Journal: Endocrinology

Article Title: Phosphorylated Progesterone Receptor Isoforms Mediate Opposing Stem Cell and Proliferative Breast Cancer Cell Fates

doi: 10.1210/en.2018-00990

Figure Lengend Snippet: PR-A expression enhanced breast CSC populations and target gene expression. (A) ALDH+ activity in T47D (Y, YA, YB) tumorspheres was assessed using flow cytometry. (B) CD44+/CD24− populations from T47D (Y, YA, YB) tumorspheres. (C) CD49f+/CD24− populations from T47D-YA and T47D-YB tumorspheres. (D) mRNA levels of select genes (e.g., PTGES, p21, FOXO1, WNT4, KLF4, NOTCH2) in T47D-YA and T47D-YB cells. Cells were cultured in tumorsphere conditions and treated with vehicle (veh), R5020 (10 nM), or RU486 (100 nM). (E) ChIP assays showing PR recruitment to a PRE-containing region of the WNT4, KLF4, or NOTCH2 promoter. T47D-YA and T47D-YB cells were stimulated with vehicle (EtOH) or R5020 (10 nM) for 1 hour. Fixed lysates were subjected to ChIP assays using specific antibodies targeting PR (or IgG control). Graphed data represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Immunoblotting was performed with the following antibodies: phosphorylated PR [Ser294 (custom), Thermo Fisher], PR (H190; Santa Cruz Biotechnology; RRID: AB_2164331 ) ( 37 ), PR (F-4; Santa Cruz Biotechnology; RRID: AB_2166687 ) ( 38 ), phosphorylated FOXO1 (Ser256; Cell Signaling; RRID: AB_329831 ) ( 39 ), FOXO1 (C29H4; Cell Signaling; RRID: AB_2106495 ) ( 40 ), phosphorylated Akt (Ser473; Cell Signaling; RRID: AB_329825 ) ( 41 ), Akt (Cell Signaling; RRID: AB_329827 ) ( 42 ), glyceraldehyde 3-phosphate dehydrogenase (0411; Santa Cruz Biotechnology; RRID: AB_627678 ) ( 43 ), goat anti-rabbit IgG-horseradish peroxidase (BioRad; RRID: AB_11125142 ) ( 44 ), and goat anti-mouse IgG-horseradish peroxidase (BioRad; RRID: AB_11125547 ) ( 45 ).

Techniques: Expressing, Targeted Gene Expression, Activity Assay, Flow Cytometry, Cell Culture, Control

Journal: Endocrinology

Article Title: Phosphorylated Progesterone Receptor Isoforms Mediate Opposing Stem Cell and Proliferative Breast Cancer Cell Fates

doi: 10.1210/en.2018-00990

Figure Lengend Snippet: Ser294 contributes to PR-A–induced breast CSC phenotypes. (A) Primary tumorspheres from T47D (EV, WT PR-A, and S294A PR-A) cells. (B) ALDH activity in T47D (EV, WT PR-A, and S294A PR-A) primary tumorspheres. (C) Soft agar colony formation of T47D (EV, WT PR-A, and S294A PR-A) cells. (D) CD44+/CD24− populations in T47D (EV, WT PR-A, and S294A PR-A) primary tumorspheres. (E) mRNA levels of select genes (e.g.,PTGES, p21, FOXO1, WNT4, KLF4, NOTCH2) in T47D (EV, WT PR-A, and S294A PR-A) cells cultured in tumorsphere conditions. Cells were treated with vehicle (veh; EtOH) or R5020 (10 nM). (F) ChIP assays showing phosphorylated PR and (G) total PR recruitment to PRE-containing regions of the WNT4, KLF4, or NOTCH2 promoter. T47D (WT PR-A and S294 PR-A) cells were stimulated with vehicle (EtOH) or R5020 (10 nM) for 1 hour. Fixed lysates were subjected to ChIP assays using specific antibodies targeting phosphorylated PR (S294) or IgG control. Graphed data represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. Avg, average.

Article Snippet: Immunoblotting was performed with the following antibodies: phosphorylated PR [Ser294 (custom), Thermo Fisher], PR (H190; Santa Cruz Biotechnology; RRID: AB_2164331 ) ( 37 ), PR (F-4; Santa Cruz Biotechnology; RRID: AB_2166687 ) ( 38 ), phosphorylated FOXO1 (Ser256; Cell Signaling; RRID: AB_329831 ) ( 39 ), FOXO1 (C29H4; Cell Signaling; RRID: AB_2106495 ) ( 40 ), phosphorylated Akt (Ser473; Cell Signaling; RRID: AB_329825 ) ( 41 ), Akt (Cell Signaling; RRID: AB_329827 ) ( 42 ), glyceraldehyde 3-phosphate dehydrogenase (0411; Santa Cruz Biotechnology; RRID: AB_627678 ) ( 43 ), goat anti-rabbit IgG-horseradish peroxidase (BioRad; RRID: AB_11125142 ) ( 44 ), and goat anti-mouse IgG-horseradish peroxidase (BioRad; RRID: AB_11125547 ) ( 45 ).

Techniques: Activity Assay, Cell Culture, Control

Journal: Endocrinology

Article Title: Phosphorylated Progesterone Receptor Isoforms Mediate Opposing Stem Cell and Proliferative Breast Cancer Cell Fates

doi: 10.1210/en.2018-00990

Figure Lengend Snippet: FOXO1 promoted breast CSC outgrowth in PR isoforms. (A) Western blot of FOXO1 (phosphorylated and total) and Akt (phosphorylated and total) in HeLa cells transfected with green fluorescent protein–tagged PR-A and with pcDNA FOXO1. Cells were treated with vehicle (veh; EtOH) or R5020 (10 nM) for the indicated times. shFOXO1 was included as a control. (B) Primary tumorsphere assays in T47D-YA and T47D-YB cells with FOXO1 knockdown. (C) Primary tumorsphere assays in T47D (YA and YB) cells stably expressing FOXO1. (D) ALDH activity in primary tumorspheres formed from T47D (YA and YB) FOXO1+ cells was assessed by flow cytometry. (E) Secondary tumorspheres in T47D (YA and YB) cells expressing WT FOXO1 or FOXO1-AAA. (F) Secondary tumorspheres in T47D (YA and YB) FOXO1+ cells treated with AS1842856 (100 nM). (Right) T47D (YA and YB) cells expressing FOXO1 were pretreated with AS1842856 (100 nM) for 60 minutes, followed by the indicated treatments with R5020 (10 nM). (G) Secondary tumorspheres in T47D (YA and YB) cells treated with AS1842856. (Right) T47D (YA and YB) cells were pretreated with AS1842856 (100 nM) for 60 minutes, followed by treatment with R5020 (10 nM). (H) MCF-7 and BT-474 primary tumorspheres treated with E2 (1 nM) or R5020 (10 nM), respectively, in the presence of onapristone (100 nM) or AS1842856 (100 nM), or both. Graphed data represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. Avg, average.

Article Snippet: Immunoblotting was performed with the following antibodies: phosphorylated PR [Ser294 (custom), Thermo Fisher], PR (H190; Santa Cruz Biotechnology; RRID: AB_2164331 ) ( 37 ), PR (F-4; Santa Cruz Biotechnology; RRID: AB_2166687 ) ( 38 ), phosphorylated FOXO1 (Ser256; Cell Signaling; RRID: AB_329831 ) ( 39 ), FOXO1 (C29H4; Cell Signaling; RRID: AB_2106495 ) ( 40 ), phosphorylated Akt (Ser473; Cell Signaling; RRID: AB_329825 ) ( 41 ), Akt (Cell Signaling; RRID: AB_329827 ) ( 42 ), glyceraldehyde 3-phosphate dehydrogenase (0411; Santa Cruz Biotechnology; RRID: AB_627678 ) ( 43 ), goat anti-rabbit IgG-horseradish peroxidase (BioRad; RRID: AB_11125142 ) ( 44 ), and goat anti-mouse IgG-horseradish peroxidase (BioRad; RRID: AB_11125547 ) ( 45 ).

Techniques: Western Blot, Transfection, Control, Knockdown, Stable Transfection, Expressing, Activity Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: Activation of AQP2 by water deprivation. Changes in urinary volume ( a ) and urine osmolality ( b ) in mice at baseline and after 24 h of water deprivation. Each dot represents a mouse. * p < 0.05 (Welch’s t -test). ( c ) Western blot analysis for Ser256−phosphorylated and total AQP2 in kidney tissue from control mice and mice under water deprivation. β−actin is also shown as a loading control. ( d ) Quantification of the signal intensities expressed as a ratio of phosphorylated AQP2 to total AQP2. N = 6. *** p < 0.001 (Unpaired t -test). ( e ) Representative immunofluorescence images for Ser256−phosphorylated AQP2 (red) in kidney sections from control and water−deprived mice. The scale bar indicates 20 μm. ( f ) Quantification of the ratio of phosphorylated AQP2 signal intensities to the tubular area. The analysis was based on at least three microscopic fields of tissue from each group of mice. * p < 0.05 (Unpaired t -test).

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Activation Assay, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: Uromodulin signals detected at the apical surface of the collecting duct. Representative immunofluorescence images for Ser256−phosphorylated AQP2 ( red ), uromodulin ( green ), and merged image with 4′,6−diamidino−2−phenylindole (DAPI) ( blue ) on Carnoy’s solution−fixed kidney sections from a wild−type mouse deprived of water for 24 h. Apical and cytosolic uromodulin signals were observed in the thick ascending limb of the loop of Henle (arrowheads). Uromodulin signals can be detected at the apical surface of the collecting duct (arrow). The scale bar indicates 20 μm.

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: AQP2 activation is altered in uromodulin−deficient mice. ( a ) Western blot analysis for uromodulin, Ser256−phosphorylated AQP2 and total AQP2 in kidney tissue from WT and Umod−KO mice after 24 h of water deprivation. β−actin was used as a loading control. ( b ) Quantification of signal intensities expressed as the ratio of phosphorylated AQP2 to total AQP2. n = 6. ** p < 0.01, *** p < 0.001 (Unpaired t −test). ( c ) Representative immunofluorescence images for Ser256−phosphorylated AQP2 (red) in kidney sections from WT and Umod−KO mice, with strong signals in WT mouse kidney tissue. The scale bar indicates 20 μm. ( d ) Quantification of the ratio of phosphorylated AQP2 signal intensities to the tubular area. The analysis was based on at least three microscopic fields of tissue from each group of mice. * p < 0.05 (Unpaired t −test).

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Activation Assay, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: Effect of uromodulin on AQP2 activation. ( a ) Western blot analysis for Ser256-phosphorylated AQP2 and total AQP2 in lysates of mouse collecting duct cells. Cell monolayer on a filter membrane was treated with 10 −9 M dDAVP on the basolateral side and 10 −6 µg/mL uromodulin on the apical side for 24 h. β-actin was used as a loading control. ( b ) Quantification of the signal intensities expressed as a ratio of phosphorylated AQP2 to total AQP2. Analyses were based on at least three independent experiments. * p < 0.05 (Unpaired t -test).

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Activation Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: Uromodulin may activate AQP2 through the suppression of endocytosis. ( a ) Representative immunofluorescence images for Ser256−phosphorylated AQP2 in mouse collecting duct cells treated with 10 −9 M dDAVP with or without 10 −6 µg/mL uromodulin and 100 µM simvastatin for 24 h. Uromodulin-treated cells showing strong signals for phosphorylated AQP2 (red). Strong linear signals for phosphorylated AQP2 at the membrane can be observed in cells treated with simvastatin, regardless of uromodulin treatment. ( b ) Intensity profiles for Ser256-phosphorylated AQP2 along axis across the cell membrane. Quantifications are based on ten axes (yellow lines) in each condition and showed as mean and SEM. Bars indicate 10 μm. DAPI, 4’,6−diamidino−2−phenylindole; umod, uromodulin; simva, simvastatin.

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Immunofluorescence

Journal: Biochemical Journal

Article Title: Sphingolipid biosynthesis upregulation by TOR complex 2–Ypk1 signaling during yeast adaptive response to acetic acid stress

doi: 10.1042/bcj20160565

Figure Lengend Snippet: Figure 1. Exposure to an inhibitory sublethal acetic acid concentration stimulates TORC2-dependent Ypk1 activation

Article Snippet: Primary antibodies and dilutions used in this study were: 1:5000 mouse anti-FLAG (Sigma–Aldrich), 1:500 rabbit anti-pSGK (Thr256) (to recognize Ypk1 phosphorylated at Thr504) (Santa Cruz Biotechnology), 1:20 000 rabbit anti-Ypk1 (p-Thr662) (a gift from Ted Powers, University of California, Davis, Davis, CA, U.S.A.) and 1:1000 goat anti-Ypk1 (Santa Cruz Biotechnology).

Techniques: Concentration Assay, Activation Assay

Journal: Biochemical Journal

Article Title: Sphingolipid biosynthesis upregulation by TOR complex 2–Ypk1 signaling during yeast adaptive response to acetic acid stress

doi: 10.1042/bcj20160565

Figure Lengend Snippet: Figure 2. Exposure to an inhibitory sublethal acetic acid concentration triggers Ypk1-mediated phosphorylation of proteins whose modification stimulates sphingolipid production and is important for survival under this stress.

Article Snippet: Primary antibodies and dilutions used in this study were: 1:5000 mouse anti-FLAG (Sigma–Aldrich), 1:500 rabbit anti-pSGK (Thr256) (to recognize Ypk1 phosphorylated at Thr504) (Santa Cruz Biotechnology), 1:20 000 rabbit anti-Ypk1 (p-Thr662) (a gift from Ted Powers, University of California, Davis, Davis, CA, U.S.A.) and 1:1000 goat anti-Ypk1 (Santa Cruz Biotechnology).

Techniques: Concentration Assay, Phospho-proteomics

Journal: Biochemical Journal

Article Title: Sphingolipid biosynthesis upregulation by TOR complex 2–Ypk1 signaling during yeast adaptive response to acetic acid stress

doi: 10.1042/bcj20160565

Figure Lengend Snippet: Figure 3. The acetic acid-tolerance determinant protein kinase Hrk1 is not required for either TORC2 activation or Ypk1

Article Snippet: Primary antibodies and dilutions used in this study were: 1:5000 mouse anti-FLAG (Sigma–Aldrich), 1:500 rabbit anti-pSGK (Thr256) (to recognize Ypk1 phosphorylated at Thr504) (Santa Cruz Biotechnology), 1:20 000 rabbit anti-Ypk1 (p-Thr662) (a gift from Ted Powers, University of California, Davis, Davis, CA, U.S.A.) and 1:1000 goat anti-Ypk1 (Santa Cruz Biotechnology).

Techniques: Activation Assay