Fig. 4: Atoh1 regulates expression of Kif21b and affects microtubule stability under hypoxia.

a Upper: Venn diagram shows co-altered genes analyzed by the transcriptomics sequencing data (blue), the Atoh1-null sequence dataset (yellow), and the Atoh1-CHIP sequence dataset (green). bottom: sequence logo diagram shows a prediction of conserved motif of Atoh1. b Upper: Transcriptional factor binding site (TFBS) of Atoh1 predicted by the JASPAR database (relative score > 0.85). Bottom: Bar graph shows transcription of Kif21b by Atoh1 verified by real time-PCR in N2A cells (All groups, n = 3) (control + vector vs control + Si-Atoh1, t = 8.54, **p = 0.0011, df = 4; model + vector vs model + Si-Atoh1, t = 2.84, *p = 0.047, df = 4) (1.00 ± 0.07 vs 0.40 ± 0.07 vs 7.65 ± 1.14 vs 3.99 ± 1.42). c, Protein fractions extracted from N2A cells after transfection and hypoxia were tested with Kif21b and Atoh1. Upper panels show representative bands of proteins mentioned above. α-tubulin was used as internal control. Bar graphs show percentage changes of proteins, *p vs control + vector; #p vs model + vector (All groups, n = 3) (For statistics, see Table S15). d Protein fractions extracted from brains of P11.5 mice were tested with Kif21b and Atoh1. Upper panels show representative bands of proteins mentioned above. β-actin was used as internal control. Bar graphs show percentage changes of proteins relative to control (control, n = 3; model, n = 3) (For statistics, see Table S16). e Staining of N2A cells after transfection and hypoxia by antibody against MAP2. Scale bars: 20 μm. Objective 40x. Bar graphs show MFI of MAP2 staining, data were presented as relative changes (*p vs control+ vector; #p vs model+ vector; ^p vs model+ Lenti-Atoh1) (All groups, n = 6) (F = 14.14, df = (20, 3), control + vector vs model + vector, **p = 0.0010; control + vector vs model + Lenti-Atoh1, **p = 0.0010; control + vector vs model + Lenti-Atoh1 + Si-Kif21b, *p = 0.010; model + vector vs model + Lenti-Atoh1, #p = 0.039; model + Lenti-Atoh1 vs model + Lenti-Atoh1 + Si-Kif21b, ^p = 0.036) (1.00 ± 0.068 vs 0.74 ± 0.028 vs 0.61 ± 0.037 vs 0.73 ± 0.030). f Staining of cerebellar lobule IV-V by antibodies against MAP2 and NeuN in P11.5 mice after micro-injection of recombinant virus illustrated and hypoxia. The three panels below show a magnified view of dashed squares on the top. Scale bars: 50 μm (columns above, objective 20x); 20 μm (three columns below, objective 63x). Bar graphs on the left show MFI of MAP2 staining. Bar graphs on the right show MFI of MAP2 around per neuron, data were presented as relative changes (*p vs control+NC; #p vs model+NC; ^p vs model+ OE-Atoh1) (All groups, n = 6) (For statistics, see Table S17). g Protein fractions extracted from N2A cells after transfection and hypoxia were tested with acetyl-α-tubulin and total-α-tubulin. β-actin was used as internal control. Bar graphs show percentage changes of proteins, *p vs control + vector; #p vs model + vector (All groups, n = 3) (F = 35.34, df = (12, 5), vs control + vector, from left to right, p = 0.71, **p = 0.0010, **p = 0.0010, p = 0.30, **p = 0.0010; vs model + vector, from left to right, ##p = 0.0010, ##p = 0.0010, p = 0.577) (1.00 ± 0.10 vs 1.04 ± 0.050 vs 1.56 ± 0.057 vs 1.93 ± 0.060 vs 1.10 ± 0.059 vs 1.61 ± 0.049). All data were shown as mean ± SEM. *p < 0.05, **p < 0.01.