1. Expression of CDCA3 Is a Prognostic Biomarker and Potential Therapeutic Target in Non–Small Cell Lung Cancer 
Mark N. Adams, PhD, Joshua T. Burgess, PhD, Yaowu He, PhD, Kathy Gately, PhD, Cameron Snell, PhD, Shu-Dong Zhang, PhD, John D. Hooper, PhD, Derek J. Richard, PhD, Kenneth J. O’Byrne, MD 
Journal of Thoracic Oncology 
Volume 12, Issue 7, Pages (July 2017)
DOI: /j.jtho
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

2. Figure 1
Cell division cycle associated 3 protein (CDCA3) transcripts are increased in NSCLC and are associated with poor patient outcome. (A) Scatter plot analysis of CDCA3 transcripts comparing nonmalignant (Non-M) and corresponding NSCLC tissue from the data set GSE (B) Scatter plots comparing CDCA3 transcript levels in Non-M tissue and stage IA, IB, and II NSCLC tissue. (C) Scatter plot analysis of data set GSE19188 comparing CDCA3 transcript levels in Non-M tissue with adenocarcinoma (ADC), large cell neuroendocrine carcinoma (LCNEC), and squamous cell carcinoma (SCC) tissue. (D) Kaplan-Meier analysis of overall survival of 1145 NSCLC cases comparing high versus low CDCA3 transcript levels split by median expression. The red lines indicate the median (****p ≤ ). HR, hazard ratio; CI, confidence interval.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

3. Figure 2
Cell division cycle associated 3 protein (CDCA3) expression in tissue from patients with NSCLC and in vitro. (A–C) Representative images of negative (0), weak (1), moderate (2), and high (3) scores for CDCA3 staining by immunohistochemistry in adenocarcinoma (ADC) (A), squamous cell carcinoma (SCC) (B), and nonneoplastic cases (C). (D) Western blot analysis of CDCA3 expression in five cases of matched normal and tumor tissue from patients with ADC and SCC. (E) Quantitative real-time polymerase chain reaction analysis of CDCA3 transcript levels relative to the 7SL housekeeping gene across three immortalized epithelial cell lines (HBEC3-5) and seven NSCLC cell lines. (F) Western blot analysis of CDCA3, total p53 and actin in whole cell lysates across three immortalized epithelial cell lines (HBEC3-5) and seven NSCLC cell lines. LCNEC, large cell neuroendocrine carcinoma.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

4. Figure 3
Cell division cycle associated 3 protein (CDCA3) depletion impairs NSCLC cell proliferation. (A) Western blot analysis of CDCA3 depletion using two individual small interfering RNAs (siRNAs) (siCDCA3-1 [light gray line] and siCDCA3-2 [dark gray line]) versus a control siRNA (siCon) (black line) across a panel of cell lines including three immortalized lung epithelial cell lines (HBEC3, HBEC4, and HBEC5) and seven NSCLC cell lines. (B–K) Proliferation analysis of HBEC3 (B), HBEC4 (C), HBEC5 (D), A549 (E), H2228 (F), H460 (G), SKMES-1 (H), EBC-1 (I), HTB-182 (J), and CRL-5889 (K) cells depleted of CDCA3 (siCDCA3-1 or siCDCA3-2) compared with control transfected cells (siCon) using the Incucyte Zoom live cell imaging system.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

5. Figure 4
Cell division cycle associated 3 protein (CDCA3) depletion in NSCLC cell lines leads to defective cell cycle progression. (A) Flow cytometry analysis of the DNA content profile for a panel of cell lines including three immortalized lung epithelial cell lines (HBEC3, HBEC4, and HBEC5) and seven NSCLC cell lines that were transfected with either a control small interfering RNA (siCON) or two small interfering RNAs targeting CDCA3 (siCDCA3-1 or siCDCA3-2 [shown only in (A)]). DNA content was determined by staining fixed cells with propidium iodide. (B) Quantification of (A) using FloJo X software to assess the proportion of cells within the G2/M phase of the cell cycle in control transfected cells or CDCA3-depleted cells. **p ≤ 0.01, ****p ≤ n.s., not significant.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

6. Figure 5
Depletion of cell division cycle associated 3 protein (CDCA3) induces cellular senescence. (A) Flow cytometry analysis of cell size (forward scatter height [FSC-H]) across a panel of control (red line) or CDCA3-depleted cells (blue line), including three immortalized lung epithelial cell lines (HBEC3, HBEC4, and HBEC5) and seven NSCLC cell lines. (B) Staining of control siRNA-transfected or CDCA3-depleted panel of cell lines used in (A) for senescence-associated β-galactosidase (SA-β-Gal) (×20 magnification light microscopy). (C) Quantification of (B) by determining the number of SA-β-Gal–positive cells within each field of view. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ siCon, control small interfering RNA; siCDCA3, small interfering RNA targeting CDCA3; n.s., not significant.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

7. Figure 6
Cell division cycle associated 3 protein (CDCA3) depletion yields an increase in p21 independent of p53. (A) Immunofluorescent images of a panel of control (control small interfering RNA [siCON]) or CDCA3-depleted cells (siCDCA3-2), including three immortalized lung epithelial cell lines (HBEC3, HBEC4, and HBEC5) and seven NSCLC cell lines stained for 4,6-diamino-2-phenylindole (DAPI) (blue) or p21 (green). (B) Quantification of (A) by high-content microscopy analysis to determine the increase in p21-expressing cells in CDCA3-depleted cells relative to control cells. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ (C) Immunofluorescent images of A549 and H460 NSCLC cell lines transfected with a control siRNA (siCON) or siRNA targeting CDCA3 (siCDCA3-2) and stained for DAPI (blue) or p53 (green). (D) Quantification of (C) assessing the relative change in the number of p53-expressing cells (left graphic analysis) and relative change in the p53 staining intensity (right graphic analysis). n.s., not significant.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

8. Supplemental Data Figure 1
(A) Scatter plots comparing CDCA3 transcript levels in non-malignant tissue (Non-M) from non-smoker and smoker patients and NSCLC tissue (C) from non-smoker and smoker patients. The red lines indicate median (n.s., not significant. **** P ≤ ). (B) Scatter plots comparing CDCA3 transcript levels in epidermal growth factor receptor (EGFR) wild-type (WT) or mutant NSCLC tissues. The red lines indicate median (n.s., not significant). (C) Scatter plots comparing CDCA3 transcript levels in Kirsten rat sarcoma viral oncogene homolog (KRAS) wild-type (WT) or mutant NSCLC tissues. The red lines indicate median (n.s., not significant). (D - H) Univariate Kaplan-Meier analysis of overall survival restricting NSCLC cases to stage I and stage II (E), stage I only (F) and stage II (G). (H - I) Univariate Kaplan-Meier analysis of overall survival restricting NSCLC cases on the basis of histology to ADC only (H) and SCC only (I). HR, hazard ratio; CI, confidence interval.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

9. Supplemental Data Figure 2
Flow cytometry analysis of cell death (apoptosis) across a panel of control (siCON) or CDCA3 depleted cells (siCDCA3-2) including immortalized lung epithelial cell line (HBEC4) and seven NSCLC cell lines. Live cells were stained with Annexin V-488 and propidium iodide and assessed using a Gallios flow cytometer (Beckman Coulter). The percentage of cells within each gating quadrant is listed for each panel. Live cells; lower left quadrant, apoptotic cells; upper right quadrant.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions