Question

SKOV3-NV, SKOV3-C1, and SKOV3-C7 cells were treated with 0-12.5 μM cisplatin (a) or 0-5 nM paclitaxel (b) for 48 h, at which time the cells were subjected to MTS assay to measure viability. C1 and C7 were transfected with an YFG (protein) over expressing construct, while NV was transfected with only control construct.   Results are displayed as percent survival of vehicle treated cells. Error bars represent the standard deviation of biological replicates. *p < .05, **p < .005, N/S = not significant

Font Paragraph Styles Editing SKOV3: 48 h cisplatin SKOV3: 48 h Paclitaxel C7- N/S * Survival * Survival 12. 1.56 3.125 6.25 5 Concentration (M) 0 0.625 1.25 2. 55 Concentration (NM) -NV ....... 1 ---C7 -NV ...... C1 --- 67

1. Answer the following questions based on the image above (10)
   1. Propose a possible mechanism by which this protein could produce the results indicated above. Make sure to consider the mechanism of action of both compounds (2).
   2. Which of the mechanism seem more likely? Design an experiment to test this hypothesis. Show your expected data generated from this experiment (4).

I need help with both 1a and 1b please.Can you explain how you got that answer. It is cell and molecular.

Answer 1

Methods

Cell culture

SKOV3 and OVCAR8 ovarian cancer cells were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco Modified Eagle Medium (DMEM, Gibco, 11965-065) with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin and kept in a 37 °C humidified incubator with 5 % CO2.

Stable cell lines

All null vector (NV) and HE4-overexpressing stable cell lines were previously established [7]. To generate HE4-CRISPR Double Nickase stable cell lines, SKOV3-C1 cells were transfected in 6-well plates with 1 μg HE4 Double Nickase Plasmid (Santa Cruz, sc-402876-NIC) or Control Double Nickase Plasmid (Santa Cruz, sc-437281), using 5 μl Lipofectamine 2000 (Invitrogen). After 48 h, media was changed to 1 μg/ml puromycin containing media for five days, then split into larger plates and selected for an additional five days. RNA and tissue culture supernatant was collected to confirm downregulation of HE4 levels by quantitative RT-PCR (qRT-PCR) and ELISA. Cells were maintained in DMEM supplemented with 10 % FBS, 1 % penicillin/streptomycin, and 1 μg/ml puromycin.

Cell treatments

Cells were treated with the described doses of cis-diamminedichloroplatinum(II) (cisplatin, Sigma Aldrich, 1134357) or paclitaxel (Sigma Aldrich, T7402) dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich, D8418), or DMSO alone as a control. Cells were collected directly into either Trizol (Ambion, 15596018) or Cell Lysis Buffer (Cell Signaling, 9803) at the indicated time points for analysis. Cells were treated with recombinant human HE4 (rHE4, MyBioSource, MBS355616) added directly to the media to a final concentration of 20 nM. Cells were treated with recombinant human epidermal growth factor (rEGF, Calbiochem, 324831) at a concentration of 10 ng/ml.

Cell viability assays

All cells were seeded at 2 000 cells/well in 96-well plates. Cells were treated with increasing doses of cisplatin and paclitaxel as described. After 48 h, cell viability assays were performed by adding 10 μl/well of CellTiter 96® Aqueous One Solution Cell Proliferation MTS Assay (Promega, G3580), incubating at 37 °C/5 % CO2 for 2 h, and reading absorbance at 492 nm. Results are displayed as percent survival of vehicle treated cells.

Microarray

SKOV3-NV and SKOV3-C1 cells were treated in triplicate at 80 % confluency with 25 μM cisplatin or DMSO vehicle. Total RNA was collected 24 h later using an RNeasy Mini Kit (Qiagen, 74104) and checked for purity by NanoDrop 2000 (Thermo Scientific). The RNA samples were randomly assigned numbers and submitted to the Brown Genomics Core Facility for Bioanalyzer (Agilent 2100) RNA quality analysis. Affymetrix HTA 2.0 Arrays were performed according to the manufacturer’s instructions at the Core Facility using 150 ng total input RNA.

DAVID analysis

Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7 [12, 13] was used to identify the top ten enriched annotation terms among 180 genes differentially expressed (1.5-fold in either direction, p < .05) between SKOV3-NV and SKOV3-C1. Default DAVID parameters were employed as follows:

Kappa Similarity: Similarity Term Overlap – 3; Similarity Threshold – 0.5

Classification: Initial Group Membership – 3; Final Group Membership – 3; Multiple Linkage Threshold – 0.5

Enrichment Threshold: EASE – 1.0

Stringency: Medium

Quantitative PCR

RNA was collected using an RNeasy Mini Kit (Qiagen, 74104) or Trizol extraction/LiCl precipication. Total RNA (500 ng) was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, 1708890) according the manufacturer’s protocol. To validate differentially expressed genes between SKOV3-NV and SKOV3-C1 cells identified by microarray, the same RNA samples used for the microarray were employed. To validate the differential cisplatin-induced upregulation of EGR1 between SKOV3-NV and SKOV3-C1/C7, microarray RNA samples were used, as well as RNA isolated from SKOV3-C7 cells that were treated in the same manner as the cells used in the microarray. Quantitative PCR was performed in triplicate by loading 1 μl cDNA reaction, 2 μl each of 5 μM custom forward and reverse primers (Invitrogen) or 1 μM forward and reverse validated primers (realtimeprimers.com), 10 μl SYBR Green (Applied Biosciences [ABI], 4367659) and 5 μl RNAse-free water to each well. Samples were run on an ABI 7500 Fast Real-Time PCR System, and data was analyzed using the ΔΔCt method. Relative expression levels were normalized to 18 s rRNA to correct for equivalent total RNA levels. Validated MAPT, CYP1B1, and EGR1 primers were purchased from realtimeprimers.com. Custom primer sequences (Invitrogen) are as follows:

AKT3 F – AAG GGA AGA ATG GAC AGA

AKT3 R – ATG GGT TGT AGA GGC ATC

NMUR2 F – CCG TTC CAC ATT GAC CGA CT

NMUR2 R – CAC CAC ATG GAC GAG GTT GA

SEPT3 F – TTG CCC TGC TTC GAG ACT TT

SEPT3 R – CTT TCC TCT GTG TCC ACG CT

18 s rRNA F – CCG CGG TTC TAT TTT GTT GG

18 s rRNA R – GGC GCT CCC TCT TAA TCA TG

Western blot

Protein was extracted from cell pellets in Cell Lysis Buffer (Cell Signaling, 9803) with 1 mM PMSF, according to the manufacturer’s protocol. Protein concentrations were determined by DC Protein Assay (Bio-Rad Laboratories, 5000116). Western blot analysis was performed by loading equal amounts of protein boiled with Novex Sample Reducing Agent (Life Technologies, NP009) and NuPAGE LDS sample buffer (ThermoFisher Scientific, NP0007) into a 4–12 % gradient NuPAGE Novex Bis-Tris gel [Life Technologies, NP0321BOX (mini), WG1402BX10 (midi)]. Protein was transferred by semi-dry transfer to methanol-activated 0.2 μm PVDF membranes (Bio-Rad, 162-0177) at 0.12-0.2 A for 1 h 15 m. Membranes were blocked in 5 % milk in phosphate-buffered saline with 0.05 % Tween 20 (PBS-T) for 30 m at room temperature, incubated in primary antibody in 5 % milk in PBS-T overnight at 4 °C, and then in secondary antibody in 5 % milk in PBS-T for 1 h at room temperature, with PBS-T washes in between. Amersham ECL Prime Western Blot Detection System (GE Healthcare, RPN2232) was used for detection of HRP-tagged secondary antibodies. Blots were developed using x-ray film in a Kodac film developer or imaged directly in a Biorad Chemidoc MP Imaging System. GAPDH was used as a loading control. Antibodies and dilutions used are as follows:

PARP (Cell Signaling, 9532, 1:1000)

phospho-p44/42 MAPK (ERK1/2) (Cell Signaling, 4370, 1:2000)

p44/42 (ERK1/2) (Cell Signaling, 9102, 1:2000)

EGR1 (Santa Cruz, sc-110, 1:200)

p38 (Cell Signaling, 9212, 1:1000)

phospho-p38 (Cell Signaling, 9215, 1:1000)

GAPDH (Cell Signaling, 2118, 1:2000)

β-tubulin (Cell Signaling, 2146, 1:2000)

α-tubulin (Cell Signaling, 2144, 1:1000)

Densitometry

Image J was used to perform densitometry analysis of western blots. Images of blots were analyzed in 8-bit TIFF format, using the “analyze gel” function. Where no band was detected, a value of “1” was assigned. Relative band densities were normalized to a loading control, or the appropriate total protein for phospho-proteins, and then the lowest value was set to 1.

Statistics

In all instances where statistics are shown, they represent n ≥ 3 independent experiments, and p-values were determined by unpaired 2-tailed Student t-test. For the microarray, Affymetrix Transcriptome Analysis Console (TAC) software was used to generate fold-changes and statistical significance, and ANOVA p-values generated by TAC were used for p-value cutoffs.

SKOV3:48 h Cisplatin ou SKOV3:48 h Paclitaxel C1 % Survival % Survival 0 12.5 0 5 1 .56 3.125 6.25 Concentration (M] –NV - 1 --- 0.625 1.25 2. 5 Concentration (M) -NV ...... ---C7 Untrx. 24h 48h 72 h E Untre 1 NV c1 cm" NV C1 C3 "NVc1c7'N - - c1 c - - NV C1 C7NV a - - - - - C7 -- PARP -cleaved PARP -PARP cleaved PAR 000000-00 GAPDH GAPDH PARP and Cleaved PARP Densitometry PARP and Cleaved PARP Densitometry T D PARP deawed Relative Band Density Normalized to GAPDH Relative Band Density Normalized to GAPDH NV-Unte CH-Unters NY-Untox C1-Unta C-Untra NV-48h 1-48 07448