1. Volume 130, Issue 2, Pages 369-376 (August 2013)
Loss of DOK2 induces carboplatin resistance in ovarian cancer via suppression of apoptosis 
Elena Lum, Michele Vigliotti, Nilanjana Banerjee, Noelle Cutter, Kazimierz O. Wrzeszczynski, Sohail Khan, Sitharthan Kamalakaran, Douglas A. Levine, Nevenka Dimitrova, Robert Lucito 
Gynecologic Oncology 
Volume 130, Issue 2, Pages (August 2013)
DOI: /j.ygyno
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2. Fig. 1
Heatmap of the differentially methylated genes from the MOMA analysis showing segregation between the platinum therapy resistant and sensitive patient groups.
This heatmap presents the results from the MOMA analysis. Hierarchical clustering was performed on the differentially methylated values from 749 probes that distinguished refractory/resistant and late sensitive patients. For these probes, hierarchical clustering using Pearson correlation as a distance metric was used. The distance metric is: 1- Pearson Correlation (x). The Ward method was utilized for finding clusters.
Gynecologic Oncology  , DOI: ( /j.ygyno )
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3. Fig. 2
Knockdown of DOK2 increases platinum resistance in ovarian cell lines.
Cell lines (A) HOSE (B) SKOV3 and (C) CAOV1 demonstrated suppression of DOK2 with lentivirus increased resistance to carboplatin at the IC20, IC50, and IC80 concentrations after 9days. Cells were plated at a density of 1x10^3 cells/well in 96-well plates in triplicate. The IC concentrations on the x-axis is plotted against percentage survival on the y-axis. The results represent the mean±SD (n=6; P<0.05) in triplicate.
Gynecologic Oncology  , DOI: ( /j.ygyno )
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4. Fig. 3 DOK2 silencing causes minimal effect on growth.
A growth assay was also done over the course of 9days showing that lentiviral DOK2 shRNA infected ovarian cell lines, (A) HOSE, (B) SKOV3, and (C) CAOV1 causes little change in growth compared to their parental counterparts. Cells were plated at 1x10^3 cells/well in 96-well plates in triplicate. The x-axis represents the time in days and the y-axis represents the cell count. The results represent the mean±SD (n=6; P<0.05) in triplicate.
(D) 3-day BrdU assay to measure actively proliferating cells was done using HOSE and SKOV3 parental and lentiviral cell lines demonstrating a slight increase with DOK2 suppression. Cells were plated at 6x10^3 cells/well in 96-well plates in triplicate. The x-axis represents the time in days and the y-axis represents the cell count. The results represent the mean±SD (n=4; P<0.05) in triplicate.
Gynecologic Oncology  , DOI: ( /j.ygyno )
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5. Fig. 4
DOK2 silencing results in decreased apoptosis/anoikis induced cell death.
(A) HOSE parental and HOSEshDOK2 were plated in triplicate on a 24 well plate at 5×10^4 cells/well and treated with carboplatin at the IC20, IC50, and IC80. Live/dead cells were measured with tryphan blue stain. The IC concentrations on the x-axis is plotted against percentage apoptotic on the y-axis. More dead cells were observed in the parental line in the presence of carboplatin. The results represent the mean±SD (n=2; P<0.05) in triplicate.
(B) HOSE parental and HOSEshDOK2 were plated in triplicate on a 96-well low binding plate at 1×10^4 cells/well and treated with carboplatin at the IC20, IC50, and IC80. Live cells were stained with the calcein AM and dead cells were stained with EthD-1 and the fluorescence signals were quantitatively measured using a fluorescence plate reader. The IC concentrations on the x-axis is plotted against percentage anoikis cells on the y-axis. A decrease in percentage of cell death was observed with loss of DOK2. The results represent the mean±SD (n=2; P<0.05) in triplicate.
Gynecologic Oncology  , DOI: ( /j.ygyno )
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6. Fig. 5 Suppression of DOK2 showed increased invasive potential.
(A) The matrigel invasion assay was used to determine invasive potential. The upper chamber was plated with 5×10^4 cells/ml of serum starved cell suspensions. The bottom chamber was filled with medium containing 15% FBS, used as a chemoattractant. After 24h there was a marked increase of invading cells through the matrigel. (B) Quantification of cell count showed suppression of DOK2 via lentivirus greatly increased number of invading cells, demonstrating increased invasive properties. Counts were based on an average cell count of 5 random areas viewed under 40× magnification. The cell count for each cell line is shown. The results represent the mean±SD (n=6; P<0.05) in triplicate.
Gynecologic Oncology  , DOI: ( /j.ygyno )
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7. Fig. S3 Validation of efficient knockdown via lentiviral DOK2 shRNA.
miR30 DOK2shRNA clones in a (A) pGIPZ lentiviral and (B) retroviral vector was validated for knockdown with QPCR to measure efficiency of DOK2 knockdown in our ovarian cell lines. mRNA Expression levels are represented on the y-axis with parental cell lines normalized to 100% as reference. The results in A) represent the average of three independent experiments in triplicate±SD (n=6; P<0.05). Results in B) represent the mean±SD (n=6; P<0.05) in triplicate.
Gynecologic Oncology  , DOI: ( /j.ygyno )
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8. Fig. S4
Knockdown of DOK2 increases platinum resistance in retroviral cell line.
Suppression of DOK2 with retrovirus in HOSE cell lines increased resistance to carboplatin at the IC20, IC50, and IC80 concentrations after 9days. Cells were plated at a density of 1×10^3 cells/well in 96-well plates in triplicate. The IC concentrations on the x-axis is plotted against percentage survival on the y-axis. The results represent the mean±SD (n=2; P<0.05) in triplicate.
Gynecologic Oncology  , DOI: ( /j.ygyno )
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9. Fig. S6
Loss of DOK2 induces transformation and increases tumorigenicity with lentiviral and retroviral shDOK2.
Soft agar plates were prepared using 1.5ml of 1% bacterial agar supplemented in a 1:1 ratio with DMEM containing 20%FBS and 1X Antibiotic/Antimycotic in a 6cm plate to form the bottom layer. Bottom plates were allowed to solidify and cool for 30min. 2.5×104 cells were seeded separately in 6ml of .35% bacterial agar, 50% DMEM, 10%FBS, 1X Antibiotic/Antimycotic to form the top layer The cell suspensions (1.5ml) were poured into triplicate 6cm plates containing the bottom layer, with a final concentration of 5×103 cells/dish. Cells were cultured for three weeks and cellular foci were photographed and foci number was quantified as a total count of 5 random fields. (A) To examine the tumorigenic potential, parental and DOK2 knockdown cells for HOSE and SKOV3 were grown on soft agar as specified above. An increase in number of colonies was observed with down-regulation of DOK2. Cell counts significantly increased in both (B) lentiviral cell lines and (C) retroviral cell lines indicating that loss of DOK2 shows transformative properties and increased tumorigenicity. The cell count for each cell line is shown. The results represent the mean±SD (n=6; P<0.05) in triplicate.
Gynecologic Oncology  , DOI: ( /j.ygyno )
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10. Fig. S7
DOK2 silenced cells shows increased migration compared to parental cell lines.
To examine migration, cells were grown to 90–95% confluent in a 10cm diameter dish. An artificial scratch was made across the cell monolayer using a 20μl pipet tip. Images were taken at T=0hrs and T=16h. NIH Image J software was used to quantify the degree of migration as measured by the change in the area of the scratch. (A) The scratch assay compares the parental cells at T=0hrs with the same scratch 16hrs later. Little change is observed after 16h. (B) The same is shown for lentiviral DOK2 silenced cells demonstrating increased migration. (C) Percent migration was quantified using ImageJ indicating that migration in lentiviral DOK2 knockdowns are more than double the parental cell lines. Results represent the mean±SD (n=6; P<0.05) in triplicate. (D) Retroviral DOK2 knockdowns were quantified for SKOV3 and CAOV1 cell lines and demonstrated increase in migration (images not shown). Results represent the mean±SD (n=4; P≤0.05) in triplicate.
Gynecologic Oncology  , DOI: ( /j.ygyno )
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