2.1. Media and Experimental Reagents
The primary antibodies for Notch-1 (ab65297), Hes-1(ab49170), EGFR (ab2430), CDK2 (ab235941,) and GAPDH (sc32233) were purchased from Abcam, UK and SantaCruz, USA. The DNA transfection reagent, Jet PEI was procured from GeneX, India. The Notch-1 inhibitor-N-[N-(3, 5-difluorophenacetyl)-Lalanyl]-S-phenylglycine t-butyl ester (DAPT) was obtained from Abcam, UK. 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide (MTT), Dimethyl sulfoxide (DMSO), Antibiotic antimycotic solution (100×), Ethanol, 2-Mercaptoethanol and 2-Propanol (Isopropanol) were obtained from Sigma, USA. Roswell Park Memorial Institute (RPMI) 1640, Dulbecco's Modified Eagle Medium (DMEM) and N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES-1M) were obtained from HiMedia Laboratories, India. The Fetal Bovine Serum (FBS) was obtained from Moregate, Australia. DNA was extracted using GF-1 Tissue DNA Extraction Kit from Vivantis Technologies, Malaysia. RNA isolation kit, Plasmid Miniprep Plus Purification Kit were procured from Gene Mark, USA. The Cycletest™ Plus DNA kit was procured from, BD Biosciences, USA. Power SYBR® Green RNA to CT TM 1-Step Kit was procured from Thermofisher, USA.
2.2 Cell lines
Adherent cervical tumor derived cell lines HPV-Negative C33A cells (HPV -ve for HPV DNA and RNA having cervical cancer phenotype)and HPV-16+ CaSki cells(with inherent HPV-16+ and HPV-18+ sequences and endogenous Notch-1), were used for transfection.
2.3. Cell culture
CaSki and C33A cell lines were procured from National Centre for Cell Sciences (NCCS), Pune. CaSki cell line was propagated in RPMI-1640 with sodium bicarbonate without L-Glutamine supplemented with 10 % fetal bovine serum and antibiotic, anti-mycotic solution. C33A cells were grown DMEM medium consisting of L-Glutamine, Sodium pyruvate, Glucose and Sodium bicarbonate.
The pLEGFPN1-(RV-Tat 86) plasmid was a gift from Dr. Francesca Peruzzi, Associate Professor, LSU Health Sciences, New Orleans LA. The pNL4.3, a full length replication competent, infectious HIV-1 subtype B, was obtained through NIH AIDS Reagent Program.
2.4.1. Cell proliferation
Briefly, CaSki and C33A cells (104/well) were seeded in 96micro well culture plates and maintained at 37°C in a CO2 incubator. Cells were exposed to serial dilutions of plasmid DNA/DAPT and incubated overnight for 24 hrs. MTT solution (5 mg/mL) was added to the plate and then incubated for 4 hrs. Supernatant was replaced with DMSO, and absorbance was measured at 550/630 nm. The viability was calculated in percentage with reference to control sets.
2.4.2. Recombinant cloning
Plasmids were transformed using DH-5α through heat shock (42–45°C) and rapid chilling on ice. After heat shock treatment, the plasmid DH-5α mixture was added to pre-warmed SOC or LB without antibiotics. LB agar plate containing ampicillin were used to grow the transformed bacteria, at 32°C. Colonies were further inoculated in 15 mL LB mixed with 65 % glycerol for stock preparation and plasmid amplification.
2.4.3 Plasmid Purification
Briefly, bacterial cells were pelleted, resuspended in 250 µL of Solution I ( as per the manufacturer's protocol-Gene Mark plasmid miniprep purification kit). With subsequent addition of 250 µL Solution II, and following 4–5 times mixing, Solution III (250 µL) was added. The spin columns containing lysate/s were centrifuged, rinsed and purged of purified DNA with preheated elution buffer. The DNA was quantified and stored at -20°C for transfection.
2.5. Transient Transfection
Transfections were carried according to the manufacturer’s instructions (jetPEI® DNA transfection reagent, USA). Briefly, CaSki and C33A cells were seeded in T-25 flasks (1 X 106) with 5 ml medium containing FCS and antibiotics. Quantified DNA-Six hundred ng/mL DNA was used for transfecting one million cells. The media was replenished the next day and cell lines maintained overnight in a CO2 incubator. Cells were detached for further analysis.
2.6. Cell cycle analysis
Transfected cells were scraped gently, washed twice in cold PBS and processed using BD Cycletest™ Plus DNA Kit as per the manufacturer's protocol. Briefly, cells were pelleted, mixed with Solution A (trypsin buffer) for 10 min followed by incubation with Solution B (trypsin inhibitor and RNase buffer) for 10 min. Finally the cells were stained with 200 µL of cold Solution C (PI stain solution) kept for 10 min in the dark at 4 ºC and analyzed for DNA content by BD FACSAria™ (BD, USA).
2.7. DNA isolation
DNA from transfected cells was isolated using the manufacturer’s protocol (Vivantis Technologies, Malaysia). Briefly, the cell pellet (5 X 106 cells) was resuspended in PBS, treated with Proteinase K and lysis enhancer in Tris buffer. After incubation at 65 ºC for 10 min the cells were transferred to the column, followed by column washing with wash buffer. DNA was eluted with preheated elution buffer and quantified using spectrophotometer.
2.8. Polymerase Chain Reaction (PCR)
Notch-1 PCR was optimized with DNA isolated from human fetal buccal mucosal (FBM) cell line procured from ACTREC, Navi Mumbai, India. Twenty µL reaction mix (1.2 µL DNA template, primer mix and PCR dye master mix (5Xia)) was subjected to the following conditions ; 95 ºC for 4 min, 45 cycles at 95 ºC for 20 secs, 60 ºC for 20 secs, two extension steps − 72 ºC for 20 secs and 2 mins. Notch-1 PCR protocol was also used for CaSki cells.
Tat PCR was performed with DNA isolated from CaSki and C33A cells using the following conditions; 95 ºC for 9 mins followed by 34 cycles at 95 ºC for 1 min, 53 ºC for 2 mins, two extension steps at 72 ºC for 1 min and 7 mins respectively. PCR products were resolved on a 2.0 % agarose gel and visualized on a UV transilluminator after staining with ethidium bromide.
2.9. Western blot analysis
C33A and CaSki cells were harvested, washed, and lysed with RIPA buffer supplemented with phenylmethylsulfonyl fluoride (PMSF). Protein extracts (30 µg) per sample were subjected to 8 % SDS‑PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, USA). Membranes were blocked with 5 % nonfat skim milk for 1 h at room temperature, probed with the appropriate primary antibodies (Notch-1, Hes-1, CDK2, EGFR and GAPDH a endogenous protein) at 1:1000 dilution, washed, and then incubated with the corresponding goat anti‑rabbit and anti-mouse secondary antibody (1:5000 dilution). The chemiluminescent signal was detected using ECL system (Intron, Seongnam, Korea). Semi‑quantitative analyses of the intensities of the protein bands were analyzed by using ImageJ software (NIH).
2.10. RNA Isolation
RNA was isolated from transfected cell lines as per the manufacturer’s protocol (GeneMark Biolab, Thailand). Briefly, transfected culture cells were lysed with lysis methanol cocktail in 70 % ethanol. The lysate was loaded on the spin column and washed. After DNAse treatment, the columns were washed again with wash buffer. Purified RNA (30–50 µL) was collected, quantified by a NanoDrop 2000c (Thermo Fisher Scientific, USA) and processed for qPCR.
2.11. Quantitative Real-time PCR (qRT-PCR)
qRT-PCR was conducted using Applied Biosystems™ Power SYBR™ Green RNA-to-CT™ One-Step Kit and ABI 7500 FAST v2.3 (ThermoFisher, Singapore) as per the manufacturer’s instructions. The qRT-PCR master mix (10 µL) consisted of a cocktail of 5 µL of 2X SYBR Green Real-time PCR Master, 2 µL primers -forward and reverse, 0.08 µL, RNA template (up to 100 ng), 2 µL, RNAse free water and 0.92 µL, RT Enzyme (125 X). The thermal cycling conditions were 48 ºC for 30 mins, followed by 1 cycle at 95 ºC for 10 min, 45 cycles at 95ºC for 15 sec, 60 ºC for 10 secs, and 72 ºC for 30 secs. Experiments were run in triplicates with β-actin as housekeeping gene. Relative concentrations were calculated using 7500 FAST software v2.3. Cycle threshold (CT ) values of transfected C33A cell line (an HPV− cell line) served as the internal control for normalizing sample variations. The forward and reverse primers used for qRT-PCR were human Notch-1, Hes-1, Hey-1, cyclin D, CDK2, p21, EGFR, and β-actin represented in supplementary Table 1.
2.12. Densitometry and statistical analysis
The results were analyzed by Graph Pad Prism 5. Student t -test was used to determine the statistical significance of the data expressed as mean ± standard deviation (SD). p values ≤ 0.05* were considered statistically significant.