Mdm2

Median overall survival of mice (n = 5 mice in vehicle and PYR-41; n = 4 mice in NSC group) inoculated with IOWA-1T xenografts were increased to 43 ± 0.5 and 39 ± 2 days with PYR-41 (PYR) and NSC-207895 (NSC), respectively, compared with a vehicle-treated control group 33 ± 1 days (*p < 0.05).

Nutlin-3a preserved p53 expression without influencing high glucose (HG)-induced podocyte impairment. A-D: cultured podocytes were pre-treated by nutlin-3a for 2 hrs before subjected to HG treatment. Western blotting gel documents (A) and summarized data (B) showing the expression of p53 and MDM2 in podocytes under HG exposure for 24 hrs. n = 4. Western blotting gel documents (C) and summarized data (D) showing the expression of Desmin in podocytes under HG exposure for 24 hrs. n = 3. *P < 0.05 vs. Ctrl, #P < 0.05 vs. Vehl + HG. Ctrl: control; Vehl: vehicle; nutlin-3a: nutlin-3a treatment.

(C) SACi2 effects are dependent on MT-kinetochore attachments. The graph shows percentage of cells undergoing forced mitotic exit by SACi2 or DMSO in various pretreatment conditions (mean ± SEM, three independent assays). Nocodazole (Noc) was used at 70 nM and 3 μM, vinblastine (Vbl) at 1 μM, taxol (Tx) at 600 nM and monastrol (Mon) at 100 μM concentrations and were added 8 h before addition of DMSO or SACi2. MG132 (20 μM) was added 1 h before nocodazole.

Spironolactone (sp.) and triptolide inhibit NER in myeloma cells. RPMI8226 cells were incubated with dimethyl sulfoxide (DMSO), spironolactone (10 μm) or triptolide (1 μm) for 6 h before NER evaluation. The figure represents the persistence of DNA-damage signal 150 min after exposure to UV (AFU: arbitrary fluorescent unit). Figure 4a shows representative merged pictures of DAPI and DDB2 proteo-probe signal (b).

 G. To evaluate effects of IKBKE/TBK1 inhibition on NF-κB signaling in Ewing, TC32 cells were incubated with CYT387 for six hours prior to stimulation with TNF-α (30 ng/mL). IκBα degradation was measured by harvesting TC32 cells thirty minutes after stimulation with TNF-α. TNF-α stimulation resulted in degradation of IκBα, and this effect was attenuated with CYT387 treatment. Parthenolide, an inhibitor of IκBα phosphorylation was used as a positive control. Similar effects of CYT387 activity were seen in HEK-293T cells which also express IKBKΕ. Nuclear extracts were prepared from TC32 cells harvested following forty-five minutes of TNF-α stimulation. Treatment with CYT387 resulted in decreased nuclear localization of NF-κB family proteins RelA/p65 and c-Rel. There was a modest impairment of p50 nuclear localization as compared to parthenolide and DMSO controls and no change in p52 nuclear localization. RelB (not shown) is not expressed in TC32 cells

Ubiquitination of p53 in vivo by BIRC6 in the presence of Nutlin-3. Cells were treated with Nutlin-3 (20 uM, 24 h) before harvest. Cell lysates were immunoprecipitated with anti-p53 antibody and immunoblotted by anti-ubiquitin antibody.

Dissociative effect of Nutlin-3a, Nutlin-3b, and RO-5963 on the preformed p53·MDM2 complex. Representative electrophoresis polyacrylamide gels in nondenaturing conditions for the analysis of the dissociation power of the inhibitors tested. (A) The reaction mixture containing 25 M p53·MDM2 complex, treated in the absence (lane 3) or in the presence (lanes 4-14) of 0.040, 0.060, 0.080, 0.12, 0.33, 0.67, 1.33, 3.33, 6.67, 33.33, and 333.33 μM Nutlin-3a, respectively, were run on a 12% nondenaturing polyacrylamide gel. Thirty micromolar MDM2 and p53 was run alone in lanes 1 and 2, respectively. (B) As in (A), but using Nutlin-3b, at concentration of (lanes 4-14) of 1.33, 6.67, 13.33, 20.00, 66.67, 133.33, 266.67, 333.33, 400.00, 833.33, and 1666.67 μM. p53 and MDM2 (25 μM both) were run alone in lanes 1 and 2, respectively. (C) As in (A), but using RO-5963, at concentration of 0.10, 0.21, 0.42, 0.83, 1.67, 3.33, 8.33, 16.67, 33.33, 166.67, and 333.33 μM (lanes 4-14), respectively. (D) The percentage of the residual p53·MDM2 complex was reported in a semilogarithmic plot, at the corresponding concentration of Nutlin-3a (empty circles), RO-5963 (triangles), and Nutlin-3b (filled circles), respectively. (E) The percentage of MDM2 dissociated from the complex was reported as a function of the inhibitor concentration. Curve fitting was done using the hyperbolic function. Symbols as in (D).

A panel of four NB cell lines were seeded in six-well plates with indicated concentrations of SAR405838 and agar, and grown for 2 to 3 weeks. Cells were stained with crystal violet for 4 hours. Colonies were counted and colony numbers were represented as % vehicle ± S.D. with P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) (Student's t-test, two-tailed) as indicated.

Median overall survival of mice (n = 5 mice in vehicle and PYR-41; n = 4 mice in NSC group) inoculated with IOWA-1T xenografts were increased to 43 ± 0.5 and 39 ± 2 days with PYR-41 (PYR) and NSC-207895 (NSC), respectively, compared with a vehicle-treated control group 33 ± 1 days (*p < 0.05).

Nutlin-3a preserved p53 expression without influencing high glucose (HG)-induced podocyte impairment. A-D: cultured podocytes were pre-treated by nutlin-3a for 2 hrs before subjected to HG treatment. Western blotting gel documents (A) and summarized data (B) showing the expression of p53 and MDM2 in podocytes under HG exposure for 24 hrs. n = 4. Western blotting gel documents (C) and summarized data (D) showing the expression of Desmin in podocytes under HG exposure for 24 hrs. n = 3. *P < 0.05 vs. Ctrl, #P < 0.05 vs. Vehl + HG. Ctrl: control; Vehl: vehicle; nutlin-3a: nutlin-3a treatment.

(C) SACi2 effects are dependent on MT-kinetochore attachments. The graph shows percentage of cells undergoing forced mitotic exit by SACi2 or DMSO in various pretreatment conditions (mean ± SEM, three independent assays). Nocodazole (Noc) was used at 70 nM and 3 μM, vinblastine (Vbl) at 1 μM, taxol (Tx) at 600 nM and monastrol (Mon) at 100 μM concentrations and were added 8 h before addition of DMSO or SACi2. MG132 (20 μM) was added 1 h before nocodazole.

Spironolactone (sp.) and triptolide inhibit NER in myeloma cells. RPMI8226 cells were incubated with dimethyl sulfoxide (DMSO), spironolactone (10 μm) or triptolide (1 μm) for 6 h before NER evaluation. The figure represents the persistence of DNA-damage signal 150 min after exposure to UV (AFU: arbitrary fluorescent unit). Figure 4a shows representative merged pictures of DAPI and DDB2 proteo-probe signal (b).

 G. To evaluate effects of IKBKE/TBK1 inhibition on NF-κB signaling in Ewing, TC32 cells were incubated with CYT387 for six hours prior to stimulation with TNF-α (30 ng/mL). IκBα degradation was measured by harvesting TC32 cells thirty minutes after stimulation with TNF-α. TNF-α stimulation resulted in degradation of IκBα, and this effect was attenuated with CYT387 treatment. Parthenolide, an inhibitor of IκBα phosphorylation was used as a positive control. Similar effects of CYT387 activity were seen in HEK-293T cells which also express IKBKΕ. Nuclear extracts were prepared from TC32 cells harvested following forty-five minutes of TNF-α stimulation. Treatment with CYT387 resulted in decreased nuclear localization of NF-κB family proteins RelA/p65 and c-Rel. There was a modest impairment of p50 nuclear localization as compared to parthenolide and DMSO controls and no change in p52 nuclear localization. RelB (not shown) is not expressed in TC32 cells

Ubiquitination of p53 in vivo by BIRC6 in the presence of Nutlin-3. Cells were treated with Nutlin-3 (20 uM, 24 h) before harvest. Cell lysates were immunoprecipitated with anti-p53 antibody and immunoblotted by anti-ubiquitin antibody.

Dissociative effect of Nutlin-3a, Nutlin-3b, and RO-5963 on the preformed p53·MDM2 complex. Representative electrophoresis polyacrylamide gels in nondenaturing conditions for the analysis of the dissociation power of the inhibitors tested. (A) The reaction mixture containing 25 M p53·MDM2 complex, treated in the absence (lane 3) or in the presence (lanes 4-14) of 0.040, 0.060, 0.080, 0.12, 0.33, 0.67, 1.33, 3.33, 6.67, 33.33, and 333.33 μM Nutlin-3a, respectively, were run on a 12% nondenaturing polyacrylamide gel. Thirty micromolar MDM2 and p53 was run alone in lanes 1 and 2, respectively. (B) As in (A), but using Nutlin-3b, at concentration of (lanes 4-14) of 1.33, 6.67, 13.33, 20.00, 66.67, 133.33, 266.67, 333.33, 400.00, 833.33, and 1666.67 μM. p53 and MDM2 (25 μM both) were run alone in lanes 1 and 2, respectively. (C) As in (A), but using RO-5963, at concentration of 0.10, 0.21, 0.42, 0.83, 1.67, 3.33, 8.33, 16.67, 33.33, 166.67, and 333.33 μM (lanes 4-14), respectively. (D) The percentage of the residual p53·MDM2 complex was reported in a semilogarithmic plot, at the corresponding concentration of Nutlin-3a (empty circles), RO-5963 (triangles), and Nutlin-3b (filled circles), respectively. (E) The percentage of MDM2 dissociated from the complex was reported as a function of the inhibitor concentration. Curve fitting was done using the hyperbolic function. Symbols as in (D).

A panel of four NB cell lines were seeded in six-well plates with indicated concentrations of SAR405838 and agar, and grown for 2 to 3 weeks. Cells were stained with crystal violet for 4 hours. Colonies were counted and colony numbers were represented as % vehicle ± S.D. with P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) (Student's t-test, two-tailed) as indicated.