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. 2016 Jun 10;5:e13374. doi: 10.7554/eLife.13374

Figure 3. Characterization of HK2 and LDHA in NPCs, differentiated neurons and neurons directly converted from fibroblasts.

(A) Immunoblotting analysis of the representative metabolic genes in glycolysis, tricarboxylic acid cycle (TCA) pathways. 20 µg of protein lysate from NPCs and from neurons differentiated for 3, 7 and 21 days (D3, D7, D21) were loaded. (B) Immunostaining analysis of HK2 and LDHA in NPCs and 3-week neurons. (C) Effects of HK2 or LDHA knockdown on NPC proliferation. NPCs at early passage (P2) were seeded in 24-well plates one day before infection. The NPC number was determined at 5 days after infection with lenti-shRNA virus against HK2 or LDHA. Two effective shRNA lentiviral vectors targeting different regions of HK2 or LDHA were used. Scramble shRNA vector was used as control. Error bars represent ± SD, n= 3. The knockdown efficiency was confirmed by immunoblotting. (D, E) Immunostaining analysis of LDHA in neurons directly converted from fibroblasts. Two protocols were applied: one was to knockdown PTB1, a single RNA-binding protein, and the other was to overexpress proneuronal transcription factors Ngn2 and Ascl1. Tuj1 (ß-III tubulin) and Tau were stained as early and mature neuronal markers, respectively. The above experiments were repeated at least three times. (F) ChIP analysis of HK2 and LDHA promoters using anti-cMYC or N-MYC antibodies and rabbit IgG as control. Chromatin were prepared from NPCs. The enrichment values are shown as percentage normalized to input. N.C. stands for non-specific control. Bars are mean ± SD, n= 3. Immunoblotting and real time PCR analysis of HK2 and LDHA expression in NPCs with inducible c-MYC. mRNA expression levels of HK2 and LDHA relative to those from non-induction control were calculated after normalization to β-actin. Bars are mean ± SD, n= 3. (G) Immunoblotting analysis of c-MYC, N-MYC and Max in NPCs and 3-week neurons. (Figure 3—source data 1).

DOI: http://dx.doi.org/10.7554/eLife.13374.017

Figure 3—source data 1. Knockdown effect on NPC proliferation and Myc control of HK2 and LDHA in NPCs.
DOI: http://dx.doi.org/10.7554/eLife.13374.018
elife-13374-fig3-data1.xlsx (10KB, xlsx)
DOI: 10.7554/eLife.13374.018

Figure 3.

Figure 3—figure supplement 1. Immunoblotting analysis of mouse HK1, HK2 and LDHA in mouse NPCs derived from embryonic stem cell (ES-E14TG2a), 2-week differentiated neurons and embryonic neurons at E18.

Figure 3—figure supplement 1.

The relative mRNA expression levels of HK1, HK2 and LDHA in neurons were calculated against the levels in NPCs. Bars are mean ± SD, n= 3. (Figure 3—figure supplement 1—source data 1).
DOI: http://dx.doi.org/10.7554/eLife.13374.019
Figure 3—figure supplement 1—source data 1. RT-PCR analysis of HK1, HK2 and LDHA during mouse neuronal differentiation.
DOI: http://dx.doi.org/10.7554/eLife.13374.020
elife-13374-fig3-figsupp1-data1.xlsx (8.3KB, xlsx)
DOI: 10.7554/eLife.13374.020
Figure 3—figure supplement 2. Immunoblotting and real time PCR analysis of HK2 expression in neurons with inducible c-MYC.

Figure 3—figure supplement 2.

mRNA expression levels of HK2 relative to those from non-induction control were calculated after normalization to β-actin. Bars are mean ± SD, n= 3. (Figure 3—figure supplement 2—source data 1).
DOI: http://dx.doi.org/10.7554/eLife.13374.021
Figure 3—figure supplement 2—source data 1. Activation of HK2 by ectopic c-Myc expression in neuron.
DOI: http://dx.doi.org/10.7554/eLife.13374.022
elife-13374-fig3-figsupp2-data1.xlsx (8.1KB, xlsx)
DOI: 10.7554/eLife.13374.022
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