Journal: Oxidative Medicine and Cellular Longevity

Article Title: Purple Corn Extract Prevents Doxo-Induced Cardiotoxicity by Counteracting AMPK Activation and p53 Acetylation in HL-1 and Primary Cardiomyocytes

doi: 10.1155/omcl/7786043

Figure Lengend Snippet: Doxo and RED effects on Topoisomerase 2β. (a) RADAR assay on HL-1 cells treated for 48 h with 0.5 μM Doxo with or without 125 µM RED. (b) Decatenation assay performed on cell extracts from HL-1 cells treated for 48 h with 0.25, 0.5, or 1 µM Doxo with or without 125 µM RED. One representative blot from three independent biological experiments is shown in Subparts (a, b). (c) Effect of Top2 silencing on DNA damage in HL-1 cells treated for 48 h with 0.5 µM Doxo with or without 125 µM RED analyzed by comet assay. Data are expressed as mean ± SEM from three independent biological experiments and were analyzed using two-way ANOVA followed by Sidak's multiple comparisons test. (d–h) qPCR analysis of the effects of Top2 silencing on p53 , Puma , p21 , Fgf21 , and Sirt1 gene expression in HL-1 cells treated for 48 h with 1 µM Doxo ± 125 µM RED. Data are expressed as mean ± SEM of three technical replicates per condition from two independent biological experiments and were analyzed using two-way ANOVA followed by Tukey's multiple comparisons test. ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, ⁣ ∗∗∗ p < 0.001, and ⁣ ∗∗∗∗ p < 0.0001 indicate significant differences versus 0 µM Doxo and between indicated pairs.

Article Snippet: The membrane was incubated with Top2β primary antibody (sc-365071, Santa Cruz, Dallas, Texas, USA, diluted 1:1000 in 1% BSA/TBST), H4 primary antibody (2935, Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000 in 1% BSA/TBST) as a loading control, and the anti-mouse secondary antibody (ab205719, Abcam, diluted 1:5000 in 1% BSA/TBST).

Techniques: Single Cell Gel Electrophoresis, Gene Expression

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 3 RG7112 interferes with the association of MDM2 with TOP2β and blocks TOP2β degradation. A, B RG7112 treatment dosage- dependently increased TOP2β levels. After incubating with VP-16 and RG7112 at different concentrations for 2 h, IB with the appropriate Abs was performed on H1299 and A549 cells. C, D RG7112 treatment prolonged the half-life of TOP2β protein after VP-16 treatment. H1299 and A549 cells were harvested at the indicated periods after administration of CHX and VP-16 or VP-16 and RG7112 (5 μM) and then subjected to IB with the appropriate Abs. E By administering RG7112, the physical interaction between MDM2 and TOP2β was inhibited. Following Flag- MDM2 transfection and 48 h of incubation with RG7112 or vehicle in HEK293 cells, IP with Flag beads and IB with the appropriate Abs were performed. F RG7112 treatment suppresses the polyubiquitination of TOP2β induced by VP-16 stimuli. HEK293 cells transfected with the specified plasmids were treated with VP-16 and MG132 (20 μM) or in association with RG7112 (5 μM) for 5 h, IP was then performed with anti- HA beads, and direct IB was performed with the selected antibodies. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the quantification results are shown. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: Ki-67 (9027, Cell Signaling Technology, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:400), TOP2β (20549-1-AP, Proteintech, 1:100).

Techniques: Transfection, Incubation

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 4 Inactivation of MDM2 increases the level of TOP2β-DNA cleavage complex. A, B MDM2 depletion occurs in a time-dependent manner, leading to an accumulation of TOP2βcc levels. H1299 cells were exposed to VP-16 for the durations specified, accompanied by fluorescence-activated cell sorting (FACS) tests to assess the levels of TOP2βcc. C, D The administration of RG7112 induced a time-dependent increase in TOP2βcc levels. TOP2βcc levels in H1299 cells were measured by FACS after administration of VP-16 monotherapy or in combination with RG7112. E–H Treatment with RG7112 or depletion of MDM2 augments the upregulation of TOP2βcc upon VP-16 exposure. VP-16 treatment was applied to H1299 cells (E, F) transfected with the specified sgRNA for 2 h. H1299 cells (G, H) with VP-16 or VP-16 and RG7112 (5 μM) for 2 h. The cells were then collected for the TARDIS assay. All experiments were independently repeated three times. Fluorescence quantification was performed with Image J, and the quantification results are shown. **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: Ki-67 (9027, Cell Signaling Technology, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:400), TOP2β (20549-1-AP, Proteintech, 1:100).

Techniques: FACS, Transfection, Fluorescence

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 5 MDM2 is involved in ICRF-193-induced TOP2β degradation. A, B Depletion of MDM2 extends the TOP2β protein’s half-life after ICRF- 193 treatment. Following the transfection of H1299 (A) and A549 (B) cells with sgRNA based on CRISPR‒Cas9-mediated knockout, they were treated with ICRF-193 and cycloheximide (CHX) for the specified time intervals. IB with the specified Abs was performed on the collected cells. C, D ICRF-193 treatment increased the interaction between TOP2β and MDM2. After 48 h of transfection, HEK293 cells were incubated with ICRF-193 (50 μM) and MG132 (20 μM) for 5 h, and then subjected to IP with Flag beads and IB with the indicated Abs. E, F MDM2 inactivation did not alter the basal level of TOP2βcc. After treatment of H1299 cells with ICRF-193 for the respective periods, the levels of TOP2βcc were determined by FACS. ns no significance. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the quantification results are shown. **p < 0.01; ***p < 0.001; ns no significant.

Article Snippet: The following antibodies were used: Ki-67 (9027, Cell Signaling Technology, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:400), TOP2β (20549-1-AP, Proteintech, 1:100).

Techniques: Transfection, Knock-Out, Incubation

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 8 Combination of RG7112 with VP-16 synergistically increased inhibition of tumor growth in BALB/c xenograft tumor model and in situ lung cancer mouse model. A–C H1299 cells were implanted subcutaneously into BALB/c nude mice. When the tumor volume reached approximately 90–110 mm3, four mice groups were established at random and injected with different drugs intravenously as described in Materials and Methods. A Representative images of each treatment group are shown. B A Vernier caliper was utilized to evaluate the changes in tumor volume. C After receiving treatment, the mice categorized into groups had their tumor weights compared. D–F Immunohistochemical analysis of Ki-67 and Cleaved-Caspase 3 was performed in tumor tissues under a bright field microscope. D Representative images of each treatment group are shown. The quantification of Ki-67 IHC score (E) and Cleaved-Caspase 3 IHC score (F) in each group was shown in the histogram. G–K An in-situ lung cancer mouse model was generated by percutaneous injection of H1299 cells into C57BL/6 mice’s right lung. Two weeks later, different drugs were administered intravenously to mice that were randomized into four groups. After a further two weeks, animals were euthanized and their lung tissue were collected and stained with H&E or the indicated antibodies (as detailed in the Materials and Methods section). G Representative photographs of H&E staining. H Representative images of Ki-67, Cleaved-Caspase 3, and TOP2β Immunohistochemical analysis. The quantification of Ki-67 IHC score (I), Cleaved-Caspase 3 IHC score (J), and TOP2β IHC score (K) in each group was shown in the histogram. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: Ki-67 (9027, Cell Signaling Technology, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:400), TOP2β (20549-1-AP, Proteintech, 1:100).

Techniques: Inhibition, In Situ, Injection, Immunohistochemical staining, Microscopy, Generated, Staining

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 9 Working model. A working model for MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53- independent manner.

Article Snippet: The following antibodies were used: Ki-67 (9027, Cell Signaling Technology, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:400), TOP2β (20549-1-AP, Proteintech, 1:100).

Techniques:

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 3 RG7112 interferes with the association of MDM2 with TOP2β and blocks TOP2β degradation. A, B RG7112 treatment dosage- dependently increased TOP2β levels. After incubating with VP-16 and RG7112 at different concentrations for 2 h, IB with the appropriate Abs was performed on H1299 and A549 cells. C, D RG7112 treatment prolonged the half-life of TOP2β protein after VP-16 treatment. H1299 and A549 cells were harvested at the indicated periods after administration of CHX and VP-16 or VP-16 and RG7112 (5 μM) and then subjected to IB with the appropriate Abs. E By administering RG7112, the physical interaction between MDM2 and TOP2β was inhibited. Following Flag- MDM2 transfection and 48 h of incubation with RG7112 or vehicle in HEK293 cells, IP with Flag beads and IB with the appropriate Abs were performed. F RG7112 treatment suppresses the polyubiquitination of TOP2β induced by VP-16 stimuli. HEK293 cells transfected with the specified plasmids were treated with VP-16 and MG132 (20 μM) or in association with RG7112 (5 μM) for 5 h, IP was then performed with anti- HA beads, and direct IB was performed with the selected antibodies. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the quantification results are shown. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies (Abs) were used: MDM2 (OP46, Calbiochem, 1:1000), MDM2 (86934 S, Cell Signaling Technology, 1:500), TOP2β (611493, BD Biosciences, 1:1000), TOP2β (sc-25330, Santa Cruz, 1:1000), TOP2α (12286, Cell Signaling Technology, 1:1000), p53 (2527 S, Cell Signaling Technology, 1:1000), p21 (2947 S, Cell Signaling Technology, 1:1000), p-CHK1 (2348, Cell Signaling Technology, 1:2000), CHK1 (sc-8408, Santa Cruz, 1:1000), ACTIN (A5441, Sigma, 1:10000), β-TrCP1 (4394, Cell Signaling Technology, 1:1000), FLAG (F1804, Sigma, 1:2000), γH2AX (05-636, Millipore, 1:1000), PARP (9542, Cell Signaling Technology, 1:1000), Caspase 3 (9665, Cell Signaling Technology, 1:1000), HA (11867423001; Roche; 1:2000).

Techniques: Transfection, Incubation

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 4 Inactivation of MDM2 increases the level of TOP2β-DNA cleavage complex. A, B MDM2 depletion occurs in a time-dependent manner, leading to an accumulation of TOP2βcc levels. H1299 cells were exposed to VP-16 for the durations specified, accompanied by fluorescence-activated cell sorting (FACS) tests to assess the levels of TOP2βcc. C, D The administration of RG7112 induced a time-dependent increase in TOP2βcc levels. TOP2βcc levels in H1299 cells were measured by FACS after administration of VP-16 monotherapy or in combination with RG7112. E–H Treatment with RG7112 or depletion of MDM2 augments the upregulation of TOP2βcc upon VP-16 exposure. VP-16 treatment was applied to H1299 cells (E, F) transfected with the specified sgRNA for 2 h. H1299 cells (G, H) with VP-16 or VP-16 and RG7112 (5 μM) for 2 h. The cells were then collected for the TARDIS assay. All experiments were independently repeated three times. Fluorescence quantification was performed with Image J, and the quantification results are shown. **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies (Abs) were used: MDM2 (OP46, Calbiochem, 1:1000), MDM2 (86934 S, Cell Signaling Technology, 1:500), TOP2β (611493, BD Biosciences, 1:1000), TOP2β (sc-25330, Santa Cruz, 1:1000), TOP2α (12286, Cell Signaling Technology, 1:1000), p53 (2527 S, Cell Signaling Technology, 1:1000), p21 (2947 S, Cell Signaling Technology, 1:1000), p-CHK1 (2348, Cell Signaling Technology, 1:2000), CHK1 (sc-8408, Santa Cruz, 1:1000), ACTIN (A5441, Sigma, 1:10000), β-TrCP1 (4394, Cell Signaling Technology, 1:1000), FLAG (F1804, Sigma, 1:2000), γH2AX (05-636, Millipore, 1:1000), PARP (9542, Cell Signaling Technology, 1:1000), Caspase 3 (9665, Cell Signaling Technology, 1:1000), HA (11867423001; Roche; 1:2000).

Techniques: FACS, Transfection, Fluorescence

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 5 MDM2 is involved in ICRF-193-induced TOP2β degradation. A, B Depletion of MDM2 extends the TOP2β protein’s half-life after ICRF- 193 treatment. Following the transfection of H1299 (A) and A549 (B) cells with sgRNA based on CRISPR‒Cas9-mediated knockout, they were treated with ICRF-193 and cycloheximide (CHX) for the specified time intervals. IB with the specified Abs was performed on the collected cells. C, D ICRF-193 treatment increased the interaction between TOP2β and MDM2. After 48 h of transfection, HEK293 cells were incubated with ICRF-193 (50 μM) and MG132 (20 μM) for 5 h, and then subjected to IP with Flag beads and IB with the indicated Abs. E, F MDM2 inactivation did not alter the basal level of TOP2βcc. After treatment of H1299 cells with ICRF-193 for the respective periods, the levels of TOP2βcc were determined by FACS. ns no significance. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the quantification results are shown. **p < 0.01; ***p < 0.001; ns no significant.

Article Snippet: The following antibodies (Abs) were used: MDM2 (OP46, Calbiochem, 1:1000), MDM2 (86934 S, Cell Signaling Technology, 1:500), TOP2β (611493, BD Biosciences, 1:1000), TOP2β (sc-25330, Santa Cruz, 1:1000), TOP2α (12286, Cell Signaling Technology, 1:1000), p53 (2527 S, Cell Signaling Technology, 1:1000), p21 (2947 S, Cell Signaling Technology, 1:1000), p-CHK1 (2348, Cell Signaling Technology, 1:2000), CHK1 (sc-8408, Santa Cruz, 1:1000), ACTIN (A5441, Sigma, 1:10000), β-TrCP1 (4394, Cell Signaling Technology, 1:1000), FLAG (F1804, Sigma, 1:2000), γH2AX (05-636, Millipore, 1:1000), PARP (9542, Cell Signaling Technology, 1:1000), Caspase 3 (9665, Cell Signaling Technology, 1:1000), HA (11867423001; Roche; 1:2000).

Techniques: Transfection, Knock-Out, Incubation

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 8 Combination of RG7112 with VP-16 synergistically increased inhibition of tumor growth in BALB/c xenograft tumor model and in situ lung cancer mouse model. A–C H1299 cells were implanted subcutaneously into BALB/c nude mice. When the tumor volume reached approximately 90–110 mm3, four mice groups were established at random and injected with different drugs intravenously as described in Materials and Methods. A Representative images of each treatment group are shown. B A Vernier caliper was utilized to evaluate the changes in tumor volume. C After receiving treatment, the mice categorized into groups had their tumor weights compared. D–F Immunohistochemical analysis of Ki-67 and Cleaved-Caspase 3 was performed in tumor tissues under a bright field microscope. D Representative images of each treatment group are shown. The quantification of Ki-67 IHC score (E) and Cleaved-Caspase 3 IHC score (F) in each group was shown in the histogram. G–K An in-situ lung cancer mouse model was generated by percutaneous injection of H1299 cells into C57BL/6 mice’s right lung. Two weeks later, different drugs were administered intravenously to mice that were randomized into four groups. After a further two weeks, animals were euthanized and their lung tissue were collected and stained with H&E or the indicated antibodies (as detailed in the Materials and Methods section). G Representative photographs of H&E staining. H Representative images of Ki-67, Cleaved-Caspase 3, and TOP2β Immunohistochemical analysis. The quantification of Ki-67 IHC score (I), Cleaved-Caspase 3 IHC score (J), and TOP2β IHC score (K) in each group was shown in the histogram. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies (Abs) were used: MDM2 (OP46, Calbiochem, 1:1000), MDM2 (86934 S, Cell Signaling Technology, 1:500), TOP2β (611493, BD Biosciences, 1:1000), TOP2β (sc-25330, Santa Cruz, 1:1000), TOP2α (12286, Cell Signaling Technology, 1:1000), p53 (2527 S, Cell Signaling Technology, 1:1000), p21 (2947 S, Cell Signaling Technology, 1:1000), p-CHK1 (2348, Cell Signaling Technology, 1:2000), CHK1 (sc-8408, Santa Cruz, 1:1000), ACTIN (A5441, Sigma, 1:10000), β-TrCP1 (4394, Cell Signaling Technology, 1:1000), FLAG (F1804, Sigma, 1:2000), γH2AX (05-636, Millipore, 1:1000), PARP (9542, Cell Signaling Technology, 1:1000), Caspase 3 (9665, Cell Signaling Technology, 1:1000), HA (11867423001; Roche; 1:2000).

Techniques: Inhibition, In Situ, Injection, Immunohistochemical staining, Microscopy, Generated, Staining

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 9 Working model. A working model for MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53- independent manner.

Article Snippet: The following antibodies (Abs) were used: MDM2 (OP46, Calbiochem, 1:1000), MDM2 (86934 S, Cell Signaling Technology, 1:500), TOP2β (611493, BD Biosciences, 1:1000), TOP2β (sc-25330, Santa Cruz, 1:1000), TOP2α (12286, Cell Signaling Technology, 1:1000), p53 (2527 S, Cell Signaling Technology, 1:1000), p21 (2947 S, Cell Signaling Technology, 1:1000), p-CHK1 (2348, Cell Signaling Technology, 1:2000), CHK1 (sc-8408, Santa Cruz, 1:1000), ACTIN (A5441, Sigma, 1:10000), β-TrCP1 (4394, Cell Signaling Technology, 1:1000), FLAG (F1804, Sigma, 1:2000), γH2AX (05-636, Millipore, 1:1000), PARP (9542, Cell Signaling Technology, 1:1000), Caspase 3 (9665, Cell Signaling Technology, 1:1000), HA (11867423001; Roche; 1:2000).

Techniques: