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: Nature Communications

Article Title: In vivo rendezvous of small nucleic acid drugs with charge-matched block catiomers to target cancers

doi: 10.1038/s41467-019-09856-w

Figure Lengend Snippet: Gene silencing and therapeutic effects of uPIC. a PLK1 mRNA level in tumour tissues harvested from subcutaneous BxPC3 tumour-bearing mice intravenously injected twice with uPIC at 24-h intervals. Data represent the means ± s.e.m. n = 5 for siLuc/uPIC and 10 for sihPLK1/uPIC. b , c PLK1 protein level in the tumour tissues harvested from subcutaneous BxPC3 tumour-bearing mice intravenously injected with PBS, uPIC, Invivofectamine ® LNP, and naked sihPLK1 thrice at 24-h intervals, as determined by western blotting ( b ), and the quantified results ( c ). Data represent the means ± s.d. n = 3. d – h Apoptosis in subcutaneous BxPC3 tumour tissues from mice treated with sihPLK1/uPIC ( d ), siLuc/uPIC ( e ), naked sihPLK1 ( f ), or PBS ( g ) based on a TUNEL assay (red: apoptosis-positive cells; blue: cell nuclei stained with Hoechst 33342; scale bar: 100 µm), and the quantified results ( h ). Data represent the means ± s.e.m. n = 4. i Antitumour activity of uPIC intravenously administered multiple times (as shown by black arrows) to BxPC3 tumour-bearing mice. Data represent the means ± s.e.m. n = 7. j Body weight change in tumour-bearing mice treated with uPIC as described in ( i ). Data represent the means ± s.e.m. n = 7. k RNAi effect of uPIC in a spontaneous pancreatic tumour model. The luminescence intensity from luciferase-expressing pancreatic tumours was determined using an IVIS instrument 24 h after single systemic injection into oncomice and normalised to that obtained from the initial value before injection. Data represent the means ± s.e.m. n = 9–11. l Kaplan–Meier survival curves of non-treated oncomice and oncomice treated with simPLK1/uPIC or siScr/uPIC. n = 10. m Number of oncomice with liver metastases at the designated days

Article Snippet: The primers comprised the hPLK1 primer (Hs00153444_m1) and an 18 S control primer (Hs99999901_s1) (Life Technologies).

Techniques: Injection, Western Blot, TUNEL Assay, Staining, Activity Assay, Luciferase, Expressing

Journal: iScience

Article Title: Identification of a neural development gene expression signature in colon cancer stem cells reveals a role for EGR2 in tumorigenesis

doi: 10.1016/j.isci.2022.104498

Figure Lengend Snippet: EGR2 is required for CSC survival and tumorigenicity (A) Proliferation of siRNA transfected patient-derived colon cancer cells in nonadherent cell culture compared to untreated control cells (mean ± SD; data from three independent experiments). ∗p value < 0.05; ∗∗p value < 0.01 ( t -test). (B) Fold expression of ALDH1A1 , EGR2 , EGR3 , HDGFRP3 , OLFML2 , OLFML3 , PCP4 , PEG10 , PLK1 , PRKACB , and THBS1 RT-PCR gene expression data (±95% confidence intervals) in siRNA transfected 278-ML-P cells (n = 3 independent cell preparations) compared to untreated control cells. Nontargeting siRNA and siRNA PLK1 were used as negative and positive controls, respectively (see also Figure S4 , Tables S3 and ). (C) Frequency of siRNA EGR2 spheroid formation in nonadherent cell culture compared to control transfected cells (mean ± SD; data from three independent experiments). ns = not significant; ∗p value < 0.05; ∗∗p value < 0.01 ( t -test). (D) Representative images of a 278-ML-P control spheroid (LHS) and a siRNA EGR2 spheroid (RHS) in nonadherent cell culture (scale bars = 100 μm). (E) Immunofluorescence staining of control (top panel) and siRNA EGR2 (lower panel) PDOs for EGR2 (yellow) and F-ACTIN (red) in Matrigel culture. Nuclei are stained blue with DAPI (scale bars, 20 μm). (F) Heatmap showing scRNA-seq generated log(nTPM) z-scores for EGR2 , stem cell genes ( ALDH1A1 , EPHB2 , OLFM2 ), Wnt signaling genes ( AXIN2 , LGR5 ), differentiation ( ATOH1 , KRT20 , MUC1 ), and proliferation genes ( MKI67 , MYC ) in neuropod cells, stem cells, mucus-secreting cells, enterocytes, granulocytes, and Paneth cells from healthy human colon tissue. Results based on Single Cell Type information from the Human Protein Atlas ( Karlsson et al., 2021 ) ( https://www.proteinatlas.org/ENSG00000122877-EGR2/single+cell+type/colon ). (G) Representative images of control virus (top panel) and shRNA EGR2 (lower panel) transduced 195-CB-P cells in non-adherent cell culture (scale bars, 100 μm). (H) Table shows results of limiting dilution transplantation of control virus transduced and shRNA EGR2 transduced 195-CB-P cells. The number of established tumors as a fraction of the number of animals transplanted is given. P values for pairwise tests of differences in CSC frequencies between control virus versus shRNA EGR2 1, shRNA EGR2 two, and shRNA EGR2 3 195-CB-P cells are 6.9 × 10 −9 , 4.9 × 10 −6 , and 6.92 × 10 −8 , respectively. (I) Growth curves for xenografts derived from control virus transduced cells and shRNA EGR2 transduced cells. (See also Table S4 ).

Article Snippet: PLK1 (Hs00983227_m1) , Thermo Fisher , 4331182.

Techniques: Transfection, Derivative Assay, Cell Culture, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Immunofluorescence, Staining, Generated, Virus, shRNA, Transplantation Assay

Journal: iScience

Article Title: Identification of a neural development gene expression signature in colon cancer stem cells reveals a role for EGR2 in tumorigenesis

doi: 10.1016/j.isci.2022.104498

Figure Lengend Snippet:

Article Snippet: PLK1 (Hs00983227_m1) , Thermo Fisher , 4331182.

Techniques: RNA Extraction, RNA Library Preparation, Viability Assay, shRNA, Transduction, Control, Software