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  • br Concordance between Rac and S pBcl expression in clinical

    2019-10-03


    3.6. Concordance between Rac1 and S70pBcl-2 expression in clinical lymphomas
    3.7. Increased levels of Rac1 and S70pBcl-2 are associated with advanced stage lymphomas
    Based on the expression analyses of Rac1, Bcl-2 and S70pBcl-2 in 34 patient lymphoma samples, we proceeded to explore the crosstalk be-tween the two proteins in relation to disease stage/severity. Of note,
    (caption on next page)
    Fig. 4. Active Rac1-induced Rac1/Bcl-2 interaction is required for the inhibition of PP2A assembly in a ROS-dependent manner.
    (a) Western blot analysis showing T183/Y185pJNK, JNK, S70pBcl-2, Bcl-2 and β-actin of M14 Melanoma cells stably expressing the Myc-tagged constitutively active Rac1 mutant, V12, or empty vector, pIRES as well as following 24-h treatment of 10 mM Tiron in M14 Melanoma cells stably expressing the Myc-tagged con-stitutively active Rac1 mutant, V12. n = 3. (b) Western blot analysis showing S70pBcl-2, Bcl-2, Rac1 and β-actin following 6-h treatment of increasing doses of FeTPPS in M14 Melanoma cells stably expressing the Myc-tagged constitutively active Rac1 mutant, V12. n = 3. (c) Western blot analysis showing the im-munoprecipitation of B56δ subunit of PP2A and immunoblotting of catalytic subunit of PP2A and their respective immunoblotted inputs of B56δ, catalytic subunit and β-actin in M14 Melanoma cells stably expressing the Myc-tagged constitutively active Rac1 mutant, V12, or empty vector, pIRES. n = 3. (d) Western blot analysis showing the immunoprecipitation of B56δ subunit of PP2A and immunoblotting of catalytic subunit of PP2A and Bcl-2 and their respective immunoblotted inputs of B56δ, catalytic subunit, β-actin, S70pBcl-2, Bcl-2 and β-actin following 48-h B56δ subunit knockdown (150 nM) in M14 Melanoma cells stably expressing the Myc-tagged constitutively active Rac1 mutant, V12. n = 3. (e) PLA showing red dot appearances (white arrow) upon interaction between B56δ and catalytic subunits of PP2A in pIRES and Rac1 V12 M14 Melanoma cells. Red dots were calculated from 30 cells from randomly selected images. Blank images indicate only secondary FLAG tag Peptide used to check for non-specific signal. n = 3. (f) Quantification of total red dots from 30 cells from randomly selected images for target and blank samples of 3 independent sets. Bar chart displaying mean and SD. One way-ANOVA and Tukey's multiple comparisons tests were used. ** indicates P-value < 0.02. Quantitated blank values in Fig. 4f is similar to that of Fig. 2c-d as both Rac1/Bcl-2 and B56δ/Catalytic of pIRES and Rac1 V12 M14 Melanoma samples were stained and concurrently compared to the similar secondary antibody blanks. (g) Western blot analysis showing the immunoprecipitation of B56δ subunit of PP2A and immunoblotting of catalytic subunit of PP2A and Bcl-2 and their respective immunoblotted inputs of B56δ, catalytic subunit, S70pBcl-2, Bcl-2 and β-actin following 24-h treatment of 1 μM ABT199 in CEM/Bcl-2 cells. n = 3. (h) Western blot analysis showing the immunoprecipitation of B56δ subunit of PP2A and immunoblotting of catalytic subunit of PP2A and Bcl-2 and their respective immunoblotted inputs of B56δ, catalytic subunit, S70pBcl-2, Bcl-2 and β-actin following 24-h treatment of 10 mM Tiron in M14 Melanoma cells stably expressing the Myc-tagged constitutively active Rac1 mutant, V12. n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) r> increased protein levels of Rac1 and S70pBcl-2 (and Bcl-2) strongly correlated with advanced stage of the disease (stage 3–4) in clinical lymphomas (Fig. 7e–g, S14); protein levels were significantly higher compared to lysates from stage 0–2 lymphomas (S70pBcl-2: p-value = 0.011; Bcl-2: p-value = 0.024; Rac1: p-value = 0.037). A similar positive correlation was observed between disease severity and Rac1/Bcl-2 in-teraction (Fig. 7h, S14-S15); however, statistical significance could not be determined due to the small sample size FLAG tag Peptide (n = 6). Furthermore, Rac1 relative mRNA level positively correlates with advanced disease stage in malignant melanoma from TCGA database (Fig. S16). Collectively, our findings strongly support the in vivo existence of a positive feed-forward loop between active Rac1 and S70pBcl-2 in an interactive and redox-dependent manner to sustain S70pBcl-2 for cancer cell survival and progression (Fig. 8).