Fig 2: EGFR modestly regulates the expression of ARID3B splice forms.QRT-PCR for ARID3B Fl and ARID3B Sh was performed on total RNA isolated from OVCA 429, OVCA 433 and DOV13 ovarian cancer cells grown in 10% serum conditions (FS) (A and C) or serum starved (SS) (B and D) for 24 h and treated with or without 20 nM of EGF or 10 nM of PDGF for an additional 24 h. QRT-PCR was performed for ARID3B Fl (A and B) and ARID3B Sh (C and D). Expression was normalized to 18 s rRNA. Fold expression was normalized to non-EGF treated cells (SS or FS) and was expressed as the mean ± SEM of triplicate measurements. Statistical analysis was performed to determine if there were any significant changes between FS vs. treatment with FS and EGF in all three ovarian cancer cell lines and for both ARID3B isoforms (A and C). We also performed statistical analysis to determine if there were any significant changes between SS vs. treatment with SS and FS or PDGF or EGF in all three ovarian cancer cell lines and for both ARID3B isoforms (B and D). E. OVCA 429 and OVCA 433 ovarian cancer cells grown in serum starved (SS) or 10% serum conditions (FS) for 24 h and treated with or without 20 nM of EGF for an additional 24 h. Whole cell lysates were analyzed by western blot using anti-ARID3B and anti-GAPDH (control) antibodies. Results were normalized to GADPH expression and compared to the expression of cell not treated with EGF (SS or FS). The densitometry evaluation of the western blot analyses for ARID3B Fl and ARID3B Sh in ovarian cancer cells were analyzed (value under blot). Statistical analysis was performed to determine if there were any significant changes between (FS) vs. (FS and EGF) and (SS) vs. (SS and EGF) in all three ovarian cancer cell lines and for both ARID3B isoforms fold expression was expressed as the mean of triplicate measurements. [*p<0.05, **p<0.005, ***p<0.0005 and #p<0.01].

Fig 3: ARID3B Fl induces apoptosis in ovarian carcinoma cells via TNFα/TRAIL pathways.Western blot using anti-Caspase 7, anti-Caspase 10, anti-TNF-R2, anti-TRAIL, anti-TRADD and anti- GAPDH (control) antibodies on transduced OVCA 429 (A) and OVCA 433 (B) cells. The densitometry evaluations of the western blots were analyzed (value under blot). Results were normalized to GAPDH expression and compared to parental ovarian cancer cells. Statistical analysis was performed to determine if there were any significant changes between parental ovarian cancer cells vs. GFP, ARID3B Sh or ARID3B Fl transduced ovarian cancer cells. [*p<0.05, ***p<0.0005, #p<0.01 and ##p<0.001].

Fig 4: EGFR regulates PEA3 binding to the ARID3B promoter.Chromatin immunoprecipitation (ChIP) analysis confirmed the binding PEA3 to the ARID3B promoter region. ChIP was performed on OVCA 429 cells serum starved (SS) for 24 h then treated with or without 20 nM EGF for 24 h. Quantitative PCR analysis was conducted for (A) a region of the ARID3B promoter containing ETS/PEA3 binding site and (B) a promoter sequence upstream of the ETS binding site. ChIP was performed using IgG (negative control) or anti-PEA3. Expression was normalized to NTC (no template control). The no template control (NTC) expression was a boundary limit (expression fold = 1), any fold-expression below this limit (expression <1) was considered to be negligible. Statistical analysis was performed to determine if there were any significant changes in chromatin binding between cells without EGF vs. cells treated with EGF. Fold expression was expressed as the mean ± SEM of triplicate measurements. [*p<0.05].

Fig 5: ARID3B Fl induces apoptosis in ovarian carcinoma cells.Flow cytometry analysis using Annexin V and 7-AAD was used to evaluate the apoptosis in OVCA 429 (A) and OVCA 433 (B) overexpressing the ARID3B splice forms. OVCA 429 and OVCA 433 cells were lentivirally transduced with ARID3B Fl, ARID3B Sh and GFP (control). Flow cytometry for Annexin V and 7-AAD was performed on OVCA 429 (A) and OVCA 433 (B) the percentage of viable cells was expressed as the mean ± SEM of triplicate measurements. C. The Caspase-Glo 3/7 assay for Caspase 3/7 activities was performed on OVCA 433 and OVCA 429 ovarian cancer cells 72 h post transduction. Caspase-Glo 3/7 activity is shown in reference to parental cell and normalized to the cell count after transduction with GFP, ARID3B Sh and ARID3B Fl for 72 h. Caspase activity of parental cells was defined as 100%. Caspase activity was expressed as the mean ± SEM of triplicate measurements. D. Cell viability analysis using MTT assay was used to confirm the apoptotic phenotype of ARID3B Fl. MTT assay was performed on OVCA 429 and OVCA 433 transduced with ARID3B Fl, ARID3B Sh and GFP cells for 72 h and the percentages of viable cells (compared to parental cells) were expressed as the mean ± SEM of triplicate measurements. E. Western blot using anti-ARID3B, anti-BIM and anti- β-actin (control) antibodies on transduced OVCA 433 and OVCA 429 cells. The densitometry evaluation of the western blot analyses for BIM was analyzed (value under blot). Results were normalized to β-actin expression and compared to parental ovarian cancer cells Statistical analysis was performed to determine if there were any significant changes between parental ovarian cancer cells vs. GFP, ARID3B Sh or ARID3B Fl transduced ovarian cancer cells. [**p<0.005, ***p<0.0005, #p<0.01,##p<0.001 and ###p<0.0001].