Article Title: mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia
Figure Lengend Snippet: ( A ) Telomere lengths of the retinal cells, which were isolated from P30 Tsc1 fl/+ and Tsc1 fl/fl mice with the indicated Cre drivers, were compared with that of control sample, and relative values are shown in the graph. Error bars denote SD. Numbers of samples analyzed are shown in the graph. All samples were obtained from independent litters. **p < 0.01; n.s., not significant. ( B ) Telomere lengths of the FACS-isolated retinal cells from P14 Tsc1 fl/+ ;Tyrp1-Cre and Tsc1 fl/fl ;Tyrp1-Cre littermate mice were compared with that of control sample, and their relative values are shown in the graph. *p < 0.05; ***p < 0.001. ( C ) Senescence markers (β-gal, p53, p21, and c-Myc) expressed in the corresponding P30 Tsc1 fl/+ ;Cre and Tsc1 fl/fl ;Cre mouse retinas were detected by Western blot (WB). Relative amounts of the proteins used in each sample were determined by WB detection of β-actin. ( D ) Expression of senescence markers (β-gal and p53) in the MG was determined by co-immunostaining with an MG marker, Sox9. The cells experienced Cre-mediated recombination were also visualized by R26tdTom reporter. Images in the right columns are the magnified versions of the boxed areas indicated by corresponding numbers in the low magnification images. ( E ) Sox9-positive MG, Pax6-positive amacrine cell (AC), and Vsx2-positive bipolar cell (BC) populations that express the senescence markers were determined and shown in the graph (Pax6 and Vsx2 staining images are provided in ). Number of samples analyzed are shown in the graph (five independent litters). n.d., not detected. ( F ) Eye and retinal structures of P30 Rptor fl/+ ;Tyrp1-Cre and Rptor fl/fl ;Tyrp1-Cre mice were investigated by hematoxylin and eosin (H&E) staining of the eye sections. Distribution of the Cre-affected cells and mTORC1 activation of the cells were examined by the immunostaining of R26tdTom and pS6, respectively. ( G ) R26tdTom-positive Cre-affected cell population in the mouse retinas were quantified by FACS and shown in the graph. ****p < 0.0001. ( H ) Telomere lengths of unsorted (whole retina) and the FACS-isolated retinal cells from P14 Rptor fl/+ ;Tyrp1-Cre and Rptor fl/fl ;Tyrp1-Cre mice were compared with that of control sample and their relative values are shown in the graph. Number of samples analyzed are shown in the graph. *p < 0.05. ( I ) Relative levels of senescence markers and mTORC1 pathway components of the mouse retinas were analyzed by WB. ( J ) Distribution of the cells expressing senescence markers and R26tdTom Cre reporter in the retinas was also examined by immunostaining.
Article Snippet: Antibody , Anti-p21/Cip1(Mouse monoclonal) , Santa Cruz Biotechnology , SC817 , WB (1:1000)IHC (1:200).
Techniques: Isolation, Western Blot, Expressing, Immunostaining, Marker, Staining, Activation Assay