Journal: Oncogene

Article Title: The CHFR mitotic checkpoint protein delays cell cycle progression by excluding Cyclin B1 from the nucleus.

doi: 10.1038/sj.onc.1208428

Figure Lengend Snippet: Figure 5 Analysis of Plk1 and Aurora-A in HCT116 neo- and CHFR-expressing cells exposed to nocodazole. The cells were synchronized in G1/S, released into the cell cycle (0 h), exposed to nocodazole (4 h) and examined 8–13 h after release from the cell cycle block, as indicated. (a,c) Plk1 and Aurora-A (Aur A) protein levels. G2/early prophase (adherent, Ad) cells were separated from prometaphase (nonadherent, NA) cells by mitotic shake-off before the extracts were prepared. (b,d,e) Immunofluorescence analysis for Plk1, Aurora-A (AurA) and Aurora-A phosphorylated on Thr288 12 h after release from the G1/S block. The centrosomes (arrow- heads) were marked by staining for g-tubulin. In (e),the cell marked with an asterisk is in prometaphase

Article Snippet: Antibodies to cell cycle markers were obtained from commercial vendors, as follows: Plk1 and H3 phosphorylated on Ser 10 from Upstate Biothechnology; Cdc2, Aurora-A, Aurora-B, Cyclin A and Cyclin B1 from Transduction Labs; Lamins A/C and B from Santa Cruz; Aurora-A phosphorylated on Thr288 and phospho-Thr-Pro from Cell Signaling Technologies.

Techniques: Expressing, Blocking Assay, Staining

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: The mitotic kinase Plk1 is overexpressed in LNCaP-AI cells. ( a ) LNCaP and LNCaP-AI PCa cells were cultured in full media (FBS) or androgen-depleted media (csFBS). Cell growth over 7 days was determined by an MTS (OD 490 nm) growth assay. Results from triplicate samples (mean ± SD) in a representative experiment from n = 3 independent experiments are shown. ( b ) Lysates (20 μg) from LNCaP and LNCaP-AI cells grown in full media were immunoblotted for the androgen receptor (AR) and mitotic proteins as shown. α-tubulin was used as a loading control. ( c ) Relative Plk1 protein levels were determined (Plk1/tubulin and normalized against the value in LNCaP cells) (mean ± SD, n = 3 experiments).

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Cell Culture, Growth Assay, Control

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Plk1 is elevated and active in LNCaP-AI cells. Cells were cultured in either full media (FBS) or androgen-depleted media (csFBS). ( a ) FACS analysis of randomly growing cells and cells synchronized by nocodazole treatment to enrich for G2/M phase population. ( b ) FACS analysis of cells costained with Plk1 and a mitotic marker, phospho-histone H3 (pH3), in randomly cycling cells and in cells synchronized by a nocodazole treatment. pH3(-) represent non-mitotic cells; pH3(+) represent mitotic cells. ( c ) Lysates (20 μg) from cells enriched in G2/M by nocodazole treatment were analyzed by immunoblotting with antibodies shown. After scanning the blots (NIH ImageJ), protein levels were measured (protein/tubulin and normalized against the value in LNCaP cells). Similar data were obtained in n = 3 experiments.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Cell Culture, Marker, Western Blot

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Differential response of LNCaP-AI cells to the Plk1 inhibitor BI2536. ( a ) Cells were cultured in androgen-depleted media and treated with increasing concentrations of BI2536 for 5 days. % Maximal growth relative to non-treated control cells (set at 100%) were analyzed in triplicates (mean ± SD) by MTS assays. Dotted line, IC50 of 0.2 nM BI2536 for LNCaP-AI cells. ( b ) FACS analysis of LNCaP-AI cells treated with or without 0.8 nM BI2536 for 5 days. ( c ) % Trypan-blue positive dead LNCaP-AI cells in (a) were counted in triplicates (mean ± SD). Similar data were obtained in n = 3 experiments.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Cell Culture, Control

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Growth inhibition of additional androgen-insensitive prostate cancer cells to the Plk1 inhibitor BI2536. ( a ) Lysates (20 μg) were analyzed for Plk1 levels by western blot analysis. α-tubulin was used as a loading control. *, nonspecific band. ( b - d ) abl, C4-2B and CWR22Rv1 cells were culture in full (FBS) or androgen-depleted (csFBS) media. Cell growth over 5 days was determined by an MTS assay. ( e - g ) Cells were cultured in androgen-depleted media and treated with increasing concentrations of BI2536 for 5 days. % Maximal growth relative to non-treated control cells (set at 100%) were analyzed in triplicates (mean ± SD) by MTS assays. Data in (b – g) are representative of n = 3 experiments.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Inhibition, Western Blot, Control, MTS Assay, Cell Culture

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Plk1 inhibition by BI2536 resulted in distinct changes in nuclear morphology and an increase in aneuploidy in LNCaP-AI cells. ( a ) Cells were treated with 0.8 nM BI2536 for 5 days in androgen-depleted media, immunostained for α-tubulin (green) and counterstained with DAPI (blue) for DNA. Arrow, cells in telophase. Bars, 10 μm. ( b ) Nuclear morphologies for cells in A were quantified as mononuclear (normal) or multinuclear (with nuclear vesicles) (mean ± SD, n = 3 experiments). N, cell number. None, no mononuclear cells were detected.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Inhibition

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Necrostatin-1 attenuates cell death by necroptosis resulting from either Plk1 depletion or Plk1 inhibition in LNCaP-AI cells. ( a ) LNCaP-AI cells were transfected with either siLuciferase or Plk1 RNAi oligos for 5 days. Cell lysates (20 μg) were analyzed for Plk1 levels. α-tubulin was used as a loading control. ( b ) Cells prepared as in (a) or treated with 0.8 nM BI2536 were analyzed after 5 days by an MTS assay. Triplicate samples (mean ± SD) from one of n = 2 experiments are shown. ( c ) Cells were treated as in (b) and counterstained with DAPI to visualize DNA, shown here in black and white for contrast. Arrows, cells in mitosis in control cultures. Single giant cells containing clusters of nuclear vesicles are shown for siPlk1 and BI2536-treated LNCaP-AI cells. Bars, 10 μm. Note differences in scale. ( d ) Cells were transfected with siPlk1 for 5 days. 7.7 μM Necrostatin-1 (Nec-1), the necroptosis inhibitor, was added 3 h prior to siPlk1 transfection and remained throughout the experiment. Triplicate samples (mean ± SD) from MTS assays from one of n = 2 experiments are shown. ( e ) Cells were treated with 0.4 or 0.8 nM BI2536 for 5 days. 5 μM Nec-1 was added 3 h prior to BI2536 addition and remained throughout the experiment. Triplicate samples (mean ± SD) from MTS assays from one of n = 3 experiments are shown. ( f ) Working model of necroptosis induction by Plk1 inhibition in androgen-insensitive PCa cells. See text.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Inhibition, Transfection, Control, MTS Assay

Journal: International journal of molecular sciences

Article Title: Proteomic Changes Induced by the Immunosuppressant Everolimus in Human Podocytes.

doi: 10.3390/ijms25137336

Figure Lengend Snippet: Figure 7. PLK1 Phosphorylation activity. (A) Dot-blot assay of PLK1 phosphorylation activity. In the dot-blot, the spots A1, B1, C1, A2, B2, and C2 correspond to positive controls; A3, B3, C3, A4, B4, and C4 to negative controls; D1, E1, F1, D2, E2, and F2 to TPT1 S46 of everolimus-treated cells (500 nM EVE); G1, H1, I1, G2, H2, and I2 to TPT1 S46 of untreated cells (CTR); D3, E3, F3, D4, E4, and F4 to CDC25C S198 of 500 nM EVE; G3, H3, I3, G4, H4, and I4 to CDC25C S198 of CTR. (B) Graphical representation of Mann–Whitney analysis applied to the phosphorylation of PLK1 substate (TPT1 and CDC25C). PLK1 activity on TPT1 and CDC25C proteins is significantly upregulated by EVE treatment.

Article Snippet: The antibodies used in this study were PLK1 purchased from Biorbyt (Cambridge, UK) and secondary goat anti-mouse IgG HRP-conjugated antibody from Novus Biologicals (Centennial, CO, USA).

Techniques: Phospho-proteomics, Activity Assay, Dot Blot, MANN-WHITNEY