Fig.S1 Inhibition of Cell Proliferation by S009-131

Supplementary Captions:

Fig.S1 Inhibition of cell proliferation by S009-131

In vitro anti-proliferative activity of S009-131 was assessed by colony formation assay. HeLa (A) and C33A (B) cells were exposed to the compound for indicated time points. Cells were then trypsinized, counted, seeded at 600 cells/ well onto a 6 welled tissue culture plate. After 14 days incubation, cellular colonies were stained with crystal violet. (C) The number of colonies was significantly reduced after exposure to S009-131. *P < 0.05, **P < 0.001 when compared to the control.

Fig.S2 Effect of S009-131 on HeLa cell xenografts

HeLa cells were inoculated (107 cells in 100 μlHBSS) at right flank of nod SCID mice. Representative images of tumour bearing mice in different experimental groups on 1st day (A) and last day (B) of the study.

Fig.S3 Apoptotic index

Apoptotic index in S009-131 treated cells was determined by DAPI staining of cellular nuclei. Cells with condensed or fragmented nuclei were considered apoptotic. Minimum 200 cells were counted for each treatment and the apoptotic index was calculated as the percentage of apoptotic cells in the population. Data presented as mean ± SE of 3 independent experiments, *P < 0.05, **P < 0.001 with respect to untreated controls.

Fig.S4S009-131 induced G2/M cell cycle arrest in HeLa and C33A cells:

HeLa (A-E) and C33A (F-J) cells were treated with S009-131 at indicated concentrations for 24 h. After harvesting in PBS, cells were fixed with ethanol, stained with PI and subjected to flow cytometry. Results are shown as percentage of cell population. Histograms showing average population of HeLa (K) and C33A (L) cells in various phases of cell cycle (mean ± S.E. of 3 independent assays). *P < 0.05, **P < 0.001 compared with untreated controls.

Fig.S5 Decrease in MMP induced by S009-131 at different concentrations:

Effect of S009-131 on mitochondrial membrane potential was measured by flow cytometry. HeLa (A) and C33A (B) cells were treated with compound at indicated concentrations for 24 h and processed with MitocaptureTM staining kit. Fluorescent cells were analyzed by flow cytometry. Bar graphs depict population of HeLa (C) and C33A (D) cells stained with MitocaptureTM dye. Significant depletion of MMP was observed in compound-treated cells (mean ± SE of 3 experiments, *P < 0.05, **P < 0.001 compared with untreated control).

Fig.S6S009-131 induced intracellular ROS level

Cells were grown in a 6 well culture plate, exposed to S009-131 at IC50 concentrations for 0 h (untreated), 3 h and 6 h, stained with DCFDA and observed under fluorescent microscope. Images, acquired with 10x objective, show peak ROS levels at 3 h.

Fig.S7 Role of cytochrome c and PUMA in S009-131 induced apoptosis.

(A) Level of cytochrome c in cytosolic fraction was examined by Western blotting and quantified based on band intensity. Significant release of Cyt c was observed in S009-131 treated cells (mean ± SE of four experiments, **P < 0.001 compared with untreated control). (B) Densitometric analysis PUMA expression in different experimental groups. Data plotted as mean ± SE of three experiments, *P < 0.05, **P < 0.001 compared with untreated control.