Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Expression and roles of p16 INK4a , p21 Cip1 and p27 Kip1 in the response of lung adenocarcinoma cell line models to long-term treatment with Dex. H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells were treated with Dex (100 nM) for the indicated number of days and whole cell lysates were probed by western blot for p16 INK4a , p21 Cip1 and p27 Kip1 with GAPDH as the loading control (Panel A). H1299GR Clone 4 cells were transduced with either non-targeted control shRNA or P21 Cip1 shRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were extracted and probed by western blot for p21 Cip1 , p27 Kip1 and GAPDH (loading control) (Panel B). In parallel, cells treated as described for Panel B, were plated for colony formation in 6-well plates in triplicates without further treatment (Panel C). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27 Kip1 siRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were probed by western blot for p27 Kip1 or GAPDH (loading control) (Panel D). In parallel, cells transfected as described for Panel D, and treated with Dex (100 nM) were plated on days 0 and 5 to measure colony formation in 6-well plates in triplicates without further treatment (Panel E) and also harvested on days 0 and 3 of treatment for measuring cell size (Panel F). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27 Kip1 siRNA and 24 h later they were treated with Dex (100 nM) or vehicle for 3 days and the cells were stained to assess expression of β- galactosidase (blue staining) (Panel G, left); in parallel, the treated cells were lysed and analyzed by western blot for p27 expression (Panel G, right). H1299GR Clone 4 cells were transduced with either non-targeted control vector or pLenti-p27 expression vector and after the indicated number of days, whole cell lysates were probed by western blot for p27 Kip1 or GAPDH (loading control) (Panel H). In parallel, cells were harvested for measuring the cell size (Panel I) and also plated to measure colony formation in 6-well plates in triplicate (Panel J). Panel C: *P, 0.0004; **P, 0.0006; § P, 0.0008; ‡ P, 0.00015. Panel E: *P, 0.016. Panel J: *P, 0.015; § P, 0.00006; ‡ P, 0.00007.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Western Blot, Control, Transduction, shRNA, Transfection, Staining, Plasmid Preparation

Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Mechanism of accumulation of p27 Kip1 in response to long-term Dex treatment in high GR expressing cells. H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or a combination of FOXO3 and FOXO4 siRNAs and treated with Dex (100 nM) for the indicated number of days, when whole cell lysates were probed by western blot for FOXO3, FOXO4, p27 Kip1 and GAPDH (loading control) (Panel A). H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells were treated with Dex (100 nM) for the indicated number of days, when RNA was extracted and relative expression levels of p27 Kip1 mRNA were measured using real time RT-PCR (Panel B). H1299GR Clone 4 cells were pre-treated with vehicle or Dex (100 nM) for 72 h. The cells were then treated with 1ug/ml of actinomycin D and at the time points indicated, RNA was harvested from the cells and p27 Kip1 mRNA was measured by real time RT-PCR to determine p27 Kip1 mRNA turnover rates. The mRNA values are plotted relative to the values at the time of addition of actinomycin D (Panel C). Panel B (H292): *P, 0.01; § P, 0.008. Panel B (A549): *P, 0.02; § P, 0.0001. Panel B (H1299GRα clone2): *P, 0.0001; ‡ P, 0.0007, § P, 0.001. Panel B (H1299GRα clone4): *P, 0.0001; ‡ P, 0.001; § P, 0.0001.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Effect of Dex on mouse tumor xenografts expressing low or high levels of GR. SCID mice bearing xenografts of parental H1299 cells (Panel A) or H1299GR Clone 4 cells (Panel B) were implanted with either placebo or Dex (2.5 mg) slow-release pellets at the time indicated by the arrow. The tumor growth was monitored via caliper measurements and tumor volumes were calculated using the formula noted in the Methods section. At the time of sacrifice, residual H1299GR Clone 4 tumors were harvested from the placebo (Panel C, left) and Dex (Panel C, right) treated mice and stained to ensure GR expression and to quantify p27 Kip1 by immunohistochemistry (Panel D). The scoring criteria based on staining intensity and percent positivity, as outlined in the Methods section. The parental H1299 cell tumor xenografts harvested at the end of placebo or Dex treatments in Panel A were also examined by immunohistochemistry for GR and p27 Kip1 expression (Panel E). Panel B: *P, 0.08; § P, 0.016; ‡ P, 0.0015.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Biomedical Optics Express

Article Title: 3D-printed biosensors in biomedical applications exploiting plasmonic phenomena and antibody self-assembled monolayers

doi: 10.1364/boe.516565

Figure Lengend Snippet: Fig. 11. (a) SPR spectra obtained by the 3D-printed biosensor for different values of p27 concentration. (b) Absolute value of the experimental variation in resonance wavelength (black marker), calculated with respect to the resonance wavelength of the blank, with relative error bars. The Langmuir fitting (red line) of the experimental values is also reported.

Article Snippet: Human recombinant p27 protein was furnished by OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Concentration Assay, Marker

Journal: Biomedical Optics Express

Article Title: 3D-printed biosensors in biomedical applications exploiting plasmonic phenomena and antibody self-assembled monolayers

doi: 10.1364/boe.516565

Figure Lengend Snippet: Fig. 12. (a) Normalized transmitted spectra obtained with the analyte (p27) and two interferents (MMP-2 and IL-6); (b) Experimental resonance wavelength variations (calculated with respect to the blank) for the three analyzed solutions.

Article Snippet: Human recombinant p27 protein was furnished by OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques:

Journal: International journal of molecular medicine

Article Title: Dihydrotestosterone induces p27 degradation via direct binding with SKP2 in ovarian and breast cancer.

doi: 10.3892/ijmm.2011.677

Figure Lengend Snippet: Figure 1. Effect of different androgens on protein levels of p21 and p27 and on the cell cycle progression. Western blot analysis demonstrated that DHT (10 nmol) down-regulated p27, but not p21, within 4 h of treatment in the MCF-7 and OVCAR-3 cell lines. The effect of DHT (10 nmol) on the cell cycle progression was detected by propidium iodide/BrdU-bivariate flow cytometric analyses. Cells were exposed to DHT for 6 h. BrdU was added for the last 30 min to label S-phase cells active in DNA replication. Values are the mean ± standard error of the mean (n=4). (C and D) Western blot analysis demonstrated that testosterone (T, 10 nmol) and DHEA (10 nmol) did not down-regulate p27 and p21 within 4 h in the MCF-7 and OVCAR-3 cell lines. (E) Down-regulation of p27 by DHT (10 nmol) was concentration-dependent, but the relationship was not linear.

Article Snippet: For the pull-down assay, the p27 gene (Origene, cA) was constructed into pgEX-6p-1 and p27-gST fusion proteins were purified with GST-sepharose beads (GE Healthcare, NY).

Techniques: Western Blot, Concentration Assay

Journal: International journal of molecular medicine

Article Title: Dihydrotestosterone induces p27 degradation via direct binding with SKP2 in ovarian and breast cancer.

doi: 10.3892/ijmm.2011.677

Figure Lengend Snippet: Figure 2. Effect of DHT in combination with different inhibitors on p27 expression in MCF-7 and OVCAR-3 cells. (A and B) Western blot analysis demon strated that actinomycin D (1.0 µg, a DNA transcription suppressor) and AZD0530 (1 µmol, a phosphorylation inhibitor) do not influence the down-regulation of p27 by DHT (10 nmol) in either cell line within 4 h. The lower panel indicates the normalization to GAPDH. (C and D) Western blot analysis showed that the proteasome inhibitors (MG132, 10 µmol; bortezomib, 1 mg) prevented the degradation of p27 by DHT (10 nmol) in MCF-7 and OVCAR-3 cell lines within 4 h. (E) DHT (10 nmol) did not have an effect on p27 mRNA expression within 4 h as determined by qPCR. (F) Cycloheximide (2 µg/ml) did not inhibit the DHT (10 nmol)-induced p27 degradation, but rather accelerated this process.

Article Snippet: For the pull-down assay, the p27 gene (Origene, cA) was constructed into pgEX-6p-1 and p27-gST fusion proteins were purified with GST-sepharose beads (GE Healthcare, NY).

Techniques: Expressing, Western Blot, Phospho-proteomics

Journal: International journal of molecular medicine

Article Title: Dihydrotestosterone induces p27 degradation via direct binding with SKP2 in ovarian and breast cancer.

doi: 10.3892/ijmm.2011.677

Figure Lengend Snippet: Figure 3. Effect of DHT treatment in combination with various steroid receptor inhibitors or with siRNAs for two different E3 ligases (SKP2 or KPC1) on p27 expression in MCF-7 and OVCAR-3 cells. (A and B) Western blot analysis showed that flutamide (an AR inhibitor) or fulvestrant (an ER inhibitor) at various concentrations did not influence the down-regulation of p27 by DHT (10 nmol) in MCF-7 or OVCAR-3 cell lines within 4 h. (C) Western blot analysis showed that SKP2 siRNA transfection successfully reduced SKP2 levels and prevented the degradation of p27 by DHT (10 nmol) in MCF-7 and OVCAR-3 cell lines within 4 h. (D) Western blot analysis showed that KPC1 siRNA transfection successfully reduced KPC1, but did not prevent the degradation of p27 by DHT (10 nmol) in MCF-7 and OVCAR-3 cell lines within 4 h.

Article Snippet: For the pull-down assay, the p27 gene (Origene, cA) was constructed into pgEX-6p-1 and p27-gST fusion proteins were purified with GST-sepharose beads (GE Healthcare, NY).

Techniques: Expressing, Western Blot, Transfection

Journal: International journal of molecular medicine

Article Title: Dihydrotestosterone induces p27 degradation via direct binding with SKP2 in ovarian and breast cancer.

doi: 10.3892/ijmm.2011.677

Figure Lengend Snippet: Figure 4. Pull-down assay performed for exogenous or endogenous p27 and SKP2. (A) Pull-down reactions were carried out at the indicated tempera tures for 30 min in the presence or absence of DHT. (B) Pull-down reactions were carried out for the indicated times with different DHT concentration at 4˚C. (C) Cell lysates from MCF-7 or OVCAR-3 were immunoprecipitated by exogenous p27 (p27-GST) and immunoblotted by SKP2, with or without DHT (10 nmol).

Article Snippet: For the pull-down assay, the p27 gene (Origene, cA) was constructed into pgEX-6p-1 and p27-gST fusion proteins were purified with GST-sepharose beads (GE Healthcare, NY).

Techniques: Pull Down Assay, Concentration Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Inhibition of mTOR Kinase by AZD8055 Can Antagonize Chemotherapy-induced Cell Death through Autophagy Induction and Down-regulation of p62/Sequestosome 1

doi: 10.1074/jbc.m111.297432

Figure Lengend Snippet: FIGURE 2. AZD8055-mediated cytoprotection is independent of cell cycle arrest. A, DLD1 and HT-29 cells were treated with rapamycin or AZD8055 at the indicated doses for 48 h and p27Kip1 expression levels were analyzed by immunoblotting. B, DLD1 cells were transduced with a lentivirus encoding GFP or p27Kip1. Cells were then treated with 5-FU or oxaliplatin for 5 days, and expression of p-H2Ax and cleaved caspase-9 and -3 proteins were measured by immunoblotting. C, AZD8055 enhances oxaliplatin-induced autophagy, as shown by LC3I-II conversion and reduced p62/SQSTM 1 expression, which is associated with attenuated DNA damage and caspase cleavage. DLD1 cells were treated with increasing doses of oxaliplatin alone or combined with AZD8055 for 5 days and expression of LC3, p62, p-H2Ax, and cleaved caspase-9 and -3 were analyzed by immunoblotting.

Article Snippet: The p27 cDNA (Origene, Rockville, MD) was cloned into a lentiviral cDNA expression vector pCDH1-puro (System Biosciences, Mountain View, CA), as previously described (29).

Techniques: Expressing, Western Blot, Transduction

Journal: Journal of Biological Chemistry

Article Title: Inhibition of mTOR Kinase by AZD8055 Can Antagonize Chemotherapy-induced Cell Death through Autophagy Induction and Down-regulation of p62/Sequestosome 1

doi: 10.1074/jbc.m111.297432

Figure Lengend Snippet: FIGURE 4. Ectopic Beclin 1 expression attenuates 5-FU-induced apoptosis and DNA damage. A, HT-29 cells stably expressing a 3tag-Beclin 1 cDNA or an emptyvectorweretreatedwith5-FUinthepresenceorabsenceofCQandproteinexpressionwasdeterminedinwholecelllysates.B,clonogenicsurvivalassay was performed using Beclin 1 expressing or control cells that were treated with 5-FU or DMSO for 3 days. Cells were then incubated in drug-free media for 7–14 days, and colonies were developed and stained with crystal violet, counted, and then normalized to vehicle (DMSO)-treated cells. Columns represent mean of experiments that were conducted in triplicate; bars, S.D. *, p 0.05; **, p 0.01.

Article Snippet: The p27 cDNA (Origene, Rockville, MD) was cloned into a lentiviral cDNA expression vector pCDH1-puro (System Biosciences, Mountain View, CA), as previously described (29).

Techniques: Expressing, Stable Transfection, Control, Incubation, Staining