Journal: Scientific Reports

Article Title: MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma

doi: 10.1038/s41598-021-82542-4

Figure Lengend Snippet: MDM4 expression is significantly elevated in HB patient samples and is correlated with increased expression of p53 target genes. ( a ) Dot plot representing expression profiles of MDM4 and MDM2 and normalized averages of the mRNA expression profiles of 22 select p53 target genes ( AEN , ALDH4A1 , CDKN1A , DDB2 , DUSP1 , EDA2R , ESR1 , FAS , FDXR , GADD45A , GADD45B , PANK1 , PERP , PTCHD4 , RPRM , RPS27L , RRM2B , SPATA18 , TRIAP1 , TRIM22 , WRAP53 , ZMAT3 ), together with the optimal linear fit. ( b ) Dot plot representing expression of MDM4 and MDM2 , together with the optimal linear fit. ( c ) Bar graph representing normalized mRNA expression of MDM2 and MDM4 analyzed with qPCR experiments with 18 patient samples in comparison to four adjacent uninvolved liver samples. Y-axis shown with a log2 scale. Error bars represent SD. Data shown in b are representative of at least three independent experiments performed with three replicate wells each time. Student’s t test (two tailed) * P < 0.05, ** P < 0.01, *** P < 0.001 representing comparison of each sample’s expression data to all expression data of uninvolved liver samples.

Article Snippet: Membranes were then incubated at 4 °C overnight with rocking with primary antibodies for MDM4 (1:1000, NBP1-28,862, Novus Biologicals, Littleton, CO, USA (Supplementary Fig. c) or 1:2000, 04–1555 (clone 8C6), Millipore Sigma, Danvers, MA, USA (Figs. c,d, c, a, Supplementary Fig. )), PARP (1:1000, 9532, Cell Signaling Technology, Danvers, MA, USA), Caspase-3 (1:500, 14,220, Cell Signaling), MDM2 (1:500, sc-965, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p53 (1:1000, sc-126, Santa Cruz Biotechnology, Inc.), phospho-p53 (serine 15, 1:1000, 9286, Cell Signaling), Bax (1:1000, sc-493, Santa Cruz Biotechnology), Puma (1:500, D30C10, Cell Signaling), p21 (1:1000, SX118, Santa Cruz Biotechnology), and β-Actin (1:30,000, A2228, Sigma-Aldrich).

Techniques: Expressing, Comparison, Two Tailed Test

Journal: Scientific Reports

Article Title: MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma

doi: 10.1038/s41598-021-82542-4

Figure Lengend Snippet: Acute MDM4 inhibition with ATSP-7041 and NSC207895 lead to upregulation of p53 activity and downstream signaling. ( a ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells treated with 10 μM ATSP-7041 for 24 h was compared to that from untreated cells (0 h). Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Student’s t test (two tailed) * P < 0.05, ** P < 0.01, *** P < 0.001. ( b ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells after treatment with 10 μM NSC207895 for 4 or 8 h was compared to that from untreated cells (0 h). Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Student’s t test (two tailed) * P < 0.05, ** P < 0.01, *** P < 0.001. ( c ) Protein lysis from cells treated with 10 μM ATSP-7041 for 24 h were compared to untreated cells (0 h). Immunoblotting was done with the indicated antibodies, including MDM4 (04–1555, Millipore). β-Actin immunoblotting was used as a loading control. Data shown are representative of at least three independent experiments. ( d ) Protein lysis from cells treated with 10 μM NSC207895 for 4 or 8 h were compared to untreated cells (0 h). Immunoblotting was done with the indicated antibodies, including MDM4 (04–1555, Millipore). β-Actin immunoblotting was used as a loading control. Data shown are representative of at least three independent experiments. Full length blots for data shown in c and d are presented in Supplementary Fig. .

Article Snippet: Membranes were then incubated at 4 °C overnight with rocking with primary antibodies for MDM4 (1:1000, NBP1-28,862, Novus Biologicals, Littleton, CO, USA (Supplementary Fig. c) or 1:2000, 04–1555 (clone 8C6), Millipore Sigma, Danvers, MA, USA (Figs. c,d, c, a, Supplementary Fig. )), PARP (1:1000, 9532, Cell Signaling Technology, Danvers, MA, USA), Caspase-3 (1:500, 14,220, Cell Signaling), MDM2 (1:500, sc-965, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p53 (1:1000, sc-126, Santa Cruz Biotechnology, Inc.), phospho-p53 (serine 15, 1:1000, 9286, Cell Signaling), Bax (1:1000, sc-493, Santa Cruz Biotechnology), Puma (1:500, D30C10, Cell Signaling), p21 (1:1000, SX118, Santa Cruz Biotechnology), and β-Actin (1:30,000, A2228, Sigma-Aldrich).

Techniques: Inhibition, Activity Assay, Expressing, Two Tailed Test, Lysis, Western Blot, Control

Journal: Scientific Reports

Article Title: MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma

doi: 10.1038/s41598-021-82542-4

Figure Lengend Snippet: Inhibition of MDM4 leads to decreases in proliferation. ( a ) HepG2 and HepT1 cells were exposed to ATSP-7041 for 120 h and MTT assays were done at 24 h intervals to assess cell number. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Error bars represent SD. Student’s t test * P < 0.05, ** P < 0.01, *** P < 0.001. ( b ) HepG2 and HepT1 cells were exposed to NSC207895 for 96 or 120 h and MTT assays were done at 24 h intervals to assess cell number. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Error bars represent SD. Student’s t test * P < 0.05, ** P < 0.01, *** P < 0.001. ( c , e ) Cells were maintained in anchorage-independent conditions on a soft agar base with ATSP-7041 or vehicle for 2 to 3 weeks before staining with 500 μl of MTT solution for 4 h to visualize colonies. Images were captured by a VersaDoc Imaging System and colonies were counted with Quantity One software. Bar graphs in c represent colony numbers in indicated conditions. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Error bars represent SD. Student’s t test * P < 0.05, ** P < 0.01, *** P < 0.001. ( d , f ) Cells were maintained in anchorage-independent conditions on a soft agar base with NSC207895 or vehicle for 2 to 3 weeks before staining with 500 μl of MTT solution for 4 h to visualize colonies. Images were captured by a VersaDoc Imaging System and colonies were counted with Quantity One software. Bar graphs in d represent colony numbers in indicated conditions. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Error bars represent SD. Student’s t test * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Membranes were then incubated at 4 °C overnight with rocking with primary antibodies for MDM4 (1:1000, NBP1-28,862, Novus Biologicals, Littleton, CO, USA (Supplementary Fig. c) or 1:2000, 04–1555 (clone 8C6), Millipore Sigma, Danvers, MA, USA (Figs. c,d, c, a, Supplementary Fig. )), PARP (1:1000, 9532, Cell Signaling Technology, Danvers, MA, USA), Caspase-3 (1:500, 14,220, Cell Signaling), MDM2 (1:500, sc-965, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p53 (1:1000, sc-126, Santa Cruz Biotechnology, Inc.), phospho-p53 (serine 15, 1:1000, 9286, Cell Signaling), Bax (1:1000, sc-493, Santa Cruz Biotechnology), Puma (1:500, D30C10, Cell Signaling), p21 (1:1000, SX118, Santa Cruz Biotechnology), and β-Actin (1:30,000, A2228, Sigma-Aldrich).

Techniques: Inhibition, Staining, Imaging, Software

Journal: Scientific Reports

Article Title: MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma

doi: 10.1038/s41598-021-82542-4

Figure Lengend Snippet: Prolonged MDM4 inhibition with ATSP-7041 and NSC207895 lead to upregulation of p53 activity and downstream signaling. ( a ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells treated with the indicated concentrations of ATSP-7041 for 48 h was compared to that from untreated cells (0 h). Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Student’s t test (two tailed) * P < 0.05, ** P < 0.01, *** P < 0.001. ( b ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells treated with the indicated concentrations of NSC207895 for 48 h was compared to that from untreated cells (0 h). Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Student’s t test (two tailed) * P < 0.05, ** P < 0.01, *** P < 0.001. ( c ) Protein lysis from cells treated with 0.5 μM ATSP-7041 or NSC207895 for 48 h were compared to untreated, control cells (0 h). Immunoblotting was done with the indicated antibodies, including MDM4 (04–1555, Millipore). β-Actin immunoblotting was used as a loading control. Data shown are representative of at least three independent experiments. Full length blots are presented in Supplementary Fig. .

Article Snippet: Membranes were then incubated at 4 °C overnight with rocking with primary antibodies for MDM4 (1:1000, NBP1-28,862, Novus Biologicals, Littleton, CO, USA (Supplementary Fig. c) or 1:2000, 04–1555 (clone 8C6), Millipore Sigma, Danvers, MA, USA (Figs. c,d, c, a, Supplementary Fig. )), PARP (1:1000, 9532, Cell Signaling Technology, Danvers, MA, USA), Caspase-3 (1:500, 14,220, Cell Signaling), MDM2 (1:500, sc-965, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p53 (1:1000, sc-126, Santa Cruz Biotechnology, Inc.), phospho-p53 (serine 15, 1:1000, 9286, Cell Signaling), Bax (1:1000, sc-493, Santa Cruz Biotechnology), Puma (1:500, D30C10, Cell Signaling), p21 (1:1000, SX118, Santa Cruz Biotechnology), and β-Actin (1:30,000, A2228, Sigma-Aldrich).

Techniques: Inhibition, Activity Assay, Expressing, Two Tailed Test, Lysis, Control, Western Blot

Journal: Scientific Reports

Article Title: MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma

doi: 10.1038/s41598-021-82542-4

Figure Lengend Snippet: Cytotoxic effects of NSC207895 are dependent on MDM4. ( a ) Immunoblotting with HepG2 and HepT1 cells transduced with MDM4 cDNA or vector control (vc) with an antibody recognizing MDM4 (04–1555, Millipore). β-Actin immunoblotting was used as a loading control. Data shown are representative of at least three independent experiments. Full length blots for data shown in a are presented in Supplementary Fig. . ( b ) Bar graphs representing normalized mRNA expression of MDM4 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells transduced with sh-MDM4-35 or sh-MDM4-37 was compared to that transduced with sh-GFP . ( c ) Cells transduced with MDM4 cDNA or vc were incubated with varying concentrations of NSC207895 for 48 h. MTT assays were performed at 48 h to asses viability. ( d ) Cells transduced with sh-MDM4 or control sh-GFP were grown for 120 h and MTT assays were done at 24 h intervals to assess cell number. Data shown in b, c, and d are representative of at least three independent experiments performed with three replicate wells each time. Error bars represent SD. Student’s t test * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Membranes were then incubated at 4 °C overnight with rocking with primary antibodies for MDM4 (1:1000, NBP1-28,862, Novus Biologicals, Littleton, CO, USA (Supplementary Fig. c) or 1:2000, 04–1555 (clone 8C6), Millipore Sigma, Danvers, MA, USA (Figs. c,d, c, a, Supplementary Fig. )), PARP (1:1000, 9532, Cell Signaling Technology, Danvers, MA, USA), Caspase-3 (1:500, 14,220, Cell Signaling), MDM2 (1:500, sc-965, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p53 (1:1000, sc-126, Santa Cruz Biotechnology, Inc.), phospho-p53 (serine 15, 1:1000, 9286, Cell Signaling), Bax (1:1000, sc-493, Santa Cruz Biotechnology), Puma (1:500, D30C10, Cell Signaling), p21 (1:1000, SX118, Santa Cruz Biotechnology), and β-Actin (1:30,000, A2228, Sigma-Aldrich).

Techniques: Western Blot, Transduction, Plasmid Preparation, Control, Expressing, Incubation

Journal: Scientific Reports

Article Title: MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma

doi: 10.1038/s41598-021-82542-4

Figure Lengend Snippet: In vivo efficacy of MDM4 inhibition with an orthotopic xenograft murine model of HB. ( a ) Timeline of the animal study. ( b ) BLI and MRI images at early and late time points show progression of the HepT1-derived tumors in the placebo animals and animals treated with NSC207895. The cell line was stably transduced with luciferase to allow the use of BLI to monitor tumor growth in living animals. ( c ) Tumor weights of the placebo and NSC207895-treated animals at time of euthanasia. Kruskal Wallis test used to show significance. ( d ) Quantification of TUNEL staining of the tumors from the placebo and treatment animals. TUNEL-positive areas from images taken on a Keyence BZ-X710 All-in-One Fluorescence Microscope were quantified using the Keyence Hybrid Cell Count Analysis Application. Error bars represent SD. Student’s t test * P < 0.05, ** P < 0.01, *** P < 0.001. ( e ) Representative pictures of H&E and TUNEL staining quantified in d. Scale bars represent 50 μm in 10X and 40X images.

Article Snippet: Membranes were then incubated at 4 °C overnight with rocking with primary antibodies for MDM4 (1:1000, NBP1-28,862, Novus Biologicals, Littleton, CO, USA (Supplementary Fig. c) or 1:2000, 04–1555 (clone 8C6), Millipore Sigma, Danvers, MA, USA (Figs. c,d, c, a, Supplementary Fig. )), PARP (1:1000, 9532, Cell Signaling Technology, Danvers, MA, USA), Caspase-3 (1:500, 14,220, Cell Signaling), MDM2 (1:500, sc-965, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p53 (1:1000, sc-126, Santa Cruz Biotechnology, Inc.), phospho-p53 (serine 15, 1:1000, 9286, Cell Signaling), Bax (1:1000, sc-493, Santa Cruz Biotechnology), Puma (1:500, D30C10, Cell Signaling), p21 (1:1000, SX118, Santa Cruz Biotechnology), and β-Actin (1:30,000, A2228, Sigma-Aldrich).

Techniques: In Vivo, Inhibition, Derivative Assay, Stable Transfection, Transduction, Luciferase, TUNEL Assay, Staining, Fluorescence, Microscopy, Cell Counting

Journal: Scientific Reports

Article Title: MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma

doi: 10.1038/s41598-021-82542-4

Figure Lengend Snippet: MDM4 inhibition with ATSP-7041 and NSC207895 lead to apoptosis of HB cell lines. ( a ) HepG2, Huh-6, HepT1, and B6-2 cells were exposed to varying indicated concentrations of ATSP-7041 for 48 h. MTT assays were performed at 48 h to assess viability. Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. ( b ) HepG2, Huh-6, HepT1, and B6-2 cells were exposed to varying indicated concentrations of NSC207895 for 48 h. MTT assays were performed at 48 h to asses viability. Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. ( c ) HepG2, Huh-6, HepT1, and B6-2 cells were exposed to varying indicated concentrations of Nutlin-3a for 48 h. MTT assays were performed at 48 h to asses viability. Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. ( d ) IC 50 values determined in the assays shown in ( a – c ). (e) Protein lysis from cells treated with 10 μM ATSP-7041 for 24 h was compared to that from untreated cells (0 h). Immunoblotting for total PARP, PARP cleavage, and Caspase-3 cleavage was done to assess apoptosis. β-Actin immunoblotting was used as a loading control. Date shown are representative of at least three independent experiments. ( f ) Protein lysis from cells treated with 10 μM NSC207895 for 4 or 8 h was compared to that from untreated cells (0 h). Immunoblotting for total PARP, PARP cleavage, and Caspase-3 cleavage was done to assess apoptosis. β-Actin immunoblotting was used as a loading control. Data shown are representative of at least three independent experiments. Full length blots for data shown in e and f are presented in Supplementary Fig. .

Article Snippet: Membranes were then incubated at 4 °C overnight with rocking with primary antibodies for MDM4 (1:1000, NBP1-28,862, Novus Biologicals, Littleton, CO, USA (Supplementary Fig. c) or 1:2000, 04–1555 (clone 8C6), Millipore Sigma, Danvers, MA, USA (Figs. c,d, c, a, Supplementary Fig. )), PARP (1:1000, 9532, Cell Signaling Technology, Danvers, MA, USA), Caspase-3 (1:500, 14,220, Cell Signaling), MDM2 (1:500, sc-965, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p53 (1:1000, sc-126, Santa Cruz Biotechnology, Inc.), phospho-p53 (serine 15, 1:1000, 9286, Cell Signaling), Bax (1:1000, sc-493, Santa Cruz Biotechnology), Puma (1:500, D30C10, Cell Signaling), p21 (1:1000, SX118, Santa Cruz Biotechnology), and β-Actin (1:30,000, A2228, Sigma-Aldrich).

Techniques: Inhibition, Lysis, Western Blot, Control