Fig. 7.

FOXG1 overexpression affects miR200 family levels in conditions of increased expression of pri-miR200 in a DDX5-dependent manner. a qRT-PCRs of pri-miR200b/a/429 of N2a cells overexpressing FOXG1 and miR200 family using pCX-miR200b/a/429 compared to an empty vector. One-sample Student’s t test. n = 3. b Representative northern blots of precursor (pre-) and mature miR200b/a/429 in N2a cells using pCX-miR200b/a/429. Shown are control-transfected and FOXG1 overexpression in N2a cells probed for pre/miR200a (upper panel), pre-miR200b (middle panel) and pre-miR429 (lower panel) as well as U6 loading control. c Densitometric quantification of the northern blot bands for precursors of miR200b (isoforms I and II) and miR429 (left panels) and mature miR200b/a/429 (right panels). Dashed lines indicate expression levels of control cells. FOXG1 overexpression does not alter expression levels of precursor and mature miR200 family members. Mean with SEM, **p < 0.01, one-sample Student’s t test. n = 3. d qRT-PCRs of pri-miR200b/a/429 of N2a cells overexpressing miR200 family using pmiRGlo-miR200b/a/429 and FOXG1 compared to empty vector. pri-miR200 levels increase upon FOXG1 overexpression. Mean with SEM, *p < 0.05, **p < 0.01, one-sample Student’s t test. n = 3–6. e qRT-PCR results confirm increased Foxg1 and unchanged Ddx5 expression. Mean with SEM, **p < 0.01, one-sample Student’s t test. n = 4. f qRT-PCRs of mature miR200b/a/429 of N2a cells overexpressing miR200 family from pmiRGlo-miR200b/a/429 plasmid and FOXG1 compared to empty vector expression. Levels of mature miR200 increase statistically significant. Mean with SEM, **p < 0.01, ***p < 0.001, one-sample Student’s t test. n = 5–6. g Reduced luciferase activity indicates an increased turnover of pri-miR200b/a/429 in the presence of FOXG1. Mean with SEM, **p < 0.01, one-sample Student’s t test. n = 4. h qRT-PCRs of pri-miR200b/a/429 of N2a cells overexpressing miR200 family using pmiRGlo-miR200b/a/429 and FOXG1 as well as simultaneous Ddx5 KD, compared to FOXG1 overexpression and scrambled control. pri-miR200 levels are unaffected by Ddx5 KD. Mean with SEM, one-sample Student’s t test. n = 3–6. i qRT-PCR results confirm decreased Ddx5 levels after Ddx5 KD and unchanged Foxg1 expression. Mean with SEM, *p < 0.05, one-sample Student’s t test. n = 4. j qRT-PCRs of mature miR200b/a/429 in N2a cells after overexpressing miR200 family from pmiRGlo-miR200b/a/429 plasmid and FOXG1 as well as simultaneous Ddx5 KD compared to FOXG1 overexpression and scrambled control. Levels of mature miR200 decrease significantly after reduction of DDX5 in FOXG1 expressing cells. Mean with SEM, *p < 0.05, **p < 0.01, one-sample Student’s t test. n = 5–6. k Reduced luciferase activity indicates an increased turnover of pri-miR200b/a/429 after Ddx5 KD and FOXG1 overexpression when compared to cells overexpressing FOXG1 and scrambled control. Mean with SEM, *p < 0.05, one-sample Student’s t test. n = 4