Figure 1.
JNK is phosphorylated during mitosis of retinal progenitor cells.
(A, B) Representative confocal photomicrographs of immunohistochemistry for phospho-JNK (red) and phospho-histone-H3 (green) in sections of retinal tissue maintained for 3 hours in vitro either in absence (A - CTR) or presence of taxol (B - TAX) showing the NBL. The sections were counterstained with DAPI (blue). Arrows indicate examples of cells double stained for phospho-JNK and phospho-histone-H3. Scale bar: 20 µm. (C) Higher magnification of mitotic cell showing the subcellular localization of phospho-JNK (red), phospho-histone-H3 (green), and DAPI (blue). Scale bar: 10 µm. (D) Numbers of phospho-JNK or phospho-histone-H3 stained cells per 100 µm of linear extent parallel to the retinal surface, along the mitotic stratum of retinal explants maintained in vitro for 3 hours, either in the absence (CTR) or presence of taxol (TAX). (E) Percentage of cells stained for phospho-JNK among all cells immunolabeled for phospho-histone-H3 in the mitotic stratum. Data are means±S.E.M. from three independent experiments. CTR - control; TAX - taxol; NBL - neuroblastic layer; *** P<0.001 versus CTR.
Figure 2.
JNK3 is absent during mitosis of retinal progenitor cells.
Representative confocal photomicrographs of immunohistochemistry for JNK3 (red) and phospho-histone-H3 (green) in sections of retinal tissue maintained for 3 hours in vitro either in absence (A - CTR) or presence of taxol (B - TAX). The sections were counterstained with DAPI (blue). Cells stained for JNK3 were found only in the ganglion cell layer (GCL), in the immature inner nuclear layer and in a few cells in the NBL. Arrows indicate examples of JNK3 positive cells. Scale bar: 20 µm. CTR - control; TAX - taxol; GCL - ganglion cell layer; NBL - neuroblastic layer.
Figure 3.
JNK1 and/or JNK2 are preferentially phosphorylated during mitosis of progenitor cells in developing retina.
(A, B) Representative confocal photomicrographs of immunohistochemistry for phospho-JNK1/2 (red) and phospho-histone-H3 (green) in sections of retinal tissue maintained for 3 hours in vitro either in absence (A - CTR) or presence of taxol (B - TAX). The sections were counterstained with DAPI (blue). Cells double stained for phospho-JNK1/2 and phospho-histone-H3 are indicated with arrows. Scale bar: 20 µm. Inset: higher magnification of mitotic cells. (C) Representative western blot of phosphorylated JNK in total protein extracts of retinal explants maintained for 3 hours in vitro either in absence (CTR) or presence of taxol (TAX). The upper blot was done with the antibody for phospho-JNK, the middle blot with an antibody for phospho-histone-H3, and the lower blot with an antibody for β-actin as loading control. (D) Densitometric analysis of the western blot of phospho-JNK. Data are means±S.E.M. from three independent experiments. CTR - control; TAX - taxol; GCL - ganglion cell layer; NBL - neuroblastic layer. * P<0.05 versus CTR.
Figure 4.
JNK is phosphorylated preferentially during early stages of mitosis in retinal progenitor cells.
(A–F) Representative confocal photomicrographs of immunolabeling for phospho-JNK (red) in sections of retinal tissue maintained 3 hours in vitro showing mitotic cells. The sections were counterstained with DAPI (blue). (A, B) Cells in prophase/prometaphase heavily labeled for phospho-JNK. (C, D) Cells in metaphase heavily labeled for phospho-JNK. (E, F) Cells in anaphase either poorly labeled or unlabeled for phospho-JNK. Scale bar: 10 µm (G) Quantification of phospho-JNK staining in different mitotic stages. Mitotic stages were classified according with chromatin morphology in prophase/prometaphase (PRO), metaphase (META) or anaphase (ANA) and phospho-JNK staining were classified in heavily labeled, poorly labeled and unlabeled. The graph represents pools of at least 250 mitotic cells in each experiment. Data are means±S.E.M. from three independent experiments.
Figure 5.
Inhibition of JNK induces mitotic arrest.
(A, B) Representative confocal photomicrographs of immunohistochemistry for phospho-JNK (red) and phospho-histone-H3 (green) in sections of retinal tissue maintained for 3 hours in vitro either in absence (A - CTR) or presence of SP600125 (B - SP). The sections were counterstained with DAPI (blue). Arrows indicate examples of phospho-histone-H3 positive cells. Scale bar: 20 µm. (C) Numbers of phospho-histone-H3 stained cells per 100 µm of linear extent parallel to the retinal surface, along the mitotic stratum of retinal explants maintained in vitro for 3 hours either in the absence (CTR - black column) or presence of SP600125 (SP - gray column). *** P<0.001 versus CTR. (D–F) Representative confocal photomicrographs of mitotic cells immunolabeled for phospho-JNK (red) in sections of retinal tissue maintained for 3 hours in vitro in the presence of SP600125. The sections were counterstained with DAPI (blue). Stages of mitosis are indicated as either prophase/prometaphase (p) or metaphase (m), while arrows show aberrant chromosome morphology. Compare the disorganized chromatin with the profiles shown in fig. 4. Scale bar: 5 µm. (G) Frequency of prophase/prometaphases (PRO), metaphases (META), anaphases (ANA) and aberrant profiles (ABE) among all mitotic cells in the outer margin of retinal explants maintained in vitro for 3 hours either in the absence (CTR - black columns) or presence of SP600125 (SP - gray columns). Data are means±S.E.M. pooled from at least 250 mitotic cells in each of six independent experiments. CTR - control; SP - SP600125; NBL - neuroblastic layer. ** P<0.01 versus CTR.