Figure 9: Lin28a induction in differentiated P19 cells results in reduction of miR-9 levels.

(a) Western blot analysis of protein extracts from control P19 cells (lane 1—d0 and lane 2—d9) and d9 of three colonies of P19 Tet-On 3G Lin28a cells. Lanes 3, 5 and 7 represent results from the corresponding cell lines without Dox. Lanes 4, 6 and 8 show results from the corresponding cell lines induced with Dox (100 ng ml⁻¹). (b) Real-time qRT–PCR analysis of mature miR-9, let-7a and miR-101 levels of Dox-induced cells after RA-mediated neuronal differentiation. The results from P19 Tet-On 3G Lin28a #2, which failed to induce Lin28a, is shown as white bars; the results from P19 Tet-On 3G Lin28a #3, which induced Lin28a, are shown as black bars. The values were normalized to miR-16 levels. The fold change was plotted relative to values derived from −Dox cells, which were set to 100. Mean values and s.d. of three independent biological replicates are shown. Statistical significance was calculated using t-test; *P⩽0.05. (c) Real-time qRT–PCR analysis of the primary miR-9-1, miR-9-2 and let-7a-1 of Dox-induced cells after RA-mediated neuronal differentiation. The results from P19 Tet-On 3G Lin28a #2, which failed to induce Lin28a, is shown as white bars; the results from P19 Tet-On 3G Lin28a #3, which induced Lin28a, are shown as black bars. The values were normalized to cyclophilin A mRNA levels. Mean values and s.d. of three independent experiments are shown; *P⩽0.05; **P⩽0.01.