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  • br caption on next page br Fig Pharmacological interventions

    2019-10-03


    (caption on next page)
    Fig. 6. Pharmacological interventions targeting the active Rac1-induced S70pBcl-2 loop sensitizes to apoptotic stimulus.
    (a–b) Measurement of cell viability (%) of CEM/Bcl-2 and Jurkat A 61603 following 24-h treatment of increasing doses of NSC23766. n = 3. (c) Measurement of cell viability of pIRES and Rac1 (V12) M14 Melanoma cells following 1-h pre-treatment of 10 mM Tiron and subsequent 24-h treatment of 0.5 μM of staurosporine. Cell viability of tiron and/or staurosporine-treated cells normalized to that of untreated cells in (%). n = 3. (d) Measurement of cell viability (%) of CEM/Bcl-2 cells following 1-h pre-treatment of 1 μM ABT199 and subsequent 24-h treatment of 50 nM of staurosporine. n = 3. (e) Measurement of cell viability of M14 Melanoma cells transiently transfected with pcDNA3.1, WT Bcl-2, S70A and S70E following 24-h treatment of 0.5 μM of staurosporine. Cell viability of staurosporine-treated cells normalized to that of untreated cells in (%). n = 3. (f) 72-h spheroid formation of pIRES and Rac1 (V12) M14 Melanoma cells following 1-h pre-treatment of 10 mM Tiron and subsequent 24-h treatment of 0.5 μM of staurosporine. n = 3. (g) Quantification of spheroid size (%) of pIRES and Rac1 (V12) M14 Melanoma cells following 1-h pre-treatment of 10 mM Tiron and subsequent 24-h treatment of 0.5 μM of staurosporine. (h) 72-h spheriod formation of M14 Melanoma cells transiently transfected with empty vector pcDNA3.1, wild-type Bcl-2, non-phosphorylatable S70A mutant Bcl-2 and phosphomimetic S70E mutant Bcl-2 following 24-h treatment of 0.5 μM of staurosporine. n = 3. (i) Quantification of spheroid size of M14 Melanoma cells transiently transfected with empty vector pcDNA3.1, A 61603 wild-type Bcl-2, non-phosphorylatable S70A mutant Bcl-2 and phosphomimetic S70E mutant Bcl-2 following 24-h treatment of 0.5 μM of staurosporine. Spheroid size of staurosporine-treated cells normalized to that of untreated cells in (%). (j) Western Blot analysis showing S70pBcl-2, Bcl-2, Rac1 and β-actin following 1-h pre-treatment of 10 mM Tiron and subsequent 24-h treatment of 0.5 μM of staurosporine in pIRES and Rac1 (V12) M14 Melanoma cells. n = 4. Bar charts showing fold change in densitometry of S70pBcl-2 normalized to Bcl-2. (k) Western Blot analysis showing S70pBcl-2, Bcl-2, Rac1 and β-actin following 1-h pre-treatment of 1 μM ABT199 and subsequent 24-h treatment of 50 nM of staurosporine. n = 3. Bar charts showing fold change in densitometry of S70pBcl-2 normalized to Bcl-2. All bar chart displaying mean and SD. One way-ANOVA and Tukey's multiple comparisons tests were used. * and ** indicates P-value < 0.05 and 0.02 respectively.
    amplifies drug sensitivity of cancer cells by inhibiting S70pBcl-2. Col-lectively, these observations indicate that targeting the feedforward loop could be an excellent chemo-sensitizer particularly in Rac1-or S70pBcl-2-driven malignancies.
    Conflict of interest disclosures
    The following authors have affiliations with following organiza-tions, with direct or indirect financial interest. Shireen Vali and Taher Abbasi are affiliated with Cellworks Group, Inc., San Jose, CA, a pre-cision medicine company using simulation modelling and Ansu Kumar works for Cellworks Research India Limited, Bangalore, India, a fully owned subsidiary of Cellworks Group Inc. The other authors express no conflicts of interest.
    CRediT authorship contribution statement
    Stephen Jun Fei Chong: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project admin-istration. Jolin Xiao Hui Lai: Methodology, Validation, Formal ana-lysis, Investigation, Writing - original draft, Visualization. Jianhua Qu: Software, Validation, Formal analysis, Investigation, Visualization. Jayshree Hirpara: Investigation, Data curation, Writing - original draft. Jia Kang: Conceptualization, Methodology, Writing - original