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. 2019 Jul 15;86(2):120–130. doi: 10.1016/j.biopsych.2019.03.977

Figure 3.

Figure 3

Knockdown of NT5C2 in human neural progenitor cells is associated with differential phosphorylation of adenosine monophostate-activated protein kinase (AMPK) and ribosomal protein S6 (RPS6). (A) The efficiency of the small interfering RNA (siRNA) transfection was determined by uptake of BLOCK-iT, a fluorescently labeled oligonucleotide. (B)NT5C2 expression was significantly reduced in knockdown cultures (linear regressions covarying for biological replicates, Tukey post hoc tests, **p < .01, *p < .05). (C, D) siRNA treatments significantly reduce NT5C2 expression in independent human neural progenitor cell cultures at the protein level (one-way analysis of variance, Tukey post hoc tests, ***p < .001). (E) The NT5C2 knockdown was associated with increased total AMPK alpha and phosphorylated AMPK alpha (pAMPK) (Thr172) in human neural progenitor cells (Kruskal-Wallis test, Dunn’s post hoc tests, **p < .01, *p < .05). (F) The knockdown did not alter total RPS6 levels but was associated with increased phosphorylated RPS6 (prpS6) (Ser235/Ser236). siRNA A was associated with a mean 23% increase in phosphorylation (Kruskal-Wallis test, Dunn’s test, *p < .05), and siRNA B was associated with a modest 10% mean increase, which was not significant after correction (p = .09). Full blots for panels (E and F) are available in Supplemental Figure S8. (G) The overexpression of NT5C2 in human embryonic kidney 293T (HEK293T) cells causes a significant decrease in phosphorylated AMPK alpha levels and in total RPS6, and a significant increase in phosphorylated RPS6 (t test, ***p < .001, *p < .05). Full blots are available in Supplemental Figure S9. ddCt, delta-delta cycle threshold; ICC, immunocytochemistry; n.s., not significant; qPCR, quantitative polymerase chain reaction.