 0.900; Total γ-enolase pcDNA3-GFP vs. ΔC γ-enolase pcDNA3-GFP, p = 0.064; Total γ-enolase pcDNA3-GFP vs. pcDNA3-GFP, p = 0.116; ΔC γ-enolase pcDNA3-GFP, vs. pcDNA3-GFP, p = 0.900; RA + BDNF – Wild type vs. Total γ-enolase pcDNA3-GFP, p = 0.042; Wild type vs. ΔC γ-enolase pcDNA3-GFP, p = 0.120; Wild type vs. pcDNA3-GFP, p = 0.623; Total γ-enolase pcDNA3-GFP vs. ΔC γ-enolase pcDNA3-GFP, p = 0.634; Total γ-enolase pcDNA3-GFP vs. pcDNA3-GFP, p = 0.123; ΔC γ-enolase pcDNA3-GFP, vs. pcDNA3-GFP, p = 0.400. ( C ) Representative western blots (top) and quantification (bottom) of the expression of β-tubulin for dopaminergic-like and cholinergic-like neuronal cells. Protein levels are normalized to GAPDH and expressed relative to the control (wild type). Data are shown as mean ± 95% CI from N = 3–4 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: Control - Wild type vs. Total γ-enolase pcDNA3-GFP, p = 0.020; Wild type vs. ΔC γ-enolase pcDNA3-GFP, p = 0.153; Total γ-enolase pcDNA3-GFP vs. ΔC γ-enolase pcDNA3-GFP, p = 0.525; RA – not significant, p = 0.998; RA + PMA - not significant, p = 0.686; RA + BDNF – not significant, p = 0.110. Data were obtained after 7 days of differentiation into dopaminergic- and cholinergic-like neuronal cells and are expressed relative to the control

Article Snippet: Two specific mouse monoclonal antibodies were used: one raised against amino acids 416–433 of γ-enolase, which detects the C-terminal region and therefore recognizes only the full-length variant (1:250; sc-21738, Santa Cruz Biotechnology, Dallas, TX, USA), and another raised against amino acids 271–285, which detects both the full-length and C-terminally truncated (ΔC) variants, representing total γ-enolase expression (1:350; sc-21737, Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Control, Western Blot

Journal: Neuromolecular Medicine

Article Title: Neuronal Subtype-Specific Expression of γ-Enolase: Its Role in Neuronal Differentiation

doi: 10.1007/s12017-025-08902-9

Figure Lengend Snippet: Morphological changes after γ-enolase silencing in differentiated Neuro-2a and LA-N-2 neuronal subtypes. ( A , B ) Representative phase-contrast images of differentiated Neuro-2a ( A ) and LA-N-2 ( B ) cells transfected with siRNA γ-enolase. Scale bars: 100 μm. Black arrows indicate cell extensions. ( C , D) Neurite lengths in differentiated Neuro-2a ( C ) and LA-N-2 ( D ) cells transfected with siRNA γ-enolase were determined in pixels when the extensions were longer than the cell diameter using ImageJ software. Data are shown as mean ± 95% CI from N = 2 independent experiments each in duplicate. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: ( C ) Control/Wild type vs. dbcAMP/Wild type, p = 0.004; dbcAMP/Wild type vs. dbcAMP/siRNA control, p = 0.203; dbcAMP/Wild type vs. dbcAMP/siRNA γ-enolase, p = 0.009; dbcAMP/siRNA control vs. dbcAMP/siRNA γ-enolase, p = 0.044; ( D ) Control/Wild type vs. RA/Wild type, p = 0.072; RA/Wild type vs. RA/siRNA control, p = 0.900; RA/Wild type vs. RA/siRNA γ-enolase, p = 0.065; RA/siRNA control vs. RA/siRNA γ-enolase, p = 0.026. Data were obtained after 4 days of differentiation into dopaminergic-like neuronal cells (Neuro-2a), and cholinergic-like neuronal cells (LA-N-2), and are expressed relative to the control

Article Snippet: Two specific mouse monoclonal antibodies were used: one raised against amino acids 416–433 of γ-enolase, which detects the C-terminal region and therefore recognizes only the full-length variant (1:250; sc-21738, Santa Cruz Biotechnology, Dallas, TX, USA), and another raised against amino acids 271–285, which detects both the full-length and C-terminally truncated (ΔC) variants, representing total γ-enolase expression (1:350; sc-21737, Santa Cruz Biotechnology).

Techniques: Transfection, Software, Control

Journal: Neuromolecular Medicine

Article Title: Neuronal Subtype-Specific Expression of γ-Enolase: Its Role in Neuronal Differentiation

doi: 10.1007/s12017-025-08902-9

Figure Lengend Snippet: The effects of γ-enolase peptide treatment on cell morphology, proliferation, and β-tubulin expression in differentiated SH-SY5Y neuronal subtypes. ( A ) Representative phase-contrast images of SH-SY5Y cells treated with γ-enolase peptide corresponding to the last 30 amino acids of the protein (γ-Eno). Scale bars: 100 μm. ( B) Neurite lengths in dopaminergic- and cholinergic-like neuronal cells treated with γ-Eno were determined in pixels when the extensions were longer than the cell diameter using ImageJ software. Data are shown as mean ± 95% CI from N = 2 independent experiments each in duplicate. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: Control/No peptide vs. RA/No peptide, p = 0.001; Control/No peptide vs. RA + PMA/No peptide, p = 0.002; Control/No peptide vs. RA + BDNF/No peptide, p = 0.006; Control/No peptide vs. Control/γ-Eno peptide, p = 0.675; RA/No peptide vs. RA/γ-Eno peptide, p = 0.418; RA + PMA/No peptide vs. RA + PMA/γ-Eno peptide, p = 0.149; RA + BDNF/No peptide vs. RA + BDNF/γ-Eno peptide, p = 0.161. ( C ) The proliferation rates of dopaminergic- and cholinergic-like neuronal cells treated with γ-Eno were assessed with carboxyfluorescein succinimidyl ester (CFSE) labeling and flow cytometry. Data are shown as mean ± 95% CI from N = 2 independent experiments, each in duplicate. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: Control/No peptide vs. RA/No peptide, p = 0.001; Control/No peptide vs. RA + PMA/No peptide, p = 0.001; Control/No peptide vs. RA + BDNF/No peptide, p = 0.005 Control/No peptide vs. Control/γ-Eno peptide, p = 0.680; RA/No peptide vs. RA/γ-Eno peptide, p = 0.769; RA + PMA/No peptide vs. RA + PMA/γ-Eno peptide, p = 0.900; RA + BDNF/No peptide vs. RA + BDNF/γ-Eno peptide, p = 0.020. ( D ) Representative western blots (top) and quantification (bottom) of the expression of β-tubulin. Protein levels are normalized to GAPDH and expressed relative to the control. Data are shown as mean ± 95% CI from N = 2 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: Control/No peptide vs. RA/No peptide, p = 0.430; Control/No peptide vs. RA + PMA/No peptide, p = 0.547; Control/No peptide vs. RA + BDNF/No peptide, p = 0.049 Control/No peptide vs. Control/γ-Eno peptide, p = 0.500; RA/No peptide vs. RA/γ-Eno peptide, p = 0.900; RA + PMA/No peptide vs. RA + PMA/γ-Eno peptide, p = 0.157; RA + BDNF/No peptide vs. RA + BDNF/γ-Eno peptide, p = 0.051. Data were obtained after 7 days of differentiation into dopaminergic- and cholinergic-like neuronal cells and are expressed relative to the control

Article Snippet: Two specific mouse monoclonal antibodies were used: one raised against amino acids 416–433 of γ-enolase, which detects the C-terminal region and therefore recognizes only the full-length variant (1:250; sc-21738, Santa Cruz Biotechnology, Dallas, TX, USA), and another raised against amino acids 271–285, which detects both the full-length and C-terminally truncated (ΔC) variants, representing total γ-enolase expression (1:350; sc-21737, Santa Cruz Biotechnology).

Techniques: Expressing, Software, Control, Labeling, Flow Cytometry, Western Blot

Journal: Neuromolecular Medicine

Article Title: Neuronal Subtype-Specific Expression of γ-Enolase: Its Role in Neuronal Differentiation

doi: 10.1007/s12017-025-08902-9

Figure Lengend Snippet: The effects of cathepsin X inhibition on cell morphology, proliferation, and expression of γ-enolase, β-tubulin in differentiated SH-SY5Y neuronal subtypes. ( A ) Representative phase-contrast images of SH-SY5Y cells treated with AMS36. Scale bars: 100 μm. ( B ) Neurite lengths in dopaminergic- and cholinergic-like neuronal cells treated with AMS36 were determined in pixels using ImageJ software when cell extensions were longer than the cell diameter. Data are shown as mean ± 95% CI from N = 2 independent experiments, each in duplicate. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: Control/No inhibitor vs. RA/No inhibitor, p = 0.003; Control/No inhibitor vs. RA + PMA/No inhibitor, p = 0.038; Control/No inhibitor vs. RA + BDNF/No inhibitor, p = 0.009; Control/No inhibitor vs. Control/AMS36, p = 0.094; RA/No inhibitor vs. RA/AMS36, p = 0.004; RA + PMA/No inhibitor vs. RA + PMA/AMS36, p = 0.614; RA + BDNF/No inhibitor vs. RA + BDNF/AMS36, p = 0.008. ( C ) The proliferation rates of dopaminergic- and cholinergic-like neuronal cells treated with AMS36 were assessed with carboxyfluorescein succinimidyl ester (CFSE) labeling and flow cytometry. Data are shown as mean ± 95% CI from N = 3 independent experiments, each in duplicate. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: Control/No inhibitor vs. RA/No inhibitor, p = 0.001; Control/No inhibitor vs. RA + PMA/No inhibitor, p = 0.001; Control/No inhibitor vs. RA + BDNF/No inhibitor, p = 0.001; Control/No inhibitor vs. Control/AMS36, p = 0.900; RA/No inhibitor vs. RA/AMS36, p = 0.156; RA + PMA/No inhibitor vs. RA + PMA/AMS36, p = 0.290; RA + BDNF/No inhibitor vs. RA + BDNF/AMS36, p = 0.088. (D) Representative western blots (top) and quantification (bottom) of the expression of β-tubulin. Protein levels are normalized to GAPDH and expressed relative to the control. Data are shown as mean ± 95% CI from N = 2 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test. Exact p-values: Control/No inhibitor vs. RA/No inhibitor, p = 0.006; Control/No inhibitor vs. RA + PMA/No inhibitor, p = 0.900; Control/No inhibitor vs. RA + BDNF/No inhibitor, p = 0.032; Control/No inhibitor vs. Control/AMS36, p = 0.900; RA/No inhibitor vs. RA/AMS36, p = 0.831; RA + PMA/No inhibitor vs. RA + PMA/AMS36, p = 0.268; RA + BDNF/No inhibitor vs. RA + BDNF/AMS36, p = 0.278. Data were obtained after 7 days of differentiation into dopaminergic- and cholinergic-like neuronal cells and are expressed relative to the control

Article Snippet: Two specific mouse monoclonal antibodies were used: one raised against amino acids 416–433 of γ-enolase, which detects the C-terminal region and therefore recognizes only the full-length variant (1:250; sc-21738, Santa Cruz Biotechnology, Dallas, TX, USA), and another raised against amino acids 271–285, which detects both the full-length and C-terminally truncated (ΔC) variants, representing total γ-enolase expression (1:350; sc-21737, Santa Cruz Biotechnology).

Techniques: Inhibition, Expressing, Software, Control, Labeling, Flow Cytometry, Western Blot