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. Author manuscript; available in PMC: 2024 Mar 2.
Published in final edited form as: Stem Cells. 2020 Apr 30;38(7):890–903. doi: 10.1002/stem.3181

FIGURE 1.

FIGURE 1

Proliferation of Plp1-expressing glial cells after knockout or overexpression of Lin28 in vitro. A, For Lin28 overexpression in Plp1-positive cells, Plp1-CreER;STOP fl; rtTA mice and Lin28a/b-TetO, Lafayette, Colorado; STOP fl; tdTm (tdTm) mice were crossed to obtain Plp1-CreER;rtTA;Lin28a/b-TetO;tdTm (Lin28-TetO) transgenic mice. Tamoxifen application initiated Cre recombinase dependent tdTm and tetracycline-controlled transactivator (rtTA) expression. In the presence of doxycycline, the tetracycline operator (TetO) was transiently activated and led to overexpression of Lin28. B, For the Lin28 knockout experiments, Plp1-CreER mice were crossed with Lin28a-fl;tdTm mice to obtain Plp1-CreER;Lin28a-fl;tdTm (Lin28−/−) transgenic mice. Application of tamoxifen led to conditional knockout of Lin28 in tdTm expressing Plp1 positive cells. C, Plp1-CreER;tdTm mice were used as controls for Lin28 overexpression and knockout experiments (control). D, Timeline for neurosphere assay from early postnatal spiral ganglion cells. Pups were dissected at P3-P4 and kept under proliferating conditions for 12 days, undergoing two passages; DIV, days in vitro. Spheres were treated after the second passage and analyzed by immunohistochemistry (red X). E, Second-generation neurospheres were treated with ethynyl-desoxyuridine (EdU) for either 6 or 24 hours and harvested for immunohistochemistry. Neurospheres of Lin28−/− (first row), control (second row), and Lin28-TetO (third row) mice were stained for EdU (white) and Sox2 (green), nuclei were labeled with DAPI (blue). Plp1-positive cells expressed tdTm (red). Merged images in the first two columns show co-localization. Scale bar = 20 μm. E′, High-magnification image of EdU-Sox2-tdTm colocalization. Scale bar = 10 μm. F, Quantification of tdTm, Sox2, and EdU-triple-positive (+) cells as percentage of the total number of tdTm+ cells after 6 or 24 hours of EdU treatment (*P < .05, mean ± SEM). G, Relative expression of Let-7 in proliferating Lin28−/− and Lin28-TetO neurospheres (mean ± SEM, *P < .05, n = 3). H-J, Quantitative RT-PCR of Lin28 (n = 7), Sox2 (n = 5), and Hmga2 (n = 6) in proliferating Lin28−/− and Lin28-TetO neurospheres compared to control (mean ± SEM, *P < .05, control set as 1)