(a) Comparison of exogenous (in SFCM) and endogenous (cell lysates) TIMP-1 in H2009, empty vector and TIMP-1 overexpressing clones: Cells were grown overnight in serum free media to assess exogenous TIMP-1 and cell lysates were collected from cells grown in complete media. The conditioned media and the cell lysates from the cell lines were assayed for TIMP-1 by ELISA. The X-axis shows H2009 cell line and its clones. The Y-axis shows concentration of TIMP-1 in ng/mL. A significant upregulation of TIMP-1 in overexpressed clones is seen (p<0.01, Student’s t test). Data represents independent triplicate determinations ± SEM (standard error of mean). (b) Effect of ABT-737 and Staurosporine on apoptosis: Cells were first treated with ABT-737 (1 μM, for 21 hours) following which Staurosporine was added at 0.5μM concentration for 3 hours (all apoptosis related experiments follow this regimen unless mentioned otherwise). Cell apoptosis was evaluated by Hoechst 33258 staining. The control cells

(a) Comparison of exogenous (in SFCM) and endogenous (cell lysates) TIMP-1 in H2009, empty vector and TIMP-1 overexpressing clones: Cells were grown overnight in serum free media to assess exogenous TIMP-1 and cell lysates were collected from cells grown in complete media. The conditioned media and the cell lysates from the cell lines were assayed for TIMP-1 by ELISA. The X-axis shows H2009 cell line and its clones. The Y-axis shows concentration of TIMP-1 in ng/mL. A significant upregulation of TIMP-1 in overexpressed clones is seen (p<0.01, Student’s t test). Data represents independent triplicate determinations ± SEM (standard error of mean). (b) Effect of ABT-737 and Staurosporine on apoptosis: Cells were first treated with ABT-737 (1 μM, for 21 hours) following which Staurosporine was added at 0.5μM concentration for 3 hours (all apoptosis related experiments follow this regimen unless mentioned otherwise). Cell apoptosis was evaluated by Hoechst 33258 staining. The control cells displayed normal nuclei. Staurosporine treatment with or without ABT-737 showed significantly more condensed and bright fluorescent nuclei with nuclear fragmentation in H2009 and HEV cells. The number of apoptotic cells were less in the HB1 and HB6 cells in panel C and D as shown by the arrows. (c) Quantitative representation of Hoechst 33258 apoptosis assay: Hoechst staining showed significantly more apoptosis in H2009 cells and the empty vector clones (HEV), compared to the TIMP-1 overexpressing clones(HB1, HB6), by One Way ANOVA (p<0.05), with or without staurosporine treatment. The control groups (H2009, HEV) showed almost 2-fold greater apoptotic morphology compared to TIMP-1 overexpressing clones. Data is representative of 3 independent experiments ± SEM. (d) TIMP-1 overexpressing cell lines show increased expression of Bcl-2 mRNA in response to staurosporine treatment: H2009 cells and its clones were treated with 0.5μM Staurosporine (S) and subjected to apoptosis specific PCR array. A significant 3 fold increase in Bcl-2 and TIMP1, was observed and confirmed by qPCR (p<0.01, One Way ANOVA with posthoc Dunnett’s test). (e) Expression of Bcl-2 and TIMP-1 in H2009 cells: The top panel shows semiquantitative reverse transcription PCR after 3hrs of Staurosporine (S) treatment. An increased amount of Bcl-2 is observed in the TIMP-1 overexpressing clones. Lower panel indicates an increased amount of Bcl-2 in the TIMP-1 overexpressing clones by western blot. The TIMP-2 level remained unchanged. The relative protein band density was normalized to β-Actin. Data is representative of triplicate independent experiments.