Journal: Cell Death and Differentiation

Article Title: A Smad3-PTEN regulatory loop controls proliferation and apoptotic responses to TGF- β in mouse endometrium

doi: 10.1038/cdd.2017.73

Figure Lengend Snippet: Smad3 deficiency results in resistance to TGF-β-induced (a) representative images of Smad2/3 immunofluorescence of 3D cultures untreated (UN) or treated with TGF-β (10 ng/ml). Nuclei were counterstained with Hoechst to show apoptotic nuclear morphology. Scale bar: 25 μm. (b) Representative cleaved caspase-3 immunostaining of endometrial 3D cultures infected with the lentiviruses carrying Smad3 shRNAs (FSV-shRNA3.1 or FSV-shRNA3.2) and UN or treated with 10 ng/ml of TGF-β for 36 h. Cultures were immunostained with tubulin to show glandular structures. Nuclei were counterstained with Hoechst to show apoptotic nuclear morphology. Scale bar: 25 μm. (c) Quantification of cleaved caspase-3-positive cells in the same culture conditions. Values are mean±S.E.M. **P<0.01, by t-test analysis. (d) Representative images and quantification of Smad3+/+ and Smad3−/− endometrial 3D cultures UN or treated with 10 ng/ml of TGF-β for 36 h and immunostained for cleaved caspase-3. Cultures were also immunostained with phalloidin to show glandular structures. Nuclei were counterstained with Hoechst to show apoptotic nuclear morphology. Scale bars: 25 μm. (e) Quantification of cleaved caspase-3-positive cells in the same culture conditions. Values are mean±S.E.M. **P<0.01, by t-test analysis. (f) RT-qPCR analysis of BIM mRNA expression in 3D cultures from Smad3+/+ and Smad3−/− or in 3D cultures infected with Smad3 shRNAs (FSV-Smad3.1 and FSV-Smad3.2) that were UN or treated with TGF-β (10 ng/ml) for 16 h. Values are mean±S.E.M. **P<0.01, by t-test analysis. (g) Quantification of cleaved caspase-3-positive cells from Smad3+/+ and Smad3−/− endometrial 3D cultures UN or treated with ABT-263. Values are mean and error bars represent mean±S.E.M. **P<0.01 by one-way ANOVA, followed by the Tukey’s multiple comparison test

Article Snippet: Antibodies to cleaved caspase-3 (#9961), PTEN (#9188), p-AKT (ser473) (#4060), cleaved caspase-9 (#9588) and p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828) and Smad2 (D43B4) XP Rabbit mAb (#5339) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Immunofluorescence, Immunostaining, Infection, Quantitative RT-PCR, Expressing

Journal: Cell Death and Differentiation

Article Title: A Smad3-PTEN regulatory loop controls proliferation and apoptotic responses to TGF- β in mouse endometrium

doi: 10.1038/cdd.2017.73

Figure Lengend Snippet: Smad3 binds PTEN promoter to enhance its transcription. (a) RT-qPCR of PTEN mRNA expression in 3D cultures infected with Smad3 shRNAs (FSV-Smad3.1 and FSV-Smad3.2). Data are from n=3 experimental replicates and values are mean±S.E.M. *P<0.01, by one-way ANOVA post hoc Tukey's multiple comparison test. (b) RT-qPCR analysis of PTEN mRNA expression of 3D cultures from four different Smad3+/+ and Smad3−/− 4-week-old littermates. Values are mean±S.E.M. *P<0.05 and **P<0.01, by t-test analysis. (c) RT-qPCR analysis showing PTEN mRNA expression in 3D cultures treated for the indicated times with 10 ng/ml of TGF-β alone or TGF-β plus SB431542. Values are mean±S.E.M. **P<0.01, by t-test analysis. (d) RT-qPCR analysis of PTEN mRNA in 3D cultures from Smad3+/+ and Smad3−/− epithelial cells treated for 12 h with 10 ng/ml of TGF-β or untreated (UN). Values are mean±S.E.M. **P<0.01, by t-test analysis. (e) ChIP analysis of Smad2/3 binding to PTEN promoter. Cell lysates from UN or TGF-β-stimulated 3D cultures (30 min) were subjected to immunoprecipitation (IP) with Smad2, Smad3 or control antibody (IgG). Immunoprecipitates were analyzed by real-time qPCR using primers to amplify three ~500 bp consecutive segments of PTEN promoter (−1/−500, −501/−986, −987/−1459). Plots show the results from qPCR analysis of PTEN promoter regions expressed as fold enrichment of PTEN promoter amplification over the input DNA. Results correspond to n=3 experimental replicates and values are mean±S.E.M. Right panel shows a representative gel image of ChIP analysis (conventional electrophoresis gel run of the samples amplified in real-time experiments)

Article Snippet: Antibodies to cleaved caspase-3 (#9961), PTEN (#9188), p-AKT (ser473) (#4060), cleaved caspase-9 (#9588) and p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828) and Smad2 (D43B4) XP Rabbit mAb (#5339) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Quantitative RT-PCR, Expressing, Infection, Binding Assay, Immunoprecipitation, Amplification, Electrophoresis

Journal: Cell Death and Differentiation

Article Title: A Smad3-PTEN regulatory loop controls proliferation and apoptotic responses to TGF- β in mouse endometrium

doi: 10.1038/cdd.2017.73

Figure Lengend Snippet: A mechanistic model for PTEN-Smad3 regulatory loop. In a wild-type cell context, TGF-β acts as a tumor suppressor by inducing apoptosis. TGF-β stimulation leads to nuclear translocation of Smad2/3. In the nucleus, Smad3 is required to transcribe PTEN, which inhibits PI3K/AKT signaling and enables TGF-β-induced apoptosis. In Smad3- or PTEN-deficient cells context TGF-β-induced upregulation of PTEN mRNA is impaired, resulting in enhanced PI3K/AKT activation, which leads to increased proliferation and hampers apoptosis triggered by TGF-β

Article Snippet: Antibodies to cleaved caspase-3 (#9961), PTEN (#9188), p-AKT (ser473) (#4060), cleaved caspase-9 (#9588) and p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828) and Smad2 (D43B4) XP Rabbit mAb (#5339) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Translocation Assay, Activation Assay

Journal: Scientific Reports

Article Title: Sustained PI3K Activation exacerbates BLM-induced Lung Fibrosis via activation of pro-inflammatory and pro-fibrotic pathways

doi: 10.1038/srep23034

Figure Lengend Snippet: Alveolar macrophages (AMs) were isolated from the lungs of myeloid PTEN knockout mice and wild-type littermates and stimulated with IL-4 (10 ng/ml), IL-13 (10 ng/ml) or both in combination. ( a ) AMs were stimulated with IL-4 and IL-13 for the indicated time points and phosphorylation of AKT and GSK3β was detected by Western Blot, which were increased in PTEN deficient macrophages. ( b ) IL-4/IL-13 stimulation overnight led to increased Arginase I (Arg1) expression in the macrophages lacking PTEN. ( c–e ) AMs were stimulated with IL-4 or IL-13 o/n, RNA was isolated and analysed by qPCR. The M2 markers Resistin like alpha (Retnla, FIZZ1), Arg1 and Chitinase-like 3 (Chil3, YM1) were determined. The Data were analysed by 2-way ANOVA, n = 4, **p < 0.01, ***p < 0.001, ****p < 0.0001. ( f ) Western Blot analysis of phospho-AKT, phospho-GSK3β, AKT and β-Actin of macrophages isolated from single cell suspension of lungs of wild-types and myeloid PTEN deficient mice 14d post BLM treatment and control mice. ( g ) Total lung of BLM treated mice was homogenized at d7 and RNA was isolated. Via qPCR the expression of the genes st a bilin1 ( stab1 ), retnla , arg1 and chil3 was measured. For statistical analysis the Mann Whitney test was performed, n ≥ 4, *p < 0.05. Expression levels were normalized to HPRT as housekeeping gene. Data are shown as fold values of BLM treated wild-type (light bars) and PTEN MyKO (grey bars) mice relative to treatment-naive mice.

Article Snippet: The antibodies against phospho-AKT (Ser473, D9E), phospho- GSK-3β (Ser9, 5B3), phospho-Smad2 (Ser465/467)/Smad3 (Ser423/467, D27F4), Smad2/3, AKT were purchased from Cell Signaling Technology, Inc., antibody against Arginase I (C-2) from Santa Cruz Biotechnology, INC. and anti-actin antibody was obtained from Sigma-Aldrich Inc, Vienna, Austria.

Techniques: Isolation, Knock-Out, Western Blot, Expressing, MANN-WHITNEY