Fig. 4: O2.− induces Mcl-1 stability through post-translational modification of phosphorylation. | Leukemia

Fig. 4: O2.− induces Mcl-1 stability through post-translational modification of phosphorylation.

From: Superoxide-mediated phosphorylation and stabilization of Mcl-1 by AKT underlie venetoclax resistance in hematologic malignancies

Fig. 4

Western blot showing Mcl-1 and tubulin levels in A RPMI8226, B MOLM14 cells following pre-treatment with DDC (100 µM) for 120 min followed by cycloheximide (CHX) 25 µg/ml, for the indicated timepoints (in minutes). Right panels: Graph plotting the densitometric analysis of RPMI8226 or MOLM14 cells treated with DDC and CHX or CHX alone. Fold change in densitometry values (Mcl-1/Tubulin) and were normalized to untreated control at 0 min of the experiment. Densitometric values were obtained using ImageJ. N = 3. C, D Western blot showing Mcl-1 and GAPDH levels in MOLM14, VEN-R MOLM14, OCI-AML2, VEN-R OCI-AML2 following treatment of cycloheximide (CHX) (25 µg/ml) for the indicated time points. Bottom panels: Graph plotting the densitometric analysis of MOLM14, VEN-R MOLM14, OCI-AML2, and VEN-R OCI-AML2 cells. Densitometric values were obtained using ImageJ. N = 3. E, F Co-Immunoprecipitation of Mcl-1 and immunoblots of Lys48 (K48) and Mcl-1 in RPMI8226 or MOLM14 cells pre-treated with MG132 (5 µM) for 1 h, followed by DDC (100 µM) for 4 h. Input showing levels of Mcl-1 and β-Actin. N = 2. G Western blot showing T163pMcl-1, S159pMcl-1, Mcl-1, Tubulin levels in MOLM14 cells after treatment with indicated doses of DDC (µM) for 4 h. N = 3. H Western blot showing T163pMcl-1, S159pMcl-1, Mcl-1, SOD1, Tubulin levels in MOLM14 cells after siRNA-mediated knockdown of SOD1 (100 nM) for 48 h. N = 3. I Western blot showing T163pMcl-1, Mcl-1, β-Actin levels in VEN-R MOLM14 or VEN-R OCI-Ly1 following treatment with O2.− scavenger, Tiron (1 mM/VEN-R MOLM14, 2.5 mM/VEN-R OCI-Ly1) for 2 h followed by VEN (0.05μM/VEN-R MOLM14, 1μM/VEN-R OCI-Ly1) for 48 h. N = 3. J Western blot showing T163pMcl-1, Mcl-1, β-Actin levels of CLL patient samples after ex vivo treatment with DDC (50 µM) for 4 h. Densitometric analyses of T163pMcl-1/β-Actin levels in CLL patient samples treated with DDC (50 µM) for 4 h, normalized to non-treated control. N = 6. Mcl-1 and β-Actin are from the same set in Fig. 2M. Paired t-test was used. K Western blot showing T163pMcl-1, Mcl-1, and SOD1 levels in 13 lymphoma patient samples. Densitometric analysis and Pearson correlation analysis of T163pMcl-1 vs SOD1 protein levels was performed from 13 lymphoma patient samples. Raw densitometric values obtained from ImageJ were used for plotting the graph. Mcl-1 and SOD1 are from the same set in Fig. 2E. N = 13.

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