Figure 5 A 1 Alternate cell lines also show diminished activation of p53 when combined with CDK4/6 inhibitor.
A. Scheme denoting the treatment scheme.
B. U-2 OS (osteosarcoma) cells were exposed to DMSO, Nutlin, PD0332991 and the combination treatment. Cells were harvested for immunoblot analysis to check for the expression levels of MDM2, p53 and p21. In both cell lines, treatment of Nutlin with PD0332991 reduced the protein levels of p53 and its target genes p21 and MDM2 in comparison to Nutlin treatment alone. β-actin serves as loading control.
C. MCF10A (non-tumorigenic breast epithelial) cells were treated with DMSO, Nutlin and PD0332991 at different concentrations. The cells were harvested for immunoblot analysis to check for the expression of p53 target genes such as p21 and MDM2. β-actin serves as loading control.
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Figure 5 A 1 Various concentrations and time points of cells treated with PD0332991.
D. SJSA cells were treated with PD0332991 at varying time points as mentioned. Immunoblot analysis revealed diminished MDM2 expression even upon 6h combined treatment with Nutlin.
E. SJSA cells were treated with different concentrations of CDK4/6 inhibitor and the cell lysates were assessed for p53 target genes- MDM2 and p21 using immunoblot analysis. We noted decreased MDM2 levels even with 1µM PD0332991 in combination with Nutlin. β-actin serves as loading control.
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Figure 5 A 2 Phosphorylation of p53 at S20 and S46 upon p53 accumulation and CDK4/6 inhibition.
A. CRL3044 cells were treated with DMSO, Nutlin, NCS alone and in combination with PD0332991 for 6 hours. Immunoblots depicting p53 and its target gene expression shows attenuated response upon combined Nutlin/NCS treatment with PD0332991. pS20 p53, a site phosphorylated upon p53 activation remained unchanged upon Nutlin treatment alone or in the combination.
B. SJSA cells were treated with DMSO, NCS, Nutlin, PD0332991 with the combinations for 6 hours and the cell lysates were harvested for immunoblot analysis to check for p53 activation and stabilization. Again, there were no differences observed in the phosphorylation of p53 at S20 with Nutlin treatment alone or in combination with PD0332991 treatment.
C. Treatment scheme for MCF10A cells.
D. To investigate changes, if any in the pS46 p53 upon Nutlin/NCS treatment alone or in combination with PD0332991, cells were harvested for immunoblot analysis. Probing for pS46 p53 did not indicate strong differences, although we still noted the decreased activation of p53 response. β-actin serves as loading control.
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Figure 5 A 3 Treatment with proteasome inhibitor partly rescued MDM2 protein expression.
A. Scheme used for treatment protocol.
B. SJSA cells were probed for p53 and its target genes upon addition of MG132, an inhibitor of the proteasome to check whether the stability of the protein is affected. Immunoblot analysis revealed only partial rescue of MDM2 protein levels upon combined addition of Nutlin and PD0332991 with MG132 in comparison to Nutlin and PD0332991 treatment alone. β-actin serves as loading control.
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Figure 5 A 3 MDM2 stability and expression remains largely unaffected upon depletion of EZH2 or p73.
C. Treatment protocol.
D. SJSA cells were depleted of EZH2 using two different siRNAs. We observe very slight decrease in protein stability of MDM2 with siEZH2-2 but not siEZH2-1 upon Nutlin treatment.
E. SJSA cells were transfected with three siRNAs towards p73. Two siRNAs – 2 and sip73-3 decreased the expression of MDM2 in combination with Nutlin treatment. However, the p5sip73-3 and p21 levels rather increased with sip73-3. β-actin serves as loading control.
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Figure 5 A 3 MDM2 stability and expression remains largely unaffected upon depletion of CDK2.
F. Scheme exhibiting the treatment protocol.
G. Exhausting the cells of CDK2 utilizing two siRNAs in combination with Nutlin and PD0332991 treatment did not rescue MDM2 levels. β-actin serves as loading control.
Figure 5 A 3 MDM2 stability and expression remains largely unaffected upon depletion of MDMX, its dimerization partner.
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H. Removal of MDMX from SJSA cells with Nutlin and PD0332991 treatment did not rescue the MDM2 protein levels as observed with control knockdown and the combination treatment. β-actin serves as loading control.
I. Three different siRNAs to MDMX showed decreased expression upon its depletion using quantitative RT-PCRs. The siRNA to MDMX used in H was a pool of the three siRNAs shown in I. RPLP0 is used as housekeeping gene.
Figure 5 A 4 Combination of MDM2 antagonist with CDK4/6 inhibitor does not largely affect pS2 and pS5 RNA polymerase II at p53 target genes.
A. Protocol for treatment of SJSA cells with MDM2 antagonist and CDK4/6 inhibitor.
B. Chromatin immunoprecipitation carried out for pS2 RNA polymerase II with IgG as negative control. Targeted qPCRs were performed at the promoters of p53 target genes. The enrichment of pS2 RNA polymerase II (an indicator of transcriptional elongation) upon combined treatment of MDM2 and CDK4/6 inhibition in comparison to Nutlin alone was similar.
C. Chromatin immunoprecipitation conducted for pS5 RNA polymerase II with IgG as negative control. Targeted ChIP-qPCRs were performed at the promoters of p53 target genes. The enrichment of pS5 RNA polymerase II (represents initiation of transcription) upon combined inhibition of MDM2 and CDK4/6 in comparison to MDM2 inhibition alone was similar.
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Figure 5 A 5. CDK4 and MDM2 inhibition leads to slightly elevated enrichment of cyclin D1 at the promoters of p53 target genes.
A-D. Upon performing chromatin immunoprecipitation in SJSA cells after 6 hours treatment with MDM2 and CDK4/6 inhibitors, we observed increased occupancy of p53 at its target genes upon combined MDM2 and CDK4/6 inhibition in comparison to single treatment alone. Moreover, the combined MDM2 and CDK4/6 inhibition resulted in reduced MDM2 enrichment and RNA polymerase II occupancy at these sites. Precipitation with cyclin D1 led to signals with enrichment just above the negative control IgG. In case of MDM2 and PIG3 promoters, the enrichment was increased in comparison to Nutlin treatment alone. Cyclin D1 has been suggested to act as a transcriptional regulator. In this case, it is tempting to suggest the role of cyclin D1 as a transcriptional repressor of p53 target genes.
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