(a): Interdependency of TIMP-1 and Bcl-2 proteins: H2009 cells and its clones were treated with 0.5μM Staurosporine (S), which showed a reduction of TIMP-1 protein expression. Treatment with a BH3 mimetic ABT-737(A) reduced TIMP-1 protein levels compared to the controls. Combined treatment with ABT-737 and Staurosporine (AS) showed a marked reduction of TIMP-1 expression, without any changes in TIMP-2. (b) TIMP-1 mRNA expression level after treatment with Staurosporine and ABT-737: HB1 and HB6 TIMP-1 levels were reduced with combined ABT-737 and Staurosporine treatment. Two Way Repeated Measure ANOVA showed p = 0.0063, indicating a very significant down regulation of relative expression of TIMP1 in the overexpressed clones after ABT+Staurosporine treatment indicating down-regulating Bcl2 affects TIMP1 level. (c) Co-immunoprecipitation of TIMP-1 and Bcl-2: A possible interaction between the Bcl-2 and TIMP-1 revealed by co-immunoprecipitation. Cell extracts from H2009 and it’s clones wer

(a): Interdependency of TIMP-1 and Bcl-2 proteins: H2009 cells and its clones were treated with 0.5μM Staurosporine (S), which showed a reduction of TIMP-1 protein expression. Treatment with a BH3 mimetic ABT-737(A) reduced TIMP-1 protein levels compared to the controls. Combined treatment with ABT-737 and Staurosporine (AS) showed a marked reduction of TIMP-1 expression, without any changes in TIMP-2. (b) TIMP-1 mRNA expression level after treatment with Staurosporine and ABT-737: HB1 and HB6 TIMP-1 levels were reduced with combined ABT-737 and Staurosporine treatment. Two Way Repeated Measure ANOVA showed p = 0.0063, indicating a very significant down regulation of relative expression of TIMP1 in the overexpressed clones after ABT+Staurosporine treatment indicating down-regulating Bcl2 affects TIMP1 level. (c) Co-immunoprecipitation of TIMP-1 and Bcl-2: A possible interaction between the Bcl-2 and TIMP-1 revealed by co-immunoprecipitation. Cell extracts from H2009 and it’s clones were immunoprecipitated (IP) with anti-Bcl-2 or normal rabbit IgG as a negative control. The immunoblot (IB) was probed for TIMP-1, TIMP-2 and Bcl-2. No TIMP-2 was pulled down with Bcl-2, showing that only TIMP-1 is associated with Bcl-2 when apoptosis is induced. Input represents 60μg of cell extract.