Figure 4. Activation of LIN28B in adult BM. (A) Adult iLIN28B mice were exposed to 1 g/liter doxycycline in drinking water or standard water for 14 d, at which time BM mononuclear cells were isolated, and Western blot analysis for human LIN28B was performed. (B) RNA was isolated from BM CMPs with and without doxycycline (Dox) exposure, and levels of mature let-7 microRNAs were measured by qPCR. For each let-7 form, results are normalized to the control, nondoxycycline-exposed condition. *, P < 0.05 by Student’s t test. n = 4 uninduced and 5 doxycycline-exposed across three experiments. (C) BM mononuclear cells from adult iLIN28B mice with and without 14 d of doxycycline exposure were isolated and immunostained, with representative flow cytometry plots presented. (D) Numbers of myeloid progenitor cells per 100,000 BM mononuclear cells are shown. *, P < 0.01 by Student’s t test. (E) Whole BM cells were analyzed for red blood cell progenitor populations by flow cytometry. Representative plots are shown. (F) Numbers of CD71+/Ter119− cells in doxycycline-exposed iLIN28B animals relative to the control are shown. *, P < 0.02 by Student’s t test. (G) BM mononuclear cells under the indicated conditions were immunostained for Gr-1 and Mac-1. Representative flow cytometry plots are shown. (H) Numbers of Gr-1+/Mac-1+ mature granulocytes per 100,000 BM mononuclear cells under the indicated conditions are presented. *, P < 0.0001 by Student’s t test. (D, F, and H) n = 8 mice for the control condition and 7 mice for the doxycycline-exposed condition across three experiments. (I and J) Morphology of BM cells (I) or splenic tissue (J) from control or doxycycline-treated iLIN28B mice is shown. Images are representative of specimens obtained over at least three independent experiments. (I) Bar, 40 µm. Arrows, neutrophils; arrowheads, erythroblasts. (J) Bar, 200 µm. RP, red pulp; WP, white pulp. Dashed lines indicate borders of these two splenic tissues. (K) BM mononuclear cells from iLIN28B mice exposed to doxycycline for 14 d or unexposed mice were cultured in methylcellulose in the presence of growth factors. After 14 d of culture, colony morphology was scored, and the mean proportions of each colony type are presented. *, P < 0.05 by Student’s t test comparing each colony type. n = 5 control mice and 4 doxycycline-treated mice across three experiments. Error bars represent SEM. CFU-M, CFU monocyte; CFU-G, CFU granulocyte; CFU-GM, CFU granulocyte–monocyte.
Figure 4.

Activation of LIN28B in adult BM. (A) Adult iLIN28B mice were exposed to 1 g/liter doxycycline in drinking water or standard water for 14 d, at which time BM mononuclear cells were isolated, and Western blot analysis for human LIN28B was performed. (B) RNA was isolated from BM CMPs with and without doxycycline (Dox) exposure, and levels of mature let-7 microRNAs were measured by qPCR. For each let-7 form, results are normalized to the control, nondoxycycline-exposed condition. *, P < 0.05 by Student’s t test. n = 4 uninduced and 5 doxycycline-exposed across three experiments. (C) BM mononuclear cells from adult iLIN28B mice with and without 14 d of doxycycline exposure were isolated and immunostained, with representative flow cytometry plots presented. (D) Numbers of myeloid progenitor cells per 100,000 BM mononuclear cells are shown. *, P < 0.01 by Student’s t test. (E) Whole BM cells were analyzed for red blood cell progenitor populations by flow cytometry. Representative plots are shown. (F) Numbers of CD71+/Ter119− cells in doxycycline-exposed iLIN28B animals relative to the control are shown. *, P < 0.02 by Student’s t test. (G) BM mononuclear cells under the indicated conditions were immunostained for Gr-1 and Mac-1. Representative flow cytometry plots are shown. (H) Numbers of Gr-1+/Mac-1+ mature granulocytes per 100,000 BM mononuclear cells under the indicated conditions are presented. *, P < 0.0001 by Student’s t test. (D, F, and H) n = 8 mice for the control condition and 7 mice for the doxycycline-exposed condition across three experiments. (I and J) Morphology of BM cells (I) or splenic tissue (J) from control or doxycycline-treated iLIN28B mice is shown. Images are representative of specimens obtained over at least three independent experiments. (I) Bar, 40 µm. Arrows, neutrophils; arrowheads, erythroblasts. (J) Bar, 200 µm. RP, red pulp; WP, white pulp. Dashed lines indicate borders of these two splenic tissues. (K) BM mononuclear cells from iLIN28B mice exposed to doxycycline for 14 d or unexposed mice were cultured in methylcellulose in the presence of growth factors. After 14 d of culture, colony morphology was scored, and the mean proportions of each colony type are presented. *, P < 0.05 by Student’s t test comparing each colony type. n = 5 control mice and 4 doxycycline-treated mice across three experiments. Error bars represent SEM. CFU-M, CFU monocyte; CFU-G, CFU granulocyte; CFU-GM, CFU granulocyte–monocyte.

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