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: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Cell surface HER2 expression on quiescent prostate cancer cells. (A) Cell surface HER2 expression on PCa cell lines (PC3, DU145, C4-2B, LNCaP) and benign prostate epithelial cells (PNT2) cultured in 10% FBS measured by flow cytometry and mean fluorescence intensity (MFI). Open histograms represent an isotype control antibody. Shaded histograms are a HER2 antibody. The vertical line and arrow represent positive expression as defined by the isotype control antibody. (B) Flow cytometry for surface HER2 and BrdU incorporated into DNA of PC3 cells (C) Histograms showing HER2 cell surface expression on the same cell lines cultured in the presence or absence of FBS. (D) Quantification of HER2 surface expression from panel (B) as defined by mean fluorescence intensity (MFI). (E) Immunofluorescence imaging of PC3 (left) or C4-2B (right) cells cultured in either 10% FBS or serum starved conditions showing endogenous p27 and HER2 expression. HER2 (red), p27 (green) and nuclei (DAPI, blue). Scale bar, 50 μm. (F) Cell surface HER2 as measured by flow cytometry of PCa cells cultured with either 1 µM abemaciclib or 0.01% ethanol vehicle. (G) Western blot for HER2 from indicated cell lines cultured for 2 days with or without FBS. β-actin was a loading control. Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown.

Article Snippet: For p27 labeling,Cells were fixed with cold 70% ethanol and stained with anti-p27 antibody (R&D systems, Cat #: IC2256V) in PBS containing 1% FBS for 30 min at room temperature.

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence, Control, Immunofluorescence, Imaging, Western Blot

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Cell surface HER2 expression is associated with p27 expression. Left: PC3, Right: C4-2B. Top: Flow cytometry histograms demonstrating cell surface (labeling before fixation) HER2 expression on PC3 and C4-2B cells. Cells were gated by lowest 10% or highest 10% of HER2 levels (HER2 low and HER2 high) populations. Middle: Example flow cytometry histograms of p27 labeling from either the lowest (blue) or highest (red) 10% of HER2 surface expression. Bottom: Quantified p27 expression in the low vs. higher HER2 populationsp27 levels were measured as mean fluorescence intensity (MFI). Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown.

Article Snippet: For p27 labeling,Cells were fixed with cold 70% ethanol and stained with anti-p27 antibody (R&D systems, Cat #: IC2256V) in PBS containing 1% FBS for 30 min at room temperature.

Techniques: Expressing, Flow Cytometry, Labeling, Fluorescence

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: Nature Communications

Article Title: C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer

doi: 10.1038/s41467-024-48742-y

Figure Lengend Snippet: a Venn diagrams show p27-upregulated and repressed genes. b Pathway analysis of p27-up (top) and down-regulated (bottom) genes. c Effects of p27CK-DD and p27CK-AA transduction in 231 cells on embryonic stem cell transcription factors (ES-TFs) expression (top) and mammosphere formation (bottom). d Effect of p27 depletion in 1833 cells on ES-TFs expression (left) and mammosphere formation (right). e Effect of p27CK- and p27CK-DD transduction in MCF12A on mammosphere formation. f Surface expression of CD44 and CD24 in MCF12A-vector, MCF12A-p27CK-, and MCF12A-p27CK-DD assayed by flow cytometry (top). Western shows CD44, p27, and p27pT198 with β-actin controls (bottom). g Effect of p27CK- and p27CK-DD transduction in MCF12A on colony formation. h Effect of p27CK-DD transduction in 231 and p27 knockdown in 1833shp27 vs 1833 on colony formation. i Gene expression heatmaps of NOS (Nanog, Oct4, and Sox2) target gene signatures in indicated lines. j – l Effects of STAT3 depletion on p27-driven increase in ES-TFs expression and sphere formation in indicated cells. All graphs show mean ± SEM from N = 3 biological replicate assays. p -values were represented by paired one-tailed Student’s T Test ( p values are shown in graphs). Source data are provided as a Source data file. For ES-TFs, all of SOX2, NANOG, MYC, KLF4 , and OCT4 were assayed, but only positive data were graphed. ESC embryonic stem cell, C control, NC negative control.

Article Snippet: Primary antibodies for p27 (610241, 1:5000 3686 s, clone D69C12, 1:1000) from Transduction Labs and from Cell Signaling; for p27pT198 (AF3994, 1:2000) from R&D Systems; for STAT3 (9139 s, clone 124H6, 1:1000), STAT3Y705 (9145 s, clone D3Z2G, 1:2000), PYK2 (3480 s, clone 5E2, 1:1000), pPYK2 (3291, 1:1000), CBP (7389, clone D6C5, 1:1000), p130Cas (13846, clone E1L9H, 1:1000), p-p130CAS (4011, 1:1000), Myc (5605 s, clone D84C12, 1:1000), Sox2 (3579 s, clone D6D9, 1:1000), Nanog (8822, clone D2A3, 1:1000), cJun (9165 s, clone 60A8, 1:1000) and cJunpS63 (2361 s, clone 54B3, 1:1000) were obtained from Cell Signaling; for β-actin (A1978, clone AC-15, 1:5000) from Sigma-Aldrich; and for PTPN12 (14735, 1:1000) from Abcam.

Techniques: Transduction, Expressing, Plasmid Preparation, Flow Cytometry, Western Blot, Knockdown, Gene Expression, One-tailed Test, Control, Negative Control

Journal: Nature Communications

Article Title: C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer

doi: 10.1038/s41467-024-48742-y

Figure Lengend Snippet: a Volcano plot of genes differentially expressed in 231DD vs. 231. b PTPN12 mRNA expression by qPCR in the indicated lines. c Western shows effects of p27CK-DD on total and activated STAT3 and Pyk2 and effects of PYK2 loss on STAT3 levels and STAT3pY705. d Western shows effects of p27pT198 (reduced by PI3K inhibitor, PF1502) and Pyk2 loss (si PYK2 ) and Pyk2 inhibition by PF431396 on STAT3 levels and activation (STAT3pY705) e p27, STAT3, and cJun co-occupied peaks at +2 kb of the PTPN12 TSS. f – k ChIP-qPCR shows p27 ( f ); STAT3 ( g ); cJun ( h ); SIN3A ( i ); YY1 ( j ) and HDAC1 ( k ) binding at +2 kb of the PTPN12 TSS where p27 peaks overlap with binding peaks of STAT3 and cJun. l cJun ChIP-Western blot (right) shows interaction on chromatin of the indicated proteins. Input is shown on the left. m Schematic representation for p27/STAT3/cJun in activator (left) and repressor (right) complexes. All graphs show mean ± SEM from N = 3 biological replicate assays. p -values were calculated by paired one-tailed Student’s T Test and are indicated in graphs. Source data are provided as a Source data file.

Article Snippet: Primary antibodies for p27 (610241, 1:5000 3686 s, clone D69C12, 1:1000) from Transduction Labs and from Cell Signaling; for p27pT198 (AF3994, 1:2000) from R&D Systems; for STAT3 (9139 s, clone 124H6, 1:1000), STAT3Y705 (9145 s, clone D3Z2G, 1:2000), PYK2 (3480 s, clone 5E2, 1:1000), pPYK2 (3291, 1:1000), CBP (7389, clone D6C5, 1:1000), p130Cas (13846, clone E1L9H, 1:1000), p-p130CAS (4011, 1:1000), Myc (5605 s, clone D84C12, 1:1000), Sox2 (3579 s, clone D6D9, 1:1000), Nanog (8822, clone D2A3, 1:1000), cJun (9165 s, clone 60A8, 1:1000) and cJunpS63 (2361 s, clone 54B3, 1:1000) were obtained from Cell Signaling; for β-actin (A1978, clone AC-15, 1:5000) from Sigma-Aldrich; and for PTPN12 (14735, 1:1000) from Abcam.

Techniques: Expressing, Western Blot, Inhibition, Activation Assay, ChIP-qPCR, Binding Assay, One-tailed Test

Journal: Nature Communications

Article Title: C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer

doi: 10.1038/s41467-024-48742-y

Figure Lengend Snippet: a Representative mammary gland whole mounts stained with Carmine red from MMTVCre controls and MMTV-Cre X TGp27CK- (p27CK-) and MMTV-Cre X TGp27CK-DD (p27CK-DD) mice show greater duct branching (top 2 panels) and hyperplasia (bottom panels) in the p27CK-DD bigenics. b Duct hyperplasia was quantitated from whole mounts as described and graphed as mean %+/−SEM. p values were calculated by one-way ANOVA with post hoc 2 by 2 comparisons using Tukey correction. N = 8 mice for MMTVCre controls, N = 7 mice for TGp27CK-, N = 6 mice for TGp27CK-DD (See also SFig. for quantitation of duct branching). c The number of mice with mammary cancers over the total number evaluated for each genotype is graphed as %. p values calculated by Fisher’s Exact Test are shown. N = 14 mice for MMTVCre controls, N = 14 mice for TGp27CK-, N = 14 mice for TGp27CK-DD. d FFPE mammary glands from the indicated genotypes were stained with hematoxylin and eosin (H&E) or with antibodies to p27pT198, to cytokeratin or to myoepithelial marker, p63, as indicated and representative photomicrographs shown. e Livers show representative micrometastases from mammary cancer-bearing MMTV Cre X p27CK-DD mouse 1668 (see also Fig. S ). Source data are provided as a Source data file.

Article Snippet: Primary antibodies for p27 (610241, 1:5000 3686 s, clone D69C12, 1:1000) from Transduction Labs and from Cell Signaling; for p27pT198 (AF3994, 1:2000) from R&D Systems; for STAT3 (9139 s, clone 124H6, 1:1000), STAT3Y705 (9145 s, clone D3Z2G, 1:2000), PYK2 (3480 s, clone 5E2, 1:1000), pPYK2 (3291, 1:1000), CBP (7389, clone D6C5, 1:1000), p130Cas (13846, clone E1L9H, 1:1000), p-p130CAS (4011, 1:1000), Myc (5605 s, clone D84C12, 1:1000), Sox2 (3579 s, clone D6D9, 1:1000), Nanog (8822, clone D2A3, 1:1000), cJun (9165 s, clone 60A8, 1:1000) and cJunpS63 (2361 s, clone 54B3, 1:1000) were obtained from Cell Signaling; for β-actin (A1978, clone AC-15, 1:5000) from Sigma-Aldrich; and for PTPN12 (14735, 1:1000) from Abcam.

Techniques: Staining, Quantitation Assay, Marker

Journal: Nature Communications

Article Title: Eukaryote initiation factor 6 modulates small-cell lung carcinoma plasticity via the integrin-FAK signaling axis

doi: 10.1038/s41467-026-69899-8

Figure Lengend Snippet: a Ribosome binding assay assessing eIF6 association with ribosome pools. Cytoplasmic lysates were fractionated into free (F1) and ribosome-associated (F2) portions via 30% sucrose cushion ultracentrifugation. Immunoblots of eIF6, RPL7, RPS15 and β-tubulin are shown. Representative of two biologically independent experiments were performed with similar results. Created in BioRender. Shen, S. (2026) https://BioRender.com/5p1ea8h . b Volcano plot of Pearson correlations between eIF6 expression and gene essentiality across ~1100 solid cancer lines from DepMap. Representative correlations for CD104 are shown for all solid tumours and SCLC lines. Two-sided p -values were computed. c Direct interaction network of eIF6 from BioGRID. d RT-qPCR of CD104 in indicated lines ( n = 3 biological replicates). Statistical significance: two-sided unpaired Wilcoxon rank-sum test. e Co-IP with anti-FAK showing interactions between CD104, eIF6, ERK1/2, and FAK in non-NE states. IgG2a as Co-IP control; β-tubulin and IgG heavy chain for input and IP loading controls. The samples derive from the same experiment, but different gels for FAK, CD104, IgG and β-tubulin, another for p-ERK, ERK and eIF6 were processed in parallel. Similar results were observed in n = 3 biologically independent experiments. PLA using anti-FAK and anti-eIF6 ( f ) or anti-CD104 ( g ) antibodies. Red fluorescent signals indicate complexes; scale bars, 10 μm. Protocol validated in three biologically independent experiments. Cell segmentation and spot quantification by CellProfiler. Created in BioRender. Shen, S. (2026) https://BioRender.com/5p1ea8h . Single-cell quantification of eIF6-FAK ( h ) and eIF6-CD104 ( i ) PLA after eIF6 knockdown. Each dot represents a single cell from one representative experiment; two additional biologically independent experiments showed similar trends. h : 490 (H69), 197 (H69M sh_NTC), 185 (H69M sh_eIF6); i : 472 (H69), 291 (H69M sh_NTC), 210 (H69M sh_eIF6). Two-sided unpaired Wilcoxon rank-sum test. j Co-IP of CD104-FAK in H69M cells with eIF6 knockdown. β-tubulin and IgG heavy chain as input/IP loading controls. The samples derive from the same experiment, but different gels for FAK, CD104, IgG, another for p-ERK, ERK, eIF6 and actin were processed in parallel. Similar results were observed in n = 3 biologically independent experiments.

Article Snippet: Briefly, slides were blocked with blocking solution for 30 minutes at RT and incubated with rabbit anti-eIF6 (Proteintech #10291-1-AP, 1:100) and mouse anti-FAK (Santa Cruz #sc1688 1:100) or rabbit anti-eIF6 and mouse anti-CD104 (R&D systems, #MAB4060, 1:100 from 500 μg ml -1 stock) at 4 °C overnight.

Techniques: Binding Assay, Western Blot, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Control, Single Cell, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Cell-permeable Carboxyl-terminal p27Kip1 Peptide Exhibits Anti-tumor Activity by Inhibiting Pim-1 Kinase

doi: 10.1074/jbc.m109.092452

Figure Lengend Snippet: FIGURE 1. Cell-permeable p27Kip1 peptide binds to and inhibits Pim-1. A, schematic representation of human p27Kip1 and its carboxyl-terminal T198wt peptide (amino acids 189–198). The amino acid sequences of cell- permeable Arg8, R8-T198wt, and R8-T198mu peptides used in the study are also shown. Amino acids are indicated by their single letter code. The mu- tated Asp194 and Asp196 residues in R8-T198mu are underlined. B, HEK293T cells were transfected with pHM6 encoding human p27Kip1 together with empty pc5FLAG (, lanes 1–5) or pc5FLAG encoding Pim-1 (, lanes 6–10). After transfection for 4 h, cells were incubated with 10 or 40 M FITC-la- beled R8-T198wt (wt), 10 or 40 M FITC-labeled R8-T198mu (mu) peptide, or without any peptide (None) for 24 h. To make HA-p27Kip1 expression level equal, the amount of transfected pHM6-p27Kip1 has decreased gradually contrary to the amount of R8-T198wt peptide (lanes 2, 3, 7, and 8). Then, cell lysates were electrophoresed and immunoblotted with the indicated anti- bodies. C, HEK293T cells were not transfected (None) or were transfected with empty pEGFP-C2 vector (GFP), pEGFP-C2-T198wt (GFP-T198wt), or pEGFP-C2-T198mu (GFP-T198mu) together with the pHM6-Pim-1 plasmid (). After transfection for 24 h, cells were lysed, and the GFP-tagged pep- tides were immunoprecipitated from the cell lysates with protein A-agar- ose-conjugated anti-GFP antibody. The immunoprecipitated proteins (IP) and cell lysates (input) were subjected to immunoblot analysis with the indi- cated antibodies.

Article Snippet: The membranes were incubated with antibodies to Pim-1 (12H8; antibody sc-13513), GFP (B-2; antibody sc-9996), and -actin (C-2-HRP; antibody sc-8432 HRP) (Santa Cruz Biotechnology); p27Kip1 (610242), human Topoisomerase II (611492), and Hsp90 (610418) (BD Biosciences); Bad (9292), phospho-Bad Ser112 (9290), caspase-3 (9662), and poly(ADPribose) polymerase (9542) (Cell Signaling); -tubulin (YL1/2) (Serotec); phospho-Thr198 p27Kip1 (AF3994) (R&D Systems); FLAG tag M2 (A8592) (Sigma); and HA tag (3F10) (Roche Applied Science).

Techniques: Transfection, Incubation, Labeling, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cell-permeable Carboxyl-terminal p27Kip1 Peptide Exhibits Anti-tumor Activity by Inhibiting Pim-1 Kinase

doi: 10.1074/jbc.m109.092452

Figure Lengend Snippet: FIGURE 2. Short term treatment of DU145 cells with cell-permeable p27Kip1 peptide up-regulates p27Kip1 protein and induces G1 arrest. A, DU145 cells were treated with 20 M FITC-labeled cell-permeable Arg8, R8-T198wt, or R8-T198mu peptide, or not treated with any peptide (None). After incubation for 24 h, cells were stained with propidium iodide. Analy- ses were performed using a flow cytometer. B, expression levels of endoge- nous p27Kip1 and -actin proteins in cells treated with 20 M FITC-labeled cell-permeable Arg8, R8-T198wt, R8-T198mu, or R8-T198-phosphopeptide, or not treated with any peptide (None). Expression of p27Kip1 and -actin were confirmed by immunoblotting. C, DU145 cells were treated with 20 M FITC-labeled Arg8 or R8-T198wt peptide. After incubation for 24 h, cells were harvested and lysed. The cytoplasmic (C) and nuclear (N) fractions were separated, electrophoresed, and immunoblotted with the indicated antibodies. Topo II, Topoisomerase II. D, localization of endogenous p27Kip1 protein in cells treated as in C was confirmed by immunostaining with anti- p27Kip1 antibody. Nuclei were detected by staining with Hoechst 33342.

Article Snippet: The membranes were incubated with antibodies to Pim-1 (12H8; antibody sc-13513), GFP (B-2; antibody sc-9996), and -actin (C-2-HRP; antibody sc-8432 HRP) (Santa Cruz Biotechnology); p27Kip1 (610242), human Topoisomerase II (611492), and Hsp90 (610418) (BD Biosciences); Bad (9292), phospho-Bad Ser112 (9290), caspase-3 (9662), and poly(ADPribose) polymerase (9542) (Cell Signaling); -tubulin (YL1/2) (Serotec); phospho-Thr198 p27Kip1 (AF3994) (R&D Systems); FLAG tag M2 (A8592) (Sigma); and HA tag (3F10) (Roche Applied Science).

Techniques: Labeling, Incubation, Staining, Flow Cytometry, Expressing, Phospho-proteomics, Western Blot, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Cell-permeable Carboxyl-terminal p27Kip1 Peptide Exhibits Anti-tumor Activity by Inhibiting Pim-1 Kinase

doi: 10.1074/jbc.m109.092452

Figure Lengend Snippet: FIGURE 4. Growth inhibitory effect of cell-permeable p27Kip1 peptide. A, DU145 cells were treated with the indicated concentrations of FITC-la- beled cell-permeable Arg8, R8-T198wt, or R8-T198mu peptide. Viable cells were counted after incubation for 24, 48, and 72 h. Each vertical bar repre- sents the mean S.D. of three independent experiments. B, RWPE-1 cells were treated as in A. Viable cells were counted after incubation for 24, 48, and 72 h. Each bar represents the mean S.D. of three independent experi- ments. C, total cell lysates of RWPE-1 and DU145 cells were electrophoresed and immunoblotted with the indicated antibodies. D, DU145 cells were in- cubated in a medium containing the indicated concentrations of the FITC- labeled R8-T198wt peptide (0–10 M) together with the indicated concen- trations of taxol (0–3 g/ml). Viable DU145 cells were counted by the trypan blue dye exclusion method. The number of viable untreated control cells was normalized to 100%. Each vertical bar represents the mean S.D. of three independent experiments. The interaction of various concentra- tions of the R8-T198wt peptide and taxol was subjected to two-way analysis of variance. *, p 0.05; **, p 0.01.

Article Snippet: The membranes were incubated with antibodies to Pim-1 (12H8; antibody sc-13513), GFP (B-2; antibody sc-9996), and -actin (C-2-HRP; antibody sc-8432 HRP) (Santa Cruz Biotechnology); p27Kip1 (610242), human Topoisomerase II (611492), and Hsp90 (610418) (BD Biosciences); Bad (9292), phospho-Bad Ser112 (9290), caspase-3 (9662), and poly(ADPribose) polymerase (9542) (Cell Signaling); -tubulin (YL1/2) (Serotec); phospho-Thr198 p27Kip1 (AF3994) (R&D Systems); FLAG tag M2 (A8592) (Sigma); and HA tag (3F10) (Roche Applied Science).

Techniques: Incubation, Labeling, Control

Journal: Journal of Biological Chemistry

Article Title: Cell-permeable Carboxyl-terminal p27Kip1 Peptide Exhibits Anti-tumor Activity by Inhibiting Pim-1 Kinase

doi: 10.1074/jbc.m109.092452

Figure Lengend Snippet: FIGURE 3. Long term treatment of DU145 cells with cell-permeable p27Kip1 peptide induces apoptosis. A, DU145 cells were treated with 20 M FITC-labeled cell-permeable Arg8, R8-T198wt, or R8-T198mu peptide or not treated with any peptide (None). After incubation for 72 h, cells were stained with propidium iodide. Analyses were performed using a flow cy- tometer. B, expression levels of endogenous caspase-3, poly(ADP-ribose) polymerase, and -actin proteins in cells treated as in A were confirmed by immunoblotting. C, HEK293T cells were cultured in serum-free medium for 24 h. Cells were then transfected with empty pc5FLAG alone () or pc5FLAG encoding Pim-1 (). After transfection for 4 h, medium was re- placed with serum-free medium containing none (None) or 40 M FITC-la- beled Arg8, R8-T198wt, or R8-T198mu peptide and incubated for an addi- tional 24 h. Then, cell lysates were electrophoresed and immunoblotted with the indicated antibodies.

Article Snippet: The membranes were incubated with antibodies to Pim-1 (12H8; antibody sc-13513), GFP (B-2; antibody sc-9996), and -actin (C-2-HRP; antibody sc-8432 HRP) (Santa Cruz Biotechnology); p27Kip1 (610242), human Topoisomerase II (611492), and Hsp90 (610418) (BD Biosciences); Bad (9292), phospho-Bad Ser112 (9290), caspase-3 (9662), and poly(ADPribose) polymerase (9542) (Cell Signaling); -tubulin (YL1/2) (Serotec); phospho-Thr198 p27Kip1 (AF3994) (R&D Systems); FLAG tag M2 (A8592) (Sigma); and HA tag (3F10) (Roche Applied Science).

Techniques: Labeling, Incubation, Staining, Expressing, Western Blot, Cell Culture, Transfection

Journal: Journal of Biological Chemistry

Article Title: Cell-permeable Carboxyl-terminal p27Kip1 Peptide Exhibits Anti-tumor Activity by Inhibiting Pim-1 Kinase

doi: 10.1074/jbc.m109.092452

Figure Lengend Snippet: FIGURE 5. In vivo anti-tumor activity of p27Kip1 peptide. A and B, DU145 cells were subcutaneously implanted into the right flank of BALB/c nude mice. Therapeutic experiments (six mice/group) were started (day 0) when the tumor reached a volume of 100 mm3. The R8-T198wt peptide was administered by intratumoral injection in 50 l of 10 mM solution on days 0 and 3. The control group received the same volume of vehicle on days 0 and 3. A, representative surface tumor morphology of the mice on day 8 after initiation of treatment (left, vehicle; right, R8-T198wt peptide). B, rela- tive tumor volume and body weight. C and D, R8-T198wt peptide (50 l of 10 mM) or the same volume of vehicle was administered intratumorally on days 0 and 5. All DU145-bearing mice were intravenously administered 60 mg/kg taxol on day 0. C, representative surface tumor morphology on day 14 after initiation of treatment (left, vehicle and taxol; right, R8-T198wt pep- tide and taxol). D, relative tumor volume and body weight. *, p 0.05; **, p 0.01.

Article Snippet: The membranes were incubated with antibodies to Pim-1 (12H8; antibody sc-13513), GFP (B-2; antibody sc-9996), and -actin (C-2-HRP; antibody sc-8432 HRP) (Santa Cruz Biotechnology); p27Kip1 (610242), human Topoisomerase II (611492), and Hsp90 (610418) (BD Biosciences); Bad (9292), phospho-Bad Ser112 (9290), caspase-3 (9662), and poly(ADPribose) polymerase (9542) (Cell Signaling); -tubulin (YL1/2) (Serotec); phospho-Thr198 p27Kip1 (AF3994) (R&D Systems); FLAG tag M2 (A8592) (Sigma); and HA tag (3F10) (Roche Applied Science).

Techniques: In Vivo, Activity Assay, Injection, Control