Additional file 5 of 5-AZA-dC induces epigenetic changes associated with modified glycosylation of secreted glycoproteins and increased EMT and migration in chemo-sensitive cancer cells

Additional file 5: Figure S1: 5-AZA-dC treatment increases resistance to cisplatin in chemo-sensitive and decreases in chemo-resistant cell lines. % viability of A2780 and A2780cis cells treated with 1 μM 5-AZA-dC and 1 μM 5-AZA-dC in combination with 1 μM cisplatin. * = P-value ≤ 0.05 or ** = P-value ≤ 0.005 (T-test). Figure S2: Glycosylation changes of cell glycans on A2780 and A2780cis chemo-sensitive/chemo-resistant pair. (A) Representative UPLC chromatograms produced from secreted N-glycans of ovarian chemo-sensitive- chemo-resistant pair (A2780, A2780cis) and their separation into 39 peaks. (B) Plotted peak areas from the cell N-glycans of these cell lines. The glycans in each peak (GP1-GP39) and features are listed in Table S2. Significant changes (p < 0.05) are starred: * = P-value ≤ 0.05 or ** = P-value ≤ 0.005. (MANOVA). Heatmap histograms indicating fold changes in 5-AZA-dC treated compared to untreated cells were created using Hierarchial Clustering Explorer HCE 3.5 software. Blue indicates decreases, and red indicates increases. The shade of colour corresponds to amounts of the decreases/increases. Figure S3: Secreted glycans differ in chemo-resistant comparing to chemo-sensitive cell lines. Plotted peak areas of GP13 and 14 from the secreted N-glycans of chemo-sensitive and chemo-resistant ovarian cancer cell lines. Significant changes (p < 0.05) are starred: * = P-value ≤ 0.05 or ** = P-value ≤ 0.005. (MANOVA). Figure S4. Representative Western blots of EMT markers in the 4 ovarian cancer cell lines and 2 TNBC cell lines post-5-AZA-dC treatment (T) compared to non-treated (UT) controls. Figure S5: Migration results are not attributable to proliferation. Migration (A) and proliferation (B) or the representative 1 uM 5-AZA-dC treated relative to the untreated A2780cis cells. Figure S6. Western blot analyses of the senescence markers p16, p21 and Rb and of cellular apoptosis (PARP cleavage) markers, post-5-AZA-dC treatment. Representative Western blots of the senescence associated proteins p21, p16, Rb and the apoptosis marker PARP of 4 ovarian cancer cell lines and 2 TNBC cell lines in 5-AZA-dC treated (T) compared to untreated (UT) controls. Figure S7. SiRNA knockdown of GATA2/3, impacts on the protein expression levels of MGAT5 and ST3GAL4. Western blot analyses of siRNA knockdown of GATA2/3 in 4 ovarian cancer cell lines (A2780, A2780cis, PEO1 and PEO4). GATA2 was knocked down in the A2780 and A2780cis cell lines and GATA3 in the PEO1 and PEO4 cell lines. Results are shown comparing (i) untreated controls (UT) with siRNA GATA knockdown UT (siG2/3) (left) with (ii) 5-AZA-dC treated (T) with siRNA GATA knockdown treated T (siG + T). All results are in triplicates (n = 3, biological replicates).