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( A ) Immunoblot of nuclear c-MYC and Neurogenin-2 (NGN2) in unedited patient 1 NPCs treated with DMSO or 20 μM nocodazole (Noc) for 7 days. TBP was used as a loading control. ( B ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO or 20 μM Noc at the NPC stage and throughout 14 day neuronal differentiation. ( C ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs transduced with lentivirus expressing control <t>shRNA</t> (shSCR) or shMYC. ( D ) Quantification of neurite lengths in patient 1 NPCs transduced with shSCR or shMYC. The dashed line denotes the mean neurite length in corrected neurons. ( E ) Representative recordings of intracellular Ca 2+ dynamics in neurons transduced with shSCR ( n = 29) or shMYC ( n = 24), captured every 10 seconds using Fura-2 ratio imaging during 45 mM KCl application. Neurons were differentiated for 47 days. ( F ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs treated with DMSO or 50 μM EN4 for the indicated durations. ( G ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO, EN4 throughout differentiation, EN4 at the NPC stage and throughout differentiation, or EN4 only at the NPC stage. The dashed line indicates the corrected neuron mean. ( H ) Representative Ca 2+ recordings in neurons treated with DMSO ( n = 28) or 50 μM EN4 ( n = 26), acquired as in E . For B , D , and G , neurite lengths were measured using the SNT plug-in in ImageJ. Data are shown as mean ± 1 SEM. Significance was determined by unpaired 2-tailed Student’s t test for B and D or 1-way ANOVA with Tukey’s HSD test for G .
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( A ) Immunoblot of nuclear c-MYC and Neurogenin-2 (NGN2) in unedited patient 1 NPCs treated with DMSO or 20 μM nocodazole (Noc) for 7 days. TBP was used as a loading control. ( B ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO or 20 μM Noc at the NPC stage and throughout 14 day neuronal differentiation. ( C ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs transduced with lentivirus expressing control <t>shRNA</t> (shSCR) or shMYC. ( D ) Quantification of neurite lengths in patient 1 NPCs transduced with shSCR or shMYC. The dashed line denotes the mean neurite length in corrected neurons. ( E ) Representative recordings of intracellular Ca 2+ dynamics in neurons transduced with shSCR ( n = 29) or shMYC ( n = 24), captured every 10 seconds using Fura-2 ratio imaging during 45 mM KCl application. Neurons were differentiated for 47 days. ( F ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs treated with DMSO or 50 μM EN4 for the indicated durations. ( G ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO, EN4 throughout differentiation, EN4 at the NPC stage and throughout differentiation, or EN4 only at the NPC stage. The dashed line indicates the corrected neuron mean. ( H ) Representative Ca 2+ recordings in neurons treated with DMSO ( n = 28) or 50 μM EN4 ( n = 26), acquired as in E . For B , D , and G , neurite lengths were measured using the SNT plug-in in ImageJ. Data are shown as mean ± 1 SEM. Significance was determined by unpaired 2-tailed Student’s t test for B and D or 1-way ANOVA with Tukey’s HSD test for G .
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( A ) Immunoblot of nuclear c-MYC and Neurogenin-2 (NGN2) in unedited patient 1 NPCs treated with DMSO or 20 μM nocodazole (Noc) for 7 days. TBP was used as a loading control. ( B ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO or 20 μM Noc at the NPC stage and throughout 14 day neuronal differentiation. ( C ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs transduced with lentivirus expressing control <t>shRNA</t> (shSCR) or shMYC. ( D ) Quantification of neurite lengths in patient 1 NPCs transduced with shSCR or shMYC. The dashed line denotes the mean neurite length in corrected neurons. ( E ) Representative recordings of intracellular Ca 2+ dynamics in neurons transduced with shSCR ( n = 29) or shMYC ( n = 24), captured every 10 seconds using Fura-2 ratio imaging during 45 mM KCl application. Neurons were differentiated for 47 days. ( F ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs treated with DMSO or 50 μM EN4 for the indicated durations. ( G ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO, EN4 throughout differentiation, EN4 at the NPC stage and throughout differentiation, or EN4 only at the NPC stage. The dashed line indicates the corrected neuron mean. ( H ) Representative Ca 2+ recordings in neurons treated with DMSO ( n = 28) or 50 μM EN4 ( n = 26), acquired as in E . For B , D , and G , neurite lengths were measured using the SNT plug-in in ImageJ. Data are shown as mean ± 1 SEM. Significance was determined by unpaired 2-tailed Student’s t test for B and D or 1-way ANOVA with Tukey’s HSD test for G .
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( A ) Immunoblot of nuclear c-MYC and Neurogenin-2 (NGN2) in unedited patient 1 NPCs treated with DMSO or 20 μM nocodazole (Noc) for 7 days. TBP was used as a loading control. ( B ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO or 20 μM Noc at the NPC stage and throughout 14 day neuronal differentiation. ( C ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs transduced with lentivirus expressing control <t>shRNA</t> (shSCR) or shMYC. ( D ) Quantification of neurite lengths in patient 1 NPCs transduced with shSCR or shMYC. The dashed line denotes the mean neurite length in corrected neurons. ( E ) Representative recordings of intracellular Ca 2+ dynamics in neurons transduced with shSCR ( n = 29) or shMYC ( n = 24), captured every 10 seconds using Fura-2 ratio imaging during 45 mM KCl application. Neurons were differentiated for 47 days. ( F ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs treated with DMSO or 50 μM EN4 for the indicated durations. ( G ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO, EN4 throughout differentiation, EN4 at the NPC stage and throughout differentiation, or EN4 only at the NPC stage. The dashed line indicates the corrected neuron mean. ( H ) Representative Ca 2+ recordings in neurons treated with DMSO ( n = 28) or 50 μM EN4 ( n = 26), acquired as in E . For B , D , and G , neurite lengths were measured using the SNT plug-in in ImageJ. Data are shown as mean ± 1 SEM. Significance was determined by unpaired 2-tailed Student’s t test for B and D or 1-way ANOVA with Tukey’s HSD test for G .
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( A ) Immunoblot of nuclear c-MYC and Neurogenin-2 (NGN2) in unedited patient 1 NPCs treated with DMSO or 20 μM nocodazole (Noc) for 7 days. TBP was used as a loading control. ( B ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO or 20 μM Noc at the NPC stage and throughout 14 day neuronal differentiation. ( C ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs transduced with lentivirus expressing control <t>shRNA</t> (shSCR) or shMYC. ( D ) Quantification of neurite lengths in patient 1 NPCs transduced with shSCR or shMYC. The dashed line denotes the mean neurite length in corrected neurons. ( E ) Representative recordings of intracellular Ca 2+ dynamics in neurons transduced with shSCR ( n = 29) or shMYC ( n = 24), captured every 10 seconds using Fura-2 ratio imaging during 45 mM KCl application. Neurons were differentiated for 47 days. ( F ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs treated with DMSO or 50 μM EN4 for the indicated durations. ( G ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO, EN4 throughout differentiation, EN4 at the NPC stage and throughout differentiation, or EN4 only at the NPC stage. The dashed line indicates the corrected neuron mean. ( H ) Representative Ca 2+ recordings in neurons treated with DMSO ( n = 28) or 50 μM EN4 ( n = 26), acquired as in E . For B , D , and G , neurite lengths were measured using the SNT plug-in in ImageJ. Data are shown as mean ± 1 SEM. Significance was determined by unpaired 2-tailed Student’s t test for B and D or 1-way ANOVA with Tukey’s HSD test for G .
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Image Search Results


( A ) Immunoblot of nuclear c-MYC and Neurogenin-2 (NGN2) in unedited patient 1 NPCs treated with DMSO or 20 μM nocodazole (Noc) for 7 days. TBP was used as a loading control. ( B ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO or 20 μM Noc at the NPC stage and throughout 14 day neuronal differentiation. ( C ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs transduced with lentivirus expressing control shRNA (shSCR) or shMYC. ( D ) Quantification of neurite lengths in patient 1 NPCs transduced with shSCR or shMYC. The dashed line denotes the mean neurite length in corrected neurons. ( E ) Representative recordings of intracellular Ca 2+ dynamics in neurons transduced with shSCR ( n = 29) or shMYC ( n = 24), captured every 10 seconds using Fura-2 ratio imaging during 45 mM KCl application. Neurons were differentiated for 47 days. ( F ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs treated with DMSO or 50 μM EN4 for the indicated durations. ( G ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO, EN4 throughout differentiation, EN4 at the NPC stage and throughout differentiation, or EN4 only at the NPC stage. The dashed line indicates the corrected neuron mean. ( H ) Representative Ca 2+ recordings in neurons treated with DMSO ( n = 28) or 50 μM EN4 ( n = 26), acquired as in E . For B , D , and G , neurite lengths were measured using the SNT plug-in in ImageJ. Data are shown as mean ± 1 SEM. Significance was determined by unpaired 2-tailed Student’s t test for B and D or 1-way ANOVA with Tukey’s HSD test for G .

Journal: The Journal of Clinical Investigation

Article Title: l -2-Hydroxyglutarate impairs neuronal differentiation through epigenetic activation of MYC expression

doi: 10.1172/JCI197010

Figure Lengend Snippet: ( A ) Immunoblot of nuclear c-MYC and Neurogenin-2 (NGN2) in unedited patient 1 NPCs treated with DMSO or 20 μM nocodazole (Noc) for 7 days. TBP was used as a loading control. ( B ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO or 20 μM Noc at the NPC stage and throughout 14 day neuronal differentiation. ( C ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs transduced with lentivirus expressing control shRNA (shSCR) or shMYC. ( D ) Quantification of neurite lengths in patient 1 NPCs transduced with shSCR or shMYC. The dashed line denotes the mean neurite length in corrected neurons. ( E ) Representative recordings of intracellular Ca 2+ dynamics in neurons transduced with shSCR ( n = 29) or shMYC ( n = 24), captured every 10 seconds using Fura-2 ratio imaging during 45 mM KCl application. Neurons were differentiated for 47 days. ( F ) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs treated with DMSO or 50 μM EN4 for the indicated durations. ( G ) Quantification of neurite lengths in patient 1 NPCs treated with DMSO, EN4 throughout differentiation, EN4 at the NPC stage and throughout differentiation, or EN4 only at the NPC stage. The dashed line indicates the corrected neuron mean. ( H ) Representative Ca 2+ recordings in neurons treated with DMSO ( n = 28) or 50 μM EN4 ( n = 26), acquired as in E . For B , D , and G , neurite lengths were measured using the SNT plug-in in ImageJ. Data are shown as mean ± 1 SEM. Significance was determined by unpaired 2-tailed Student’s t test for B and D or 1-way ANOVA with Tukey’s HSD test for G .

Article Snippet: MYC knockdown was performed using lentiviral shRNA constructs targeting human MYC (OriGene), with a nontargeting shRNA (shSCR) as control.

Techniques: Western Blot, Control, Transduction, Expressing, shRNA, Imaging