Formalin Fixed Paraffin Embedded (FFPE) post-surgical human prostate tissue samples were collected from patients diagnosed with either prostatic carcinoma (PCa), N=13) or benign prostatic hyperplasia (PHp, N=18) confirmed by prostatic biopsy and other clinico-pathological tests, following all medical and ethical regulations, and with formal approval from the ethical committee of both CSIR-IICB and Park Clinic (source). The modal age is 66 years, the range being 61 - 69.
Immunohistochemistry and Image Scoring
For the purpose of Immunohistochemical staining, 5 micron thick sections were obtained from FFPE blocks using a microtome (Leica; RM2235) on clean Poly-L-lysine (P8920, Sigma, St. Louis, MO, USA) coated glass slides (Riviera; #72910-135). Sections were then deparaffinised and rehydrated using Xylene and Alcohol grades respectively. After heat induced antigen retrieval in Citrate Buffer (pH 6.0), sections were blocked using Power Block (BS-1310-25, Biogenex, Fremont CA, U.S.A.). Primary antibodies were incubated overnight at 4°C at a dilution of 1:200 in all the cases. HRP-linked secondary antibodies were incubated for 2 hours at a dilution of 1:500. 3, 3-Diaminobenzidine (DAB) reagent (#550880, BD Pharmingen, San Jose, CA, USA) was used to detect the positivity of the staining. Mayer's Hematoxylin (MHS16, P8920, Sigma, St. Louis, MO, USA) was used as a counter stain for nucleus. Slides were finally mounted by cover slips (Riviera; #72920-2222) with DPX (#06522, Sigma, St. Louis, MO, USA) and viewed under microscope (BX61, Olympus, Japan) using X20 and X60 OI magnification. Images were captured using DP71 (Olympus, Japan) camera and Image Pro Plus imaging software (Media Cybernetics, Bethesda, USA).
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A semi quantitative scoring method57 was employed for assessing the intensity of staining described as follows. Multiple randomly chosen fields were captured from each section of both PCa and PHp to assess equal number of glandular cells (1000 in our case), degree of staining ranging from 0% to 100% , which were multiplied by the dominant intensity pattern of staining (1, negative or trace; 2, weak; 3, moderate; 4, intense). Therefore an overall H-score ranging from 0 to 400 was achieved. Cell nuclei with scores 0 to 200, 201 to 300, and 301 to 400 were categorized as having negative or low, intermediate, and high levels of expression respectively. The mean H-score was calculated for each slide and incorporated for further statistical analysis. Two observers (A.C and U.C) independently evaluated the immunostaining results. The concordance ratio was more >95%. A consensus assessment was obtained by endorsing the opinion of a third evaluator (M.G). Since the phenomenon mentioned in our study chiefly concentrates on the nuclear action of CK2, and rest of the proteins (viz. PML, pAKT and pFOXO3a) are predominantly nuclear, so only positive staining for nucleus have been taken into consideration for all sorts of quantitation and Image scoring.
For the purpose of Fluorescence-Immunohistochemical staining, 5 micron thick sections were obtained from FFPE blocks using a microtome on clean Poly-L-lysine coated glass slides. Sections were then deparaffinised and rehydrated using Xylene and Alcohol grades respectively. After heat induced antigen retrieval in Citrate Buffer (pH 6.0), sections were blocked using Power Block. An intermediate step of permeabilization was engaged with 1.25% Triton-X-100 in 1X PBS for 15 mins to facilitate proper and uniform antibody entry. Primary antibodies were incubated overnight at 4°C at a dilution of 1:200 in all the cases. Fluorochrome conjugated secondary antibodies were incubated for 2 hours at a dilution of 1:500 in a humidified chamber under darkness. Slides were finally mounted by cover slips with Fluoroshield® DAPI and viewed under microscope (BX61, Olympus, Japan) using X60 OI magnification. Images were captured using DP71 (Olympus, Japan) camera with Image Pro Plus imaging software.
Cell Culture, Transfections and Treatments
HEK293 and PC3 cells were procured from ATCC and cultured in Dulbecco's Modified Eagle's Medium (DMEM), (Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 10% Fetal Bovine Serum (FBS) (Invitrogen, Life Technologies, Grand Island, NY, USA) maintained at 5% CO2 and 37° C.
Penicillin/ Streptomycin (Invitrogen, Life Technologies, Grand Island, NY, USA) cocktail and Gentamycin (Invitrogen, Life Technologies, Grand Island, NY, USA) were added as prescribed antibiotic at the recommended dose.
For HEK293 cells, Calcium Phosphate method as described previously58 while for PC3 cells Lipofectamine® 2000 reagent (Invitrogen, USA) was used for transfection of DNA plasmids.
For siRNA transfection (100pmol/well), HiPerFect™ reagent (#301705; Qiagen, Valencia, CA, USA) was used in all cases. Transfection of DNA plasmids were followed up to 36 hours, while in the case of siRNA, 72 hours was determined as the optimum time limit. TBCA and LY294002 treatment was done for 2 hours at the mentioned doses in all experiments mentioned separately otherwise. 0.5M d -Sorbitol (in water) for 1 hour was used to stimulate CK2 activity as it has been described previously23, 59.
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pGZdx21-GFP-CK2α plasmid construct was generated through sub-cloning from pcDNA6.1-WT-CK2α-V5-His plasmid construct. (In every experiment, both pGZdx21-GFP-CK2α construct and pcDNA6.1-WT-CK2α-V5-His construct has been abbreviated as WT-CK2 only, otherwise separately mentioned). pGZdx21-AKT1 (S129D)-GFP plasmid construct was generated from pGZ-AKT1-GFP (cloned from human cDNA) by PCR Stitching method of Site Directed Mutagenesis (described separately below). pECE-HA-FOXO3a-TM (T32A, S253A, and S315A) plasmid was procured from ADDGENE. All constructs were verified by sequencing.
Site-Directed Mutagenesis by PCR method (Stitching Method):
The protocol is adapted from60.In brief, at first two different PCR reactions were ran, one containing the sub-cloning forward primer (with restriction enzyme site) and mutagenic reverse primer, the other containing the sub-cloning reverse primer and mutagenic forward primer. The PCR products were subsequently ran and isolated from soft agarose gel. The concentration of the isolated products were spectrophotometrically determined and mixed into 1:1 ratio, 100 ng each. Another PCR reaction was ran with this mixture and this time sub-cloning forward and reverse primers were used. The product from this reaction was similarly gel isolated and used for ligation and transformation subsequently.
Confirmation of the SDM:
The mutation was also confirmed by PCR amplification method, where two primers (one serving as the reverse primer for the mutated gene [C] and the other serving as the forward primer [Cf] of the mutated gene) were designed with the mutated nucleotide(s) at their 3' ends. Two separate PCR reactions were ran with either sub-cloning forward primer (AF) and sub-cloning reverse primer (AF) in the required combinations with the mutated 3' end primers (Cf and Cr).
Negative control PCR reactions were ran with primers having original/unmutated nucleotide(s) at their 3' ends (Wf and Wr). Confirmatory result yielded two products of different sizes (417 bps and 1089 bps) while there were absolutely no amplifications in the negative control reactions. The representative picture of the agarose gel with the PCR products is provided as supplementary information (S-2b). The primer sequences used for both SDM and confirmation has been provided in a separate table given in the same supplementary (S2-a). Apart from this, the mutation was also confirmed by sequencing methods, which not only confirmed the mutation, but also supported the PCR method of SDM confirmation to be valid and applicable.
HEK293 and PC3 cells were plated over sterile cover slips placed inside 35mm tissue culture dishes and cultured in prescribed condition. After 24 hours of initial seeding, required treatments (as described in the result section below) were carried out for predetermined time points. Cells were then harvested, washed with 1X ice cold phosphate buffered saline (PBS); fixed with 3.4% Paraformaldehyde (PFA) followed by permeabilization with 0.25% Trition X-100 solution in PBS. After blocking with 1% BSA solution containing 3M Glycine in PBS-T (used to quench excess aldehyde), cells were incubated with respective primary antibodies at required dilution at 4°C for overnight. On the next day, following secondary antibody (fluorochrome tagged) incubation, coverslips were mounted on glass slides with Fluoroshield® DAPI, and viewed under microscope (BX61, Olympus, Japan) in X60 OI magnification. Images were captured using DP71 camera, (Olympus, Japan) and Image Pro Plus imaging software (Media Cybernetics, Bethesda, USA).
For the time lapse co-localization experiment, cells were treated with appropriate drugs for defined time points. Cells were then fixed, processed and viewed for fluorescence as stated above. Co-localization and Coefficient of co-localization at various time points were determined using JACoP v2.0 in Image J software (http://rsbweb.nih.gov).
Immuno Blot analyses and Co- Immunoprecipitation
Prior to harvesting cells were washed twice with ice cold 1X PBS, spun down and re-suspended in Tris lysis buffer ((50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, 1 mM Na3VO4, 30 mM ß-glycerophosphate, 10 mM NaF and 0.1M Phenylmethanesulfonylfluoride (PMSF)) containing a protease inhibitor cocktail (#539136; Calbiochem, MA, USA). The whole cell lysates thus obtained were photometrically quantified and subjected to SDS-PAGE, followed by Immunoblotting. For preparation of Cytoplasmic-Nuclear extract cells were harvested as described above and then first re-suspended in harvest buffer containing 10 mM HEPES (pH 7.9), 50 mM NaCl, 0.5 M Sucrose, 0.1 mM EDTA, 0.5% Triton-X 100 and protease inhibitor cocktail (539136; Calbiochem, MA, USA). After obtaining the cytoplasmic extract, the pellet was washed thrice with wash buffer/ Buffer A (10 mM HEPES (pH 7.9), 10 mM KCL, 0.1 mM EDTA and 0.1 mM EGTA), then re-suspended in Buffer C (10 mM HEPES (pH 7.9), 500 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% NP40 and protease inhibitor cocktail (#539136; Calbiochem, MA, USA) to extract the nucleus.
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For immunoblotting, respective primary antibodies were used in 1:1000 dilutions in 1% BSA, and HRP tagged secondary antibodies were incubated at a dilution of 1:2000 also in 1% BSA. For primary antibody, overnight incubation at 4°C was used, and for secondary antibody, 2 hour incubation at RT was followed.
For Immunoprecipitation experiments, cells were harvested in Immunoprecipitation Lysis Buffer (50 mM HEPES, pH 7.2, 250 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1.0 mM EDTA, 0.5 mM DTT ,0.1M PMSF and protease inhibitor cocktail (#539136; Calbiochem, MA, USA). 1 mg of total protein was used for actual immunoprecipitation in all cases. The rest of the Immunoprecipitation procedure was performed as per standard protocol58 using Protein A Sepharose CL-4B beads (GE Healthcare, Piscataway, NJ, USA).
RNA preparation and Quantitative Real-Time PCR
Total RNA was extracted from HEK293 and PC3 cells by using Trizol reagent (#15596-026; Invitrogen, NY, USA) as per manufacturer's protocol. cDNA was prepared by using RevertAid™ First Strand cDNA Synthesis Kit (#K1622; Fermentas, Glen Burnie, Maryland, USA) as per manufacturer's protocol. For qRT-PCR analysis, 1.0 uL from each cDNA sample was used as template. Real-time PCR was performed using Fast SYBR® Green Master Mix (#4385617; Applied Biosystems, Foster City, CA, USA) in 7500-Fast Real Time PCR Instrument (Applied Biosystems, Foster City, CA, USA) according to manufacturer's instructions. Each sample was analyzed in at least three independent assays. Mean values of the gene of interest were normalized against 18s rRNA levels. The primer sequences have been provided separately as supplementary Information (S2-a).
HEK293 cells were transiently transfected with 2.0 µg of WWP-p21/Waf1-Luc (procured from ADDGENE) and pGVB2-p27/Kip1-Luc reporter (a kind gift from Dr. Toshiyuki Sakai, Kyoto Prefectural University, Japan) constructs along with respective plasmid constructs of genes as per experimental interest (detail description provided in result section below) and 100 ng of Renilla luciferase vector (pRL-TK) in all cases and cultured for 24 + 36 hours. After that cells were treated with respective inhibitors and stimulators for the requisite durations (mentioned above, described below). Luciferase activity was assessed using Dual Luciferase® Reporter Assay System (#E1910, Promega, Madison, USA) and measured VICTOR X Multilabel Plate Readers (PerkinElmer, Waltham, MA, USA). Quantification was based on three independent experiments.
Cell Cycle Analysis and Annexin V Labeling Assay
Human prostate carcinoma cells, PC3 were either treated with respective inhibitors and stimulators (see figure legend) or transfected with WT-CK2alpha DNA for the requisite durations and were harvested using 0.25% Trypsin. For cell cycle analysis, cells were then processed as per standard protocol, incubated with PI-RNase solution (#550825, BD Pharmingen, San Jose, CA, USA) and analyzed in FACS Verse Instrument using FACS Suite Software (BD Bioscience, San Jose, CA, USA). For transfection, 4.0 ug of pGZdx21-GFP-CK2α plasmid DNA construct was used. For Annexin V Labeling Assay, cells were harvested after respective treatments (described below) and processed as per manufacturer's protocol. Annexin V-FITC antibody was procured from BD Pharmingen (#560931, San Jose, CA). Analysis was performed in FACS Verse Instrument using FACS Suite Software (BD Bioscience, San Jose, CA, USA). Figure supplied, is the best of three individual experiments, while quantification was based on all of them.
Paired Student's t - test was employed to determine the significance value in all other experiments (even if not mentioned separately). The evaluation of the differences in H - scores values of all the concerned proteins between PCa and PHp tissues were carried out by Mann-Whitney -U test. The Correlation test was employed to evaluate correlation between CK2 - PML protein expression levels (individual H scores) in both PCa and PHp tissues and Coefficient of Correlation (r) was estimated. All statistical analyses were performed using either PSPP (GNU-GSL, MA; USA) or Graph Pad Prism (Graph Pad Prism, San Diego, CA, USA) statistical software packages. In every case the value of p < 0.05 was considered to be statistically significant, otherwise mentioned.
The above study was conducted from the support provided by Council of Scientific and Industrial research (CSIR), Government of India, and Department of Science and Technology DST), Government of India.
We are sincerely thankful and obliged to the following scientists, for their valuable support:
Dr. Khalil Ahmed; Masonic Cancer Center, University of Minnesota, Minneapolis, USA, for kindly gifting us with the pcDNA6-CK2alpha-V5-His plasmid DNA construct.
Dr. Paul S Freemont; Division of Molecular Biosciences, Imperial College of London, UK for kindly gifting us with the pSG5-FLAG-PML plasmid DNA construct
Dr. Toshiyuki Sakai; Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Japan, for kindly gifting us with the pGVB2-p27/Kip1-Luc promoter construct.
We are also thankful to NCCS, Pune, India, for their timely support in form of supplying established human cell lines, whenever it was necessary.