Journal: Genes & Development

Article Title: Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase

doi: 10.1101/gad.242131.114

Figure Lengend Snippet: Differential binding of USP9x underlies control of FOXO3a stability by EglN2. ( A ) Immunoblot analysis of T47D cells that were infected with a lentivirus encoding the indicated USP9x shRNAs or Ctrl shRNA. ( B ) Immunoblot analysis of T47D cells that were infected with a lentivirus encoding HA-FOXO3a and then superinfected with a lentivirus encoding USP9x shRNA (364 or 064) or Ctrl shRNA. ( C ) Immunoblot analysis of 293T cells that were infected with a lentivirus encoding USP9x shRNA (364) or Ctrl shRNA and then, after drug selection, transfected with a plasmid encoding V5-tagged mouse full-length USP9x or empty vector (Ctrl). ( D ) Immunoblot (IB) assays of whole-cell extracts (WCE) and immunoprecipitates (IP) of T47D cells that were infected with a lentivirus encoding USP9x shRNA (sh128 or sh364) or Ctrl shRNA and then, after drug selection, transiently transfected with plasmids encoding Flag-ubiquitin and HA-FOXO3a followed by treatment with 10 μM MG132 for 16 h. ( E ) Immunoblot analysis of MCF-7 cells that were infected with a lentivirus encoding shRNA against either USP9x (364) or Ctrl shRNA followed by 100 μg/mL cycloheximide for the indicated duration. ( F ) Immunoblot analysis of 293T cells that were cotransfected with plasmids encoding V5-tagged USP9x (wild-type or C1556S) and HA-FOXO3a. ( G ) Immunoblot (IB) assays of whole-cell extracts (WCE) and immunoprecipitates (IP) of T47D cells that were transfected with a plasmid encoding HA-FOXO3a (wild-type or P426A;P437A) followed by treatment with 10 μM MG132 for 16 h. ( H ) Immunoblot analysis of bound USP9x recovered from lysed T47D cells that were incubated with 20 μL of Neutravidin-Sepharose preloaded with the indicated FOXO3a peptides. ( I , J ) Immunoblot analysis of T47D cells that were infected with a lentivirus encoding USP9x shRNA (364) or Ctrl shRNA and then, after drug selection, treated with the indicated drugs or siRNAs.

Article Snippet: V5-tagged mouse USP9x, catalytic-dead USP9x C1556S, and GST-tagged human USP9x (N1, N2, C1, and C2) plasmids were generously provided by Dr. Feng Cong (Novartis).

Techniques: Binding Assay, Western Blot, Infection, shRNA, Selection, Transfection, Plasmid Preparation, Incubation

Journal: Genes & Development

Article Title: Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase

doi: 10.1101/gad.242131.114

Figure Lengend Snippet: Proposed models for FOXO3a regulation by EglN2. ( A ) Estrogen binding with ER transcriptionally induces EglN2, which triggers FOXO3a hydroxylation on prolyl residue 426 and 327 sites. Hydroxylation of these sites dissociates FOXO3a from USP9x deubiquitinase, thereby facilitating FOXO3a ubiquitylation and degradation. ( B ) Depletion of EglN2 by either lack of ER activity or EglN2 siRNAs/shRNAs prevents FOXO3a from being hydroxylated. As a result, USP9x binds with FOXO3a and prevents FOXO3a from being ubiquitylated and degraded. Stabilization of FOXO3a decreases Cyclin D1 expression.

Article Snippet: V5-tagged mouse USP9x, catalytic-dead USP9x C1556S, and GST-tagged human USP9x (N1, N2, C1, and C2) plasmids were generously provided by Dr. Feng Cong (Novartis).

Techniques: Binding Assay, Activity Assay, Expressing

Journal: Human Molecular Genetics

Article Title: Deficiency of Patched 1-induced Gli1 signal transduction results in astrogenesis in Swedish mutated APP transgenic mice

doi: 10.1093/hmg/ddu370

Figure Lengend Snippet: Aβ is overexpressed and Shh levels are elevated in the hippocampus of APP23 mice. (A) Immunostaning of antibody clone 6E10 showed Aβ-positive reaction in the hippocampus of 3-, 12- and 24 months old APP23 mice. Intracellular Aβ-positive reaction in 3 months old, a few of Aβ deposit plaques in the subiculum of 12-month-old, lots of Aβ plaques, especially dense deposits in molecular layer in hippocampus were visualized in 24 months old APP23 mice. Scale bar: 50 µm. (B) The levels of soluble Aβ1–42 and Aβ1–40 levels were tested in the hippocampi of different age group of WT and APP23 mice (n = 5 in each group, Student t-test, **P < 0.01 of soluble Aβ1–42 and Aβ1–40 versus corresponding that of WT, respectively; ##P < 0.01 of soluble Aβ1–42 versus soluble Aβ1–40 amount in APP23 mice). (C) The levels of insoluble Aβ1–42 and Aβ1–40 were measured in the hippocampi of different age groups of WT and APP23 mice (n = 5 in each group, Student t-test, **P < 0.01 of insoluble Aβ1–42 and Aβ1–40 versus corresponding that of WT, respectively; ##P < 0.01 of insoluble Aβ1–42 versus insoluble Aβ1–40 levels in APP23 mice). (D) Representative micrographs showed Shh expression in the hippocampi of WT and APP23 mice. (E). A significant increase in the expression of ligand Shh with different molecular weights was observed in the hippocampus at 3, 12 and 24 months old APP23 mice (n = 10 in each group, Student's t-test, **P < 0.01 versus WT).

Article Snippet: APP23 transgenic mice were provided by Novartis Institute for Biomedical Research and the mice express mutated human βAPP (Swedish double mutation, KM670/671NL) under brain and neuron-specific murine Thy-1 promoter element.

Techniques: Expressing

Journal: Human Molecular Genetics

Article Title: Deficiency of Patched 1-induced Gli1 signal transduction results in astrogenesis in Swedish mutated APP transgenic mice

doi: 10.1093/hmg/ddu370

Figure Lengend Snippet: Shh signal components are altered in the hippocampus of both APP23 mice and AD patients. (A) Ptc1 expression (green) was visualized by immunostaining in the dentate gyrus with an increased Ptc1 expression in 12-month-old APP23 mice, especially in the cells of hilus region. Neurons were stained with red. Scale bar: 50 µm. (B) Western blot showed the protein expressions of Shh signaling components in the hippocampus of 3-month-old APP23 mice. (C) Statistical analysis showed that the Smo expression level is increased (n = 10, Student's t-test, **P < 0.01) in APP23 mice of 3 months old of age, whereas the levels of Ptc1, Ptc2, Gli1, Gli2 and Gli3 expressions were still not significantly changed. (D) Western blot showed the protein expressions of Shh signaling components in the hippocampus of 12-month-old APP23 mice. (E) Statistical analysis showed that the expression levels of Ptc1, Ptc2, Smo and Gli1 were increased (n = 10, Student's t-test, *P < 0.05, **P < 0.01 versus corresponding WT) in APP23 mice of 12 months old of age, whereas the levels of Gli2 and Gli3 expressions were not significantly changed. (F) Western blot showed the protein expressions of Shh signaling components in the hippocampus of 24-month-old APP23 mice. (G) Statistical analysis showed that the expression levels of Smo, Gli1 and Gli2 are increased (n = 10, Student's t-test, *P < 0.01 versus corresponding WT) in APP23 mice of 24 months old of age, whereas the levels of Ptc1, Ptc2 and Gli3 expressions were significantly reduced (n = 10, Student's t-test, *P < 0.05, **P < 0.01 versus corresponding WT). (H) Western blot showed that Shh signaling components were expressed in the hippocampus of isolated from human brains of HC and AD patients. (I) The protein levels of Shh, Smo, Gli1 and Gli2 expression were significantly increased, whereas the expression levels of Ptc1, Ptc2 and Gli3 were significantly reduced in comparison with corresponding health controls without dementia (n = 5, Student's t-test, *P < 0.05, **P < 0.01 versus HC).

Article Snippet: APP23 transgenic mice were provided by Novartis Institute for Biomedical Research and the mice express mutated human βAPP (Swedish double mutation, KM670/671NL) under brain and neuron-specific murine Thy-1 promoter element.

Techniques: Expressing, Immunostaining, Staining, Western Blot, Isolation

Journal: Human Molecular Genetics

Article Title: Deficiency of Patched 1-induced Gli1 signal transduction results in astrogenesis in Swedish mutated APP transgenic mice

doi: 10.1093/hmg/ddu370

Figure Lengend Snippet: The survival of GPCs decrease and GPC apoptosis increases in APP23 mice. (A) Four weeks last BrdU injection the total number of remaining BrdU+ cells was significantly reduced (n = 6 in each group, Student's t-test, **P < 0.01) in APP23 mice. (B) Four weeks last BrdU application, the total number of surviving BrdU+ cells was counted and was shown as mean ± SEM per mice. Analysis showed a significant decrease in the remaining BrdU+ nuclei at SGZ and GCL of APP23 mice compared with WT (n = 6 in each group, Student's t-test, **P < 0.01, *P < 0.05) but no significant changes in hilus and Mol regions. (C) Representative micrographs of TUNEL staining showed TUNEL-positive nuclei (green) in the DG of WT and APP23 mice at age of 3 months old. Neurons were showed in red color with NeuN antibody. Arrowheads are for TUNEL-positive nuclei. Scale bar: 50 µm. (D) TUNEL-positive nuclei were counted and the average number per section was shown as mean ± SEM. Quantification of immunoreactive structures revealed a significant increase in the number of apoptotic nuclei in 3-month-old APP23 mice compared with age-matched WT (n = 6 in each group, Student's t-test, **P < 0.01). (E) GPC number declines and astrocyte number increases in the hippocampus of APP23 mice. Double labeling showed nestin-positive cells (green) and GFAP-positive cells (red). Closed white arrowhead was for GPCs (co-labeling of both GFAP and nestin); yellow arrowhead for transit-amplifying cells (nestin-positive and GFAP-negative), and white arrows for mature astrocytes (nestin-negative and GFAP-positive). Scale bar: 20 µm. (F) Nestin+GFAP+ expressing cells (NSCs), nestin+GFAP− cells (transit-amplifying cell) and GFAP+/nestin− cells (mature astrocyte) were counted and the average number per section was shown as mean ± SEM. Statistical analysis was significant difference (n = 6 in each group, Student's t-test, **P < 0.01 versus WT). (G) Double labeling showed BrdU+ cells (green) and GFAP+ cells (red) in the hilus of 12 months old WT and APP23 mice. Scale bar: 20 µm.

Article Snippet: APP23 transgenic mice were provided by Novartis Institute for Biomedical Research and the mice express mutated human βAPP (Swedish double mutation, KM670/671NL) under brain and neuron-specific murine Thy-1 promoter element.

Techniques: Injection, TUNEL Assay, Staining, Labeling, Expressing

Journal: Human Molecular Genetics

Article Title: Deficiency of Patched 1-induced Gli1 signal transduction results in astrogenesis in Swedish mutated APP transgenic mice

doi: 10.1093/hmg/ddu370

Figure Lengend Snippet: Cell proliferation changes and Smo antagonist cyclopamine prevents Shh-induced cell dividing in APP23 mice. (A) Microphotographs showed BrdU-positive staining nuclei in the hippocampus of WT and APP23 mice at 3 months old. The dentate gyrus was counterstained with hemotaxylin. Scale bar: 50 µm. (B) At 3 months old of APP23 mice, the number of BrdU-positive cells in SGZ was significantly increased (n = 6 each group, Student's t-test, **P < 0.01) whereas there were not significant changes in the regions of Hilus, Mol and GCL, compared with age-matched WT controls. (C) With the application of cyclopamine for 7 days, the number of BrdU+ cells were significantly decreased in WT (n = 5 each group, Student's t-test, *P < 0.05) and APP23 (n = 5 each group, Student's t-test, **P < 0.01) mice of 3 months old, compared with corresponding controls. (D) At 12 months old of APP23 mice, the number of BrdU-positive cells in SGZ was counted and the total number per hippocampus was summed and shown as mean ± SEM per mice. BrdU-positive cells were significantly increased (n = 6 each group, Student's t-test, *P < 0.05) whereas there were not significant changes in the regions of Hilus, Mol and GCL. (E) At 24 months old of APP23 mice, however, the number of BrdU-positive cells in SGZ was significantly decreased (n = 6 each group, Student's t-test, **P < 0.01) whereas there was not significant changes in Hilus, Mol and GCL.

Article Snippet: APP23 transgenic mice were provided by Novartis Institute for Biomedical Research and the mice express mutated human βAPP (Swedish double mutation, KM670/671NL) under brain and neuron-specific murine Thy-1 promoter element.

Techniques: Staining

Journal: Human Molecular Genetics

Article Title: Deficiency of Patched 1-induced Gli1 signal transduction results in astrogenesis in Swedish mutated APP transgenic mice

doi: 10.1093/hmg/ddu370

Figure Lengend Snippet: Neurogensis alters in the hippocampus of APP23 mice. (A) Representative micrographs showed DCX-expressing cells in the hippocampi of 3, 12 and 24 months old WT and APP23 mice. Scale bar: 50 µm. (B) The number of DCX+ cells was counted and the average number per section was shown at 3, 12 and 24 months old of WT and APP23 mice. The statistical differences were significant (n = 6 in each group, Student's t-test, **P < 0.01 versus WT). (C) Double labeling showed cells with BrdU+ (green) and psa-NCAM+ (red) in DG of 3 months of old WT and APP23 mice. Scale bar: 20 µm. (D) Immunostaining structures were accounted and the average number per section was shown as mean ± SEM. The ratio (%) of double-labeling cells with BrdU and psa-NCAM versus psa-NCAM expressing cells was significantly increased in 3-month-old APP23 mice (n = 6 in each group, Student's t-test, *P < 0.01 versus WT). (E) Double labeling showed cells with BrdU+ (green) and NeuN+ (red) in DG of 3 months old WT and APP23 mice after 4-weeks of BrdU application. Scale bar: 20 µm. (F) Co-labeling cells were accounted and the average number per section was shown as mean ± SEM. A differentiation to neurons with double-labeling of BrdU and NeuN was significantly decreased in 3-month-old APP23 mice after 4 weeks of BrdU injection (n = 6 in each group, Student's t-test, *P < 0.05 versus WT).

Article Snippet: APP23 transgenic mice were provided by Novartis Institute for Biomedical Research and the mice express mutated human βAPP (Swedish double mutation, KM670/671NL) under brain and neuron-specific murine Thy-1 promoter element.

Techniques: Expressing, Labeling, Immunostaining, Injection

Journal: EMBO Molecular Medicine

Article Title: Increased CSF Aβ during the very early phase of cerebral Aβ deposition in mouse models

doi: 10.15252/emmm.201505026

Figure Lengend Snippet: Human Aβ exhibits a biphasic profile in CSF of APP transgenic mice A, B Aβ40 and Aβ42 concentrations in CSF of male APP23 mice (heterozygous; 3 ( n = 14), 6 ( n = 12), 8 ( n = 10), 12 ( n = 11), 19 ( n = 9), and 25 ( n = 6) months of age). CSF Aβ40 ( F (1, 56) = 22.351, P < 0.001) as well as CSF Aβ42 ( F (1, 56) = 38.597, P < 0.001) followed a significant quadratic trend. C Aβ42/40 ratio in CSF of APP23 mice showed a delayed decrease with age ( F (1, 56) = 53.894, P < 0.001). D, E Aβ40 and Aβ42 concentrations in CSF of male and female APP24 mice (homozygous; 2 ( n = 13), 3–4 ( n = 16), 7–8 ( n = 15), 18–19 ( n = 14), 24 ( n = 16), and 30 ( n = 18) months of age). CSF Aβ40 followed a significant quadratic trend ( F (1, 86) = 6.678, P = 0.011) and CSF Aβ42 best fitted a cubic trend ( F (1, 86) = 30.599, P < 0.001). F Aβ42/40 ratio in CSF of APP24 mice showed a delayed decrease with age ( F (1, 86) = 64.936, P < 0.001). G, H Aβ40 and Aβ42 in the CSF of female APP51 mice (heterozygous; 3 ( n = 6), 15 ( n = 8), and 24 ( n = 8) months of age; 22 mice in total). CSF Aβ40 ( F (1, 19) = 37.349, P < 0.001) as well as CSF Aβ42 ( F (1, 19) = 107.670, P < 0.001) followed a significant quadratic trend. I Aβ42/40 ratio in CSF of APP51 mice showed a delayed decrease with age ( F (1, 19) = 26.367, P < 0.001). Data information: Post hoc Dunnett's test was employed for group comparisons, which were always conducted between the youngest group and all other groups. (Observed CSF Aβ40 or Aβ42 changes were independent of batch.) All data are represented as group means ± SEM; * P < 0.05; ** P < 0.01; and *** P < 0.001.

Article Snippet: Male and female 2- to 30-month-old homozygous APP24 mice (Abramowski et al , ) were bred at both the Novartis Mouse facility (Basel, Switzerland) and the Hertie Institute for Clinical Brain Research (Tübingen, Germany).

Techniques: Transgenic Assay

Journal: EMBO Molecular Medicine

Article Title: Increased CSF Aβ during the very early phase of cerebral Aβ deposition in mouse models

doi: 10.15252/emmm.201505026

Figure Lengend Snippet: Human Aβ in CSF and brain of APP transgenic mice A APP23 CSF Aβ40 and Aβ42 in the same animals as shown in Fig . CSF Aβ42 and Aβ40 are expressed as percentages of levels measured in the youngest age group. B, C Aβ40 and Aβ42 (pmol/g wet brain) in the FA-soluble brain extract from the same APP23 mice showed a robust increase with age; ANOVA revealed a significant cubic trend ( F (1, 56) = 221.114, P < 0.001 and F (1, 56) = 370.947, P < 0.001, respectively). D APP24 CSF Aβ40 and Aβ42 in the same animals shown in Fig as percentage of the youngest age group. E, F Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP24 mice also showed a robust increase with age; ANOVA revealed a significant cubic trend ( F (1, 86) = 202.173, P < 0.001 and F (1, 86) = 139.941, P < 0.001, respectively). G APP51 CSF Aβ40 and Aβ42 in the same animals shown in Fig as percentages of levels in the youngest age group. H, I Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP51 mice showed a robust increase with age; ANOVA revealed a significant quadratic trend ( F (1, 19) = 12.960, P = 0.002 and F (1, 19) = 19.366, P < 0.001, respectively). Data information: Post hoc Dunnett's test group comparisons were always conducted between the youngest group and all other groups. All data are represented as group means ± SEM; * P < 0.05; ** P < 0.01; and *** P < 0.001.

Article Snippet: Male and female 2- to 30-month-old homozygous APP24 mice (Abramowski et al , ) were bred at both the Novartis Mouse facility (Basel, Switzerland) and the Hertie Institute for Clinical Brain Research (Tübingen, Germany).

Techniques: Transgenic Assay

Journal: EMBO Molecular Medicine

Article Title: Increased CSF Aβ during the very early phase of cerebral Aβ deposition in mouse models

doi: 10.15252/emmm.201505026

Figure Lengend Snippet: Brain sAPPβ shows an age-related increase in APP23, APP24, and APP51 mice sAPPβ was measured in Triton X-100 brain extracts from largely the same mice as analyzed in Figs and and is expressed as percentages of levels measured in the youngest age group. Swedish sAPPβ showed an age-dependent increase in APP23 mice following a linear trend ( F (1, 83) = 52.914, P < 0.001); APP23 from two independent batches were included in this analysis (see Materials and Methods and Supplementary Fig for details). Swedish sAPPβ showed an age-dependent increase in APP24 mice following a linear trend ( F (1, 84) = 11.264, P = 0.001). Human wild-type sAPPβ showed an age-dependent increase in APP51 following a quadratic trend ( F (1, 18) = 68.980, P < 0.001). Data information: Post hoc Dunnett's test group comparisons were always conducted between the youngest group and all other groups. All data are represented as group means ± SEM; * P < 0.05; ** P < 0.01; and *** P < 0.001. For absolute values, see Supplementary Fig .

Article Snippet: Male and female 2- to 30-month-old homozygous APP24 mice (Abramowski et al , ) were bred at both the Novartis Mouse facility (Basel, Switzerland) and the Hertie Institute for Clinical Brain Research (Tübingen, Germany).

Techniques:

Journal: Translational Oncology

Article Title: Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET 1

doi:

Figure Lengend Snippet: Everolimus decreases FDG uptake by human H596 lung tumor xenografts. Human tumors were created by s.c. implantation of viable H596 tumor tissue in Harlan athymic mice. (A) For efficacy, mice were treated with vehicle or with everolimus (10 mg/kg p.o.) for 21 days. Results show mean ± SEM. (B and C) A separate cohort of tumor-bearing mice was studied by FDG-PET as described in the Materials and Methods section, immediately before and 2 days after treatment with everolimus. Results show the individual SUVs for tumors (n = 10 per treatment group) and the mean ± SEM fractional effect of treatment, where *P < .05, **P <.01. (D) Histology and IHC of ablated tumors on day 2 after completion of the second PET scan.

Article Snippet: Female Brown-Norway rats and C57BL/6 mice were obtained from Charles River (France) and female Harlan Hsd:Npa nu/nu (nude) athymic mice were obtained from the Novartis breeding stock (Basel, Switzerland).

Techniques:

Journal: Translational Oncology

Article Title: Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET 1

doi:

Figure Lengend Snippet: Everolimus inhibits growth of insensitive human tumors but does not affect FDG uptake. Human HCT116 (colon) and KB31 (cervical) were created by s.c. injection of cells in Harlan athymic mice. In all cases, mice were treated daily p.o. with vehicle or everolimus (10 mg/kg). (A and B) Efficacy in HCT116 tumors for 1 week (A) or 2 weeks (B) showing the mean ± SEM with the associated T/CTVol at the end point. (C and D) FDG-PET in HCT116 tumors in the respective experiment showing the mean fractional change for each treatment compared with day 0 (C) or the individual tumor SUV at two different time points (D). (E and F) Efficacy in KB31 tumors with the fractional change in FDG-PET compared with day 0 for each treatment, where *P < .05, ***P < .001.

Article Snippet: Female Brown-Norway rats and C57BL/6 mice were obtained from Charles River (France) and female Harlan Hsd:Npa nu/nu (nude) athymic mice were obtained from the Novartis breeding stock (Basel, Switzerland).

Techniques: Injection

Journal: Translational Oncology

Article Title: Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET 1

doi:

Figure Lengend Snippet: Everolimus decreases FLT uptake by human H596 lung tumor xenografts. Human tumors were created by s.c. implantation of viable H596 tumor tissue in Harlan athymic mice. Tumors were studied by FLT-PET as described in the Materials and Methods section, immediately before and 2 days after treatment with everolimus (10 mg/kg p.o.). (A) Representative experiment showing horizontal whole-body images of mice with tumors (blue arrow) before and after treatment with everolimus; red arrow indicates the bladder. (B) Individual SUVs for tumors (n = 6 per treatment group), and the mean ± SEM fractional effect of treatment, where **P < .01. (C) Histology and IHC of ablated tumors on day 2 after completion of the second PET scan.

Article Snippet: Female Brown-Norway rats and C57BL/6 mice were obtained from Charles River (France) and female Harlan Hsd:Npa nu/nu (nude) athymic mice were obtained from the Novartis breeding stock (Basel, Switzerland).

Techniques:

Journal: Translational Oncology

Article Title: Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET 1

doi:

Figure Lengend Snippet: Everolimus does not affect FLT uptake by human HCT116 colon tumor xenografts. Human HCT116 tumors were created by s.c. injection of cells in Harlan athymic mice. (A and B) Tumors were studied by FLT-PET as described in the Materials and Methods section, immediately before (day 0) and on days 3 and 10 after treatment with vehicle or everolimus (10 mg/kg p.o.). Results show (n = 6 per treatment group) the individual SUVs for tumors (A) and the mean ± SEM fractional effect of treatment (B). (C) Histology and IHC of ablated tumors on day 10 after completion of the second PET scan.

Article Snippet: Female Brown-Norway rats and C57BL/6 mice were obtained from Charles River (France) and female Harlan Hsd:Npa nu/nu (nude) athymic mice were obtained from the Novartis breeding stock (Basel, Switzerland).

Techniques: Injection