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  • br draft Kunchithapadam Swaminathan Conceptualization

    2019-10-04

    
    draft. Kunchithapadam Swaminathan: Conceptualization,
    Methodology, Validation, Supervision, Resources, Writing - review & editing. Thomas Loh: Resources, Formal analysis, Investigation. Ansu Kumar: Software, Formal analysis, Visualization. Shireen Vali: Software, Formal analysis, Visualization. Taher Abbasi: Software, Formal analysis, Visualization. Shazib Pervaiz: Conceptualization, Methodology, Validation, Writing - original draft, Writing - review & editing, Resources, Supervision, Project administration, Funding ac-quisition.
    Acknowledgments
    This work was supported by the National Medical Research Council of Singapore (NMRC/CIRG/1433/2015) and Ministry of Education Tier 2 of Singapore (MOE2013-T2-2-130). We thank Marie V. Clement (NUS) for providing us the human M14 melanoma pIRES and Rac1V12 cell lines. We thank Roberta A. Gottlieb (Cedars-Sinai) for providing us the CEM/Neo and CEM/Bcl-2 cell lines. We thank David M. Virshup (DUKE-NUS) for providing us the Rabbit polyconal anti-B56δ (UT98) antibody. We thank Elizabeth Yang (Vanderbilt University) for providing us the pcDNA3.1 vector plasmid. We thank Askhaya Krishnagopal for providing technical support to this study.
    (caption on next page)
    Fig. 7. Rac1 and S70pBcl-2 Methylpiperidino pyrazole as well as S70pBcl-2 and Rac1/Bcl-2 interaction are positively correlated and implicated in more advanced stage lymphomas. (a) Western Blot analysis showing S70pBcl-2, Bcl-2, Rac1 and GAPDH of 34 patient lymphoma samples. Jurkat cell lysate is used as a control. n = 1. A more detailed clinical diagnosis of these lymphoma samples is displayed in Supplementary Fig. S14. (b) Scatter plot showing the positive correlation of protein band densities (arbitrary unit) normalized to their respective control proteins from Jurkat lysate of Rac1 and S70pBcl-2 of 34 patient lymphoma samples as well as their respective Pearson correlation coefficient, R, and P values. P-value is calculated using ANOVA. Raw values are displayed in Supplementary Fig. S14. (c) Western blot analysis showing the immunoprecipitation of Bcl-2 and immunoblotting of Rac1 of 6 lymphoma patient samples. Input is displayed in Fig. 7a labelled in red patient numbers. n = 1. (d) Scatter plot showing the positive correlation of protein band densities (arbitrary unit) normalized to their respective control proteins from Jurkat lysate of S70pBcl-2 against density of Rac1 from immunoprecipitated Bcl-2 of 6 patient lymphoma samples as well as their respective Pearson correlation coefficient, R, and P-values. P-value is calculated using ANOVA. Raw values are displayed in Supplementary Figs. S14 and S15. (e–h) Box plots showing the correlations of S70pBcl-2, Bcl-2, Rac1 and Rac1/Bcl-2 Binding to tumor stages of lymphoma patient samples. Raw values are displayed in Supplementary Figs. S14 and S15. Box plot displaying median, inter-quartiles and whiskers in horizontal lines and mean in “x”. P-value is calculated using paired T test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Fig. 8. Diagram depicting the crosstalk between active Rac1 and S70pBcl-2.
    (1) Active Rac1 binds to Bcl-2, thereby permitting Rac1 to be in close proximity of Bcl-2-bound B56δ for (2) NOX/O2.-- and ONOO−-mediated nitration and (3) inhibition of PP2A assembly. (4) This reciprocally induces the accumulation of kinase (JNK)-induced S70pBcl-2, which in turn (5) further secures the binding between Bcl-2 and active Rac1, resulting to a positive feedforward loop. This loop potentially sustains S70pBcl-2 and thereby (6) inhibits cell death.
    Appendix A. Supplementary Methylpiperidino pyrazole data
    References
    T. Uruno, DOCK1 inhibition suppresses cancer cell invasion and macropinocytosis induced by self-activating Rac1(P29S) mutation, Biochem. Biophys. Res. Commun. 497 (2018) 298–304.