Cyclin D1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1 (2021)
Figure 4

Figure 4 Phosphorylation of S309 inhibits further phosphorylation at S328A of ATOH1

A. CHX assay with S309A. N2a cells transfected with either S309A or S309A+4D were treated with CHX for 0, 4 or 8 hrs before harvest. Lysates were subjected to immunoblotting with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Error bars are representing as Mean ± SEM from three trials of each condition.

B. Lysates of N2a cells transfected with either WT or a phospho-mimic form of ATOH1 (S309D) were immunoblotted with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Mean± s.e.m. Student T-test between WT and S309D was performed. ***p < 0.001. Three slots of each experimental condition represent the three biological replicants of each condition.

C. GST-pull down assay of WT and WT with 4D. GST-Atoh1 plasmids were transfected with or without Ccnd1 and Cdk4 plasmids (4D) in N2a cells. Cell lysates were pull-downed with glutathione-conjugated beads after a 6 hr treatment of 10µM MG132 and then immunoblotted with anti-poly Ubiquitin. Quantification of relative levels of poly-ubiquitin (right panel). Bars and error bars are representing as Mean ± SEM from three trials of each condition. Student T-test between WT with MG132 and WT+4D with MG132. **p < 0.01.

D. Lysates of N2a cells transfected with either WT or S309D were immunoblotted to detect Atoh1 S328 phosphorylation (p-S328-Ab), Atoh1 or β-actin. Quantification of relative levels of phosphorylated ATOH1 at S328 to the total ATOH1 (right panel). Mean± s.e.m. Student T-test between WT and S309D was performed. **p < 0.01. Three slots of each experimental condition represent the three biological replicants of each condition.

E. Lysates of N2a cells transfected with either WT or S328A were immunoblotted with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Bars and error bars are representing as Mean ± SEM from three trials of each condition. Student T-test between WT and S328A was performed. Three slots of each experimental condition indicated the biological replicants.

F-K. Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1. (F-I) Scramble (Control), Atoh1 shRNA (Atoh1 KD), shRNA and WT (KD + WT) or shRNA and S309A (KD + S309A) plasmids were introduced to P5 mice EGL with H2B-EGFP plasmid and immunostaining for NEUROD1(magenta) was performed at P7. Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest which GFP signals are not co-localized with NEUROD1 (AT+GCPs). (J) Proportions of NEUROD1- cells (AT+GCPs) among the electroporated cells. (K) Proportions of KI67+ cells (total GCPs). Tukey-Kramer test was performed among the control (N=3), Atoh1 KD (N=3), Atoh1 KD +WT (N=3) and Atoh1 KD +S309A (N=3) mice. ***p < 0.001, *p < 0.05, N.S.: Not Significant. Bars and error bars are representing as Mean ± SEM. Number of cells: 138, 147 and 124 from N=3 mice (Control), 82, 75 and 117 from N=3 mice (KD), 127, 269 and 347 from N=3 mice (KD+WT), 195, 71 and 111 from N=3 mice (KD+S309A).

Data information: Scale bars: 20µm(F-I)