PMC

Panel A shows weight in grams of R26-M2rtTA;TRE-Tight-Cdkn1b (n = 22) and R26-M2rtTA control (n=21) littermates during neonatal development when mothers were placed on 0.25 mg/ml doxycycline on birth of the pups (error bars indicate s.d.). Panel B shows comparison of an R26-M2rtTA;TRE-Tight-Cdkn1b (Cdkn1b+) with an R26-M2rtTA (Cdkn1b−) control littermate at 4 weeks of development following continuous treatment with 0.25 mg/ml doxyxcycline. Panel C shows the same pair of animals following 24 weeks of continuous doxycycline administration. Panels D–K compare mice treated for various times with 0.25 mg/ml doxycycline either as neonates (panels D–G) or when treatment was begun at 2 months of age (panels H–K) where panels D and H are 4 weeks of treatment, panels E and I are 6 weeks of treatment, panels F and J are 8 weeks of treatment, and panels G and K are 12–13 weeks of treatment.

The phenotype of an R26-M2rtTA;TRE-Tight-Cdkn1b mouse treated with 0.75 mg/ml doxycycline from 2 through 9 months of age followed by removal from doxycycline for 3 additional months is shown in Panel A (where the same animal is shown prior to removal from doxycycline in Figure 1, panel G). Panels B–I show images of histological sections from abdominal skin of this mouse where B is HE stained and taken with a 10× objective, D–E are immuno-fluorescence images stained for smooth muscle actin (red) and DAPI (blue) and taken with a 20 × objective. Fand G are unstained phase contrast images taken with a 20× objective. H and I are immuno-fluorescence images stained for IgG (green) and DAPI (blue) and taken with a 20 × objective. Panels C, F and H are controls prepared from an R26-M2rtTA mouse treated with doxycycline, panel D is an R26-M2rtTA;TRE-Tight-Cdkn1b mouse treated continuously with doxycycline and panels E, G, and I are from an R26-M2rtTA;TRE-Tight-Cdkn1b mouse following treatment with and removal from doxycycline as described above.

M2rtTA;TRE-Tight-Cdkn1b mice were either untreated (panel A), or treated at two months of age with 1 mg/ml doxycycline continuously for 3d (panels B and E), 2 weeks (panels C and F) or 5 months (panel D). Paraffin sections were prepared and stained for CldU incorporation (green) and Cdkn1b (red) in panels A–D and F, or CldU (green) and IdU (red) incorporation in panel E, and images were captured using a 20× objective. In panel G the frequency with which Mcm2+ nuclei (see ) at various positions from the bases of the crypts label with CldU following a 2 hour pulse is quantified for control (blue squares) and Cdkn1b over-expressing (red triangles) mice. In panels H–K the distribution of crypts containing different numbers of nuclei incorporating CldU is plotted for untreated (Panel H) and 3d (Panel I), two week (Panel J) and 5 month (Panel K) doxycycline treated mice.

For this set of experiments R26-M2rtTA;TRE-Tight-Cdkn1b (panels B, D and F) and control (R26-M2rtTA+ litermates that do not carry the TRE-Tight-Cdkn1b transgene, panels A, C, and E) mice were treated, beginning at two months of age, with 0.25 mg/ml doxycycline continuously for a period of 5 months. Histological sections were prepared from abdominal skin (panels A and B), gastrocnemius muscle (panels C and D) and abdominal muscle (panels E and F) and stained with hematoxylin and eosin prior to image capture using a 10× objective. Various layers of the skin are indicated for panels A and B. In panel G, diameters were determined for gastrocnemius and abdominal musle fibers (n=50, error bars indicate s.d.) from control and R26-M2rtTA;TRE-Tight-Cdkn1b mice treated, beginning at two months of age, with 0.25 mg/ml doxycycline continuously for a period of 5 months and show a decrease in Cdkn1b over-expressing mice (gastrocnemius ρ = 7.1×10⁻⁷; abdominal ρ = 4.8×10^{−2`1</sup>; two-tailed t-test). Panel H shows muscle fiber diameters determined for gastrocnemeus muscle of control and R26-M2rtTA;TRE-Tight-Cdkn1b mice (n=50, error bars indicate s.d.) treated beginning at two months of age with 0.5 mg/ml doxycycline continuously for a period of 8 months and also show a decrease in Cdkn1b over-expressing mice (ρ = 6.2 × 10<sup>−2`7}; two-tailed t-test).

Panel A shows western blots for pp53(ser18) (a full-length image of this blot is shown in Supplementary Figure 10, panel B) and actin in lung, intra-abdominal fat (IA Fat), and skin of R26-M2rtTA transgenic mice that either carry or do not carry the TRE-Tight-Cdkn1b transgene as indicated in the figure where, starting at two months of age, mice were continuously treated with 1 mg/ml doxycycline for two months and assessed at 4 months of age. Additionally, Cdkn1b expression in the same tissues from a control 24 month old mouse is included for comparison. Arrows indicate the positions of size markers in kDa. Panels B and C show immune-fluorescence staining for γ-H2AX in epidermal sections of control (B) and Cdkn1b over-expressing (C) mice. Panels D and E show immune-fluorescence staining for TP53BP1 in epidermal sections of control (D) and Cdkn1b over-expressing (E) mice. Panels F and G demonstrate reduced frequency of γ-H2AX (n = 9, error bars indicate s.d., ρ = 0.04, two-tailed t-test) and TP53BP1 (n = 10, error bars indicate s.d , ρ = 3.7 × 10⁻⁵, two-tailed t-test) foci, respectively, in epidermal sections from Cdkn1b over-expressing mice relative to controls. Panel H shows SA-β galactosidase staining of intra-abdominal fat (IAT) for R26-M2rtTA control and R26-M2rtTA;TRE-Tight-Cdkn1b transgenic mice that were treated with 1 mg/ml doxycycline either from birth through 3 months of age (left panels) or starting at 2 months of age and through 8 months of age (right panels). Bars = 5 mm. Quantitative assessment of SA-β galactosidase activity was made using ONPG as a substrate and results from this assay are shown below each panel. Additionally, the weight of total IAT recovered from each animal is given in grams (g). Qualitatively similar SA-β galactosidase staining was observed in a two additional sets of control and R26-M2rtTA;TRE-Tight-Cdkn1b mice treated with 0.25 mg/ ml doxycycline from months 2–7 although quantitative measures were not performed.

Supraventricular zones (SVZs) from R26-M2rtTA;TRE-Tight-Cdkn1b mice either untreated (A and B) or treated with 1 mg/ml doxycycline between months 2–7 (C and D) or treated between months 2–7 but removed from doxycycline treatment for 24 hours (E and F). IdU was administered for 3 days (A–D) or 1 day (E and F) and CldU 2 hours prior to sacrifice. A, C, and E are stained for IdU (red), CldU (green), and DAPI (blue). B, D, and F are the same sections stained for Mcm2 (green). Images were captured using a 40× objective. G, percent of Mcm2+ cells that were also positive for IdU incorporation in SVZ sections from untreated (n = 4) or treated with 1 mg/ml doxycycline for 3 d (n = 6), 2 wk (n = 5), 5 mo (n = 7), or 5 mo followed by removal from doxyxcline for 1 day (5mo 1dR, n = 6) R26-M2rtTA;TRE-Tight-Cdkn1b mice. Error bars indicate s.d. IdU incorporation is reduced in treated relative to control mice (3 d ρ = 0.00014, 2 wk ρ = 0.00017, 5 mo ρ = 0.00017; two-tailed t-tests) but returns to levels that are not significantly different from control on doxyxcline removal (ρ = 0.067; two-tailed t-tests). H shows clonogenic neurosphere assays where the percent of wells in 96 well plates (n = 4, error bars indicate s.d.), cultured in the absence of doxycycline, that contained neurospheres after 1 or 3 passages is shown for R26-M2rtTA-control (blue bars) and R26-M2rtTA;TRE-Tight-Cdkn1b (red bars) mice that had been continuously treated with 1 mg/ml doxycycline from 2 months through 16 months of age. More neurospheres are recovered from Cdkn1b over expressing mice than controls (passage 1 ρ = 0.032; passage 3 ρ = 0.013; two-tailed t-tests). I–L, muscle fibers from the gastrocnemius muscles of R26-M2rtTA control (I and K) and R26-M2rtTA;TRE-Tight-Cdkn1b (J and L) mice treated with 0.25 mg/ml doxycycline from mo 2–7 and IdU during the final three weeks. I and J, Pax7 (red) and DAPI (blue) overlain on a bright-field image (arrows, Pax7+ nuclei). K and L, IdU (red) and DAPI (blue) (arrows, IdU positive nuclei). N, total, Pax7 negative, and Pax7+ nuclei per myofiber (M) and percent of nuclei labeled with IdU (N) were quantified.

Panel A : Western blot showing Cdkn1b (p27) and actin in thymuses of mice carrying the R26-M2rtTA transgene with or without the TreTight-Cdkn1b transgene or treatment with 1 mg/ml doxycycline (DOX) for 24 hours (a full-length image of this blot is shown in , panel A). Panel B shows Cdkn1b protein levels in lung, skin and intra-abdominal fat of R26-M2rtTA transgenic mice that either carry or do not carry the TRE-Tight-Cdkn1b transgene where, starting at two months of age, mice were continuously treated with 1 mg/ml doxycycline for two months and assessed at 4 months of age. Additionally, Cdkn1b expression in the same tissues from a control 24 month old mouse is included for comparison. Panel C shows a titration of doxyxycline concentration verses Cdkn1b expression in the thymus where mice were treated with the indicated concentrations of doxycycline for 3 days. A western blot is shown on the left and quantified by densitometry of Cdkn1b levels corrected for loading based on the signal from actin on the right. Arrows to the right of panels A–C indicate the positions of size markers in kDa. Panel D shows gross phenotypic changes in an R26-M2rtTA-control (left, mouse 4608) and R26-M2rtTA;TreTight-Cdkn1b (right, mouse 4605) mice continuously treated with 1 mg/ml doxycycline beginning at 2 months of age for a period of three weeks. The same R26-M2rtTA;TreTight-Cdkn1b mouse is also shown at 6 weeks (panel E) or 5 months (panel F) of continuous doxycycline administration. Panel G shows a mouse treated, beginning at two months of age, continuously for 9 months with 0.75 mg/ml doxycycline. Panel H shows survival in days of R26-M2rtTA;TreTight-Cdkn1b mice treated with various concentrations of doxycycline as indicated in the figure where the number of mice per group is given in parentheses. None of 40 Cdkn1b over-expressing mice that have died during doxycycline treatments ranging from 0.25 to 1.0 mg/ml had overt tumors on necropsy (a rate of <2.5%). This is much lower than the expected rate for this strain (40%). In the present study, one of the 4 control animals that died (25%) succumbed to thymic lymphoma.