Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 exhibits differential cytotoxicity to cancer cells with p53MT compared with healthy (noncancerous) cells. a, clinically relevant models were used to analyze the safety of HO-3867 toward normal body cells while simultaneously observing its anticancer efficiency in human cancer-derived cell populations. The graphical representation shows the isolation of heterogeneous cell populations from breast, colon, and liver cancer samples. In addition, noncancerous healthy cells such as fibroblasts from stromal tissue adjacent to breast cancer and radio- and chemosensitive cells from lymphoid and GI tract tissue were used. All of these cells were treated with HO-3867, cisplatin, or vehicle alone. b, a cancer-specific pro-apoptotic effect of HO-3867 was observed in all cells depicted in panel a. Cells were treated with HO-3867 (10 μm), cisplatin (10 μm), or vehicle (DMSO) alone, and apoptosis was measured using annexin V flow cytometry. HO-3867 selectively induced apoptosis in tumor-derived cells and minimal apoptosis in primary culture from normal tissue of different origins as well as tumor-adjacent stroma-derived fibroblasts and radiosensitive lymphoid and GI tract tissue. Cisplatin nonspecifically killed a significantly higher percentage of cells derived from normal tissues (n = 3 for all experiments; p values are as indicated, ANOVA was used for p value calculations, and error bars indicate standard deviation). c, p53 mutational analysis of breast, colon, and liver cancer samples used for the cell cultures shown in b confirms the presence of DNA-binding domain mutations. The exact nucleotide sequence point mutations and resulting amino acid sequence changes are depicted. d, the ability of HO-3867 to induce apoptosis in cancer cells with a p53MT genotype was determined using annexin V flow cytometry. p53MT cells (A431, MDA-MB-468, WRO, and DU-145) and p53−/− cells (MCF-7p53−/− and HCT7p53−/−) were used in the analysis. Cellular apoptosis was not observed in untreated p53MT and p53−/− cells (bars 1–6). shRNA-mediated p53 knockdown and the exogenous addition of p53MT cDNA were used as controls in untreated cells (bars 7–18). In the experimental set, all cell lines were treated with HO-3867, and p53MT cells showed a significant increase in cellular apoptosis (bars 19–22). HO-3867–treated p53null cells did not show a marked increase in apoptosis (bars 23–24). shRNA-mediated p53MT knockdown abolished the HO-3867–induced increase in apoptosis (bars 25–30). The exogenous addition of p53MT cDNA alongside HO-3867 treatment significantly increased apoptosis in both p53MT and p53−/− cells (bars 31–36) (n = 3; mean ± S.D. shown). p values are shown on the figure; standard ANOVA test). Inset, the efficiency of lentiviral particles coding for p53MT cDNA or p53 shRNA was demonstrated using immunoblotting of MCF-7 p53−/− or MCF-7 cells. Untreated MCF-7 p53−/− samples showed no expression of p53 (lane 1). Overexpression of increasing amounts of p53MT cDNA led to increased p53 protein levels. p53 shRNA treatment showed effective knockdown of p53 expression (a representative image from n = 3 replicates is shown).

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Derivative Assay, Isolation, Flow Cytometry, Standard Deviation, Binding Assay, Sequencing, shRNA, Knockdown, Western Blot, Expressing, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 covalently binds to p53MT in the DNA-binding domain. a, 1H-15N HSQC NMR spectrum of the p53MT-Y220C core domain (75 μm) after incubation with (green) or without (red) 1000 μm HO-3867 for 70 min. Chemical shift perturbations were observed for residues in proximity to the solvent-exposed cysteine 277. b, an NMR-generated model depicting the putative sites of interaction between p53MT and HO-3867. c, 1H-15N-HSQC NMR spectrum of the p53MT-Y220C core domain (75 μm) after incubating with (green) or without (red) 430 μm HO-3867 for 20 or 150 min. Chemical shift perturbations were time-dependent, suggesting a covalent binding mode. d, ESI (ES+) mass spectra of the p53MT-Y220C core domain (50 μm) after incubation without (left) or with HO-3867 (right) for 4 h at room temperature. HO-3867 treatment led to a mass increase of 713 or 1426 Da, confirming covalent binding to the p53 core domain by HO-3867. e, ESI (ES+) mass spectra of p53 DBD (50 μm) C182S/C277S, C124S/C277S, and C124S/C182S mutants after incubation without (left) or with HO-3867 (right) for 4 h at room temperature. HO-3867 treatment led to a mass increase of 713 Da only for the C124S/C277S and C124S/C182S mutants, confirming selective covalent modification of Cys-277 and Cys-182 by HO-3867.

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Binding Assay, Incubation, Solvent, Generated, Modification

Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 shows anticancer efficacy in both p53MT and p53WT tumor xenografts by inducing p53MT–RE interaction and induces expression of p53 downstream effectors. a, the anticancer effect of HO-3867 on genetically tractable tumor xenografts of p53WT (MCF-7), p53MT (A-431), and p53−/− (MCF7 p53−/−) cells was observed (n = 3). In row 1, the excised tumors for untreated p53WT, p53MT, and p53−/− xenografts after 4 weeks are shown. In row 2, all of the tumors were treated with HO-3867 along with lentivirus-assisted overexpression of p53WT. A reduction in the tumor volumes of all tumor types was observed in row 2 when compared with the control (row 1). In row 3, tumors were treated with vehicle (DMSO) and lentiviral transfections. The tumor volumes in the vehicle-treated group remained unaltered. In row 4, all tumors were treated with lentivirus coding for p53 shRNA. In row 5, all tumors were treated with HO-3867, and p53WT tumors and p53MT tumors showed a decrease in tumor volume for all biological replicates. Interestingly, in p53 knockdown tumors, HO-3867 did not exhibit very high anticancer efficacy. These data suggest a role for p53 in HO-3867-mediated anticancer activity that appears to be independent of p53 mutational status. In row 6, p53WT and p53MT tumors were treated with HO-3867 along with lentiviral particles coding for p53 shRNA. p53 knockdown in these tumors reversed the anticancer effect of HO-3867, and all biological replicates in both experimental groups showed larger tumor volumes. In rows 6 and 7, p53 null tumor xenografts were treated with HO-3867 and lentiviral particles coding for p53MT cDNA (p53R175H (row 6); p53R273H (row 7)). Interestingly, HO-3867 reduced tumor growth in the presence of p53MT cDNA (compare tumor volumes in row 5 with rows 6 and 7) (n = 3) (HO-3867 treatment started at week 0 in the plot). b, tumor growth curves showing the volume of MCF-7 p53WT, A-431 p53MT, and MCF-7 p53−/− tumors in the eight treatment groups over the course of 4 weeks. In both MCF-7 p53WT and A-431 p53MT tumors, treatment with HO-3867 and HO-3867+ p53WT cDNA led to the greatest reduction in tumor volume. Treatment of MCF-7 p53−/− tumors with HO-3867, HO-3867+p53R175H cDNA, and HO-3867+p53R273H cDNA led to a significant reduction in tumor volume compared with control. In the insets, the efficiency of lentiviral particles coding for p53 shRNA, p53WT cDNA, or p53MT cDNA was demonstrated in MCF-7 p53WT or A-431 p53MT cells using immunoblotting with the indicated antibodies. MCF-7 p53WT and A-431 p53MT samples treated with p53 shRNA showed no expression of p53 (lane 2). p53 shRNA showed effective knockdown of p53 expression. MCF-7 p53−/− cells were treated with p53WT, p53R175H, and p53R273H cDNA and blotted with anti-p53 antibody or anti-GAPDH antibody (loading control). Overexpression of p53WT cDNA or p53MT cDNA led to increased p53 protein expression in MCF-7 p53−/− cells (HO-3867 treatment started at week 0 in the plot. n = 3 for all experiments; p values are labeled on the figure, and two-factor ANOVA with repeated measures was performed for p value calculations).

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Expressing, Over Expression, Control, Transfection, shRNA, Knockdown, Activity Assay, Western Blot, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 converts mutant p53 conformation to its wildtype form. a, model depicting sites of mutagenesis in the p53 gene in a panel of 29 cell lines. All mutations are present in the p53 DNA-binding domain. b, a Fluidigm digital qPCR-based gene expression analysis of a panel of 14 genes (Fig. S3) was conducted in a panel of 29 control and HO-3867–treated cell lines. Consistent with qChIP analysis, p53-regulated genes were overexpressed in all HO-3867–treated p53MT cell lines; this effect was reversed upon p53 shRNA treatment. Cisplatin (10 μm) was used as a positive control for p53 activation. (n = 5 for all experiments; p values are labeled on the figure, and ANOVA was performed for p value calculations). c, ChIP analysis was conducted in a genetically tractable system of p53MT (A-431) and p53−/− (MCF-7p53−/−) cell lines to measure the binding of p53MT to its REs at the bax (left) and p21 (right) promoters. The results were analyzed using the QIAxcel advanced instrument platform (Qiagen). Input (lane 1), no antibody (lane 2), actin antibody (lane 3), and p53 shRNA (lanes 5 and 11) were used as controls. The data show the presence of p53 on the bax and p21 promoters in HO-3867–treated p53WT and p53MT cell lines but not p53−/− cell lines (lane 8). Exogenous addition of either p53WT (lane 12) or p53MT (lane 13) cDNA resulted in significant binding of p53 at its respective REs in HO-3867–treated p53MT and p53−/− cell lines. d, up-regulation of two important p53 target genes, p21 and Noxa, was confirmed at the protein level by Western blotting. A genetically tractable system of p53WT (MCF-7), p53MT (A-431), and p53−/− (MCF-7 p53−/−) cells was used to study the effect of HO-3867 treatment (10 μm) in p53MT cells (lanes 1–6). Lane 7, both p21 and Noxa Western blotting show less expression in MCF-7 p53−/− cells transfected with p53MT cDNA. However, the same combination in the presence of HO-3867 significantly increases p21 and Noxa expression (lane 8).

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Mutagenesis, Binding Assay, Gene Expression, Control, shRNA, Positive Control, Activation Assay, Labeling, Western Blot, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 converts mutant p53 conformation to its wildtype form. a, the p53MT and p53WT forms were immunoprecipitated using Ab 240 or Ab 1620, respectively, and immunoblotted using a polyclonal anti-p53 antibody (FL393) in p53MT (A-431), p53WT (MCF-7), and MCF-7p53−/− tumors. Input (lane 1), actin antibody (lane 2), and p53 shRNA (lanes 5 and 6) were used as controls for all tumors. In untreated MCF-7 tumors, p53 was recognized by Ab 1620 (lane 3) and to a minor extent by Ab 240 (lane 4). In untreated A-431 tumors, p53 was exclusively recognized by Ab 240 (lane 4). No signal was detected in MCF-7p53−/− tumors (third row). Overexpression of p53WT and p53MT cDNA in all three tumors resulted in a strong signal for Ab 1620 (lane 7) and Ab 240 (lane 10), respectively. HO-3867 treatment in MCF-7 tumors significantly increased detection by Ab 1620 (compare lanes 3 with 11). HO-3867 treatment in A-431 tumors resulted in a change in the p53 conformation from an Ab 1620–recognized form to an Ab 1620–recognized form (compare lanes 4 and 11). HO-3867 had no effect on MCF-7p53−/− tumors. Exogenous addition of p53WT cDNA in HO-3867–treated A-431, MCF-7, and MCF-7p53−/− tumors showed the strong presence of p53 in the Ab 1620–recognized form (lanes 15 and 16). Exogenous addition of p53MT cDNA in HO-3867–treated A-431, MCF-7, and MCF-7p53−/− tumors again showed the strong presence of p53 in the Ab 1620–recognized form (lanes 17 and 18) (n = 3). b, wildtype and mutant forms of p53 were immunoprecipitated using Ab 1620 and Ab 240, respectively, and immunoblotted for p53 protein (FL393) in p53WT (MCF-7 and HCT) or p53MT (A-431, DU-145, and MDA-MB-231) cell lines. Input (lane 1) and actin antibody (lane 2) were used as controls. In untreated p53WT cells, p53 was recognized by Ab 1620 (lane 3, rows 1 and 3). p53−/− (MCF-7p53−/− and HCTp53−/−) cells served as negative controls and showed no p53 signal (rows 2 and 4). In untreated p53MT cells, p53 existed exclusively in an Ab 240–recognized form (lane 4, rows 5–7), which upon HO-3867 treatment converted to an Ab 1620–recognized form (compare conversion from 240 to 1620 form, lanes 4 and 5) (n = 3). c, graphical representation of the experimental design for conducting in vitro transcription assays (top). The synthetic DNA template consisted of a poly(6)-p53 DNA-binding site followed by an adenovirus major late core promoter, a transcription start site, a G-less cassette as the coding region, and a poly(A) tail coding region (for qPCR-based detection) followed by a CCT stop signal. Nuclear extracts from p53null (H1299) cells were the source of the RNA polymerase machinery. Lack of reverse transcriptase to convert synthetic transcripts to a qPCR-detectable form in the reaction mix served as a negative control (No RT, bars 1 and 7). p53 immunoprecipitated from untreated MCF-7 cells in combination with H1299 nuclear extracts showed basal transcript synthesis (second bar). p53 from p53MT cell lines in combination with H1299 nuclear extract resulted in minimal transcript synthesis (bars 3–6). p53 immunoprecipitated from HO-3867–treated p53WT and p53MT cell lines in combination with H1299 nuclear extracts successfully generated RNA transcripts from the synthetic DNA template (blue) (n = 3 for all experiments; p values are labeled on the figure, and ANOVA was performed for p value calculations). d, luciferase-based reporter transcription assay (Cignal) was used to analyze p53-dependent transcription in HO-3867–treated p53MT cell lines in vivo. Empty vector (bars 1 and 7) was used as a negative control. Standard p53-dependent transcription was observed in p53WT MCF-7 cells. Results showed minimal p53-dependent transcription in a variety of p53MT cell lines. The effect of HO-3867 on p53-induced transcription was observed in treated p53WT and p53MT cells (n = 3 for all experiments; p values are labeled on the figure, and ANOVA was performed for p value calculations).

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Mutagenesis, Immunoprecipitation, shRNA, Over Expression, In Vitro, Binding Assay, Reverse Transcription, Negative Control, Generated, Labeling, Luciferase, Transcription Assay, In Vivo, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A , D , H Immunoblotting of KDM4C expression in U87 and U251 cells treated. Normalized cell growth ( B ) and viability ( C ) of siCtrl and siKDM4C-treated U87 cells for 72 h. The proliferation of U87 ( E ) and U251 ( F ) cells expressing a non-target control shRNA (shCtrl NT) or shRNA constructs targeting KDM4C (shKDM4C) was monitored for 10 days using an IncuCyte ZOOM system. G Microscopic images and quantified colonies formed by KDM4C knockdown in U87 and U251 cells. Data present the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. siCtrl or shCtrl NT control; Student’s t -tests.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Expressing, shRNA, Construct

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A Immunoblotting of KDM4 and histone proteins in shCtrl NT and shKDM4C expressing U87 and U251 cells. B Immunoblotting of apoptotic markers in shCtrl NT and shKDM4C expressing U87 and U251 cells. C Apoptosis analysis of shCtrl NT and shKDM4C expressing U87 and U251 cells as determined by Annexin V/PI double-staining and flow cytometry. Data showing the percentage of early apoptotic and late apoptotic cells counted from dot plots. Data present the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. shCtrl NT control; Student’s t -tests.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Expressing, Double Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A Immunoblotting of KDM4C and its target genes in shCtrl NT and shKDM4C expressing U87 and U251 cells. B RT-qPCR showing KDM4C and MDM2 mRNA levels in shCtrl NT and shKDM4C expressing U87 cells. mRNA expression levels were normalized to GAPDH mRNA levels. * p < 0.05 and ** p < 0.01 vs. shCtrl NT control; Student’s t- tests. C Schematic representation of the c-Myc promoter. D ChIP-qPCR analysis of KDM4C, H3K4me3, and H3K9me3 levels at the c-Myc promoter in 0.1% DMSO (control)- and SD70-treated U87 cells. Data present the mean ± SD ( n = 3). * p < 0.05 and ** p < 0.01 vs. DMSO control; Student’s t- tests. E GEO analysis of microarray dataset GSE36245 (GPL570, glioblastoma brain tumors, n = 35). Pearson’s correlation coefficient a n alysis was used to measure the relationship between KDM4C and c-Myc or MDM2 mRNA levels.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Microarray

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A Immunoblot analysis of KDM4C, c-Myc, and its target genes in U87 cells transfected with c-Myc (0.6 μg) without or with the treatment of SD70 (10, 20 μM). B Immunoblot analysis of KDM4C, p53, and its target genes in shCtrl NT and shp53 expressing U87 cells without or with the treatment of SD70 (5 μM).

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Transfection, Expressing

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A , B The activity of p21 and Bax luciferase reporter constructs in U87 cells treated with 0.1% DMSO (control) and SD70 following transfection with p53. *** p < 0.001 vs. pcDNA control; $$$ p < 0.001 vs. pcDNA-p53 group, ### p < 0.001 vs. DMSO + pcDNA-p53 group; one-way ANOVA. C The activity of a p21 luciferase reporter construct in U87 cells following transfection with p53 and KDM4C. D – G RT-qPCR showing KDM4s, p53, and p53 target gene mRNA levels in U87 cells treated with shRNA ( D and E ) or SD70 ( F and G ). mRNA expression levels were normalized to GAPDH mRNA levels. H Immunoblotting of KDM4C, p53, and p21 expression in U87 and U251 cells treated with shRNA or SD70 for 24 h. I ChIP-qPCR analysis of p53 and p53K372me1 levels at the PUMA promoter in 0.1% DMSO (control) and SD70 (5 µM) treated U87 cells. Data present the mean ± SD ( n = 3). * p < 0.05 and ** p < 0.01 vs. shCtrl NT or DMSO control; Student’s t- tests.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Activity Assay, Luciferase, Construct, Transfection, Quantitative RT-PCR, shRNA, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A , B Immunoblotting of KDM4C, p53, and p53K372me1 in U87 and U251 cells transfected with ( A ) shRNA and ( B ) siRNA. C , D Lysates of cells treated with SD70 (20 μM) ( C ) or HA-KDM4C ( D ) were immunoprecipitated with p53 antibody followed by immunoblotting with antibodies as indicated. Ten percent of total cell lysates used in immunoprecipitation is shown as input. The level of p53K372me1 was quantified relative to p53, and the control levels were set at 1. The asterisk indicates the expected sizes for p53K372me1.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Transfection, shRNA, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A Immunoblotting of KDM4C, c-Myc, and p53 in U87 TetOn-KDM4C cells expressing shCtrl NT or shKDM4C. B Liquid colony formation assays of U87 TetOn-KDM4C cells expressing shCtrl NT or shKDM4C. * p < 0.05 and ** p < 0.01 vs. Dox (-) group; one-way ANOVA. C – E Xenograft assays of U87 cells with inducible expression of shCtrl NT or shKDM4C ( n = 3). C Representative U87 xenograft tumors isolated from individual animals. D Increase in tumor volume over time. E Final tumor weights of the U87 xenografts. Data present the mean ± S.D. * p < 0.05 and ** p < 0.01 vs. shCtrl NT control; one-way ANOVA. F Representative image of IHC staining of KDM4C and c-Myc in tissues from U87 xenograft model. Scale bar, 100 μm. G Molecular model describing that KDM4C can regulate glioblastoma proliferation and apoptosis by modulating c-Myc and p53.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Expressing, Isolation, Immunohistochemistry

Journal: Oncology Letters

Article Title: DBC1 does not function as a negative regulator of SIRT1 in liver cancer

doi: 10.3892/ol.2012.875

Figure Lengend Snippet: DBC1 did not affect SIRT1 activity in vitro in liver cancer cells. A549 and SNU-182 (A and B) cells were transfected with siRNAs targeting SIRT1 or DBC1 (50 or 200 nM). Etoposide (20 μM) was administered for 12 h to induce p53 hyperacetylation due to DNA damage. Knockdown of SIRT1 or DBC1 and acetylation of p53 were evaluated by western blotting with the indicated antibodies. All membranes were probed for GAPDH to confirm equal protein loading. SIRT1, silent mating type information regulation 2 homolog 1; DBC1, deleted in breast cancer-1.

Article Snippet: Cell culture, transfections and treatments The human HCC cell lines, HepG2 (wt p53) and SNU-182 (mt p53), and the non-small lung carcinoma cell line A549 (wt p53) were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Activity Assay, In Vitro, Transfection, Knockdown, Western Blot

Journal: Theranostics

Article Title: Interaction between p53 and Ras signaling controls cisplatin resistance via HDAC4- and HIF-1α-mediated regulation of apoptosis and autophagy

doi: 10.7150/thno.29673

Figure Lengend Snippet: p53 collaborates with RAS signaling to modulate cell proliferation and apoptosis. A. Expression of p53 and apoptosis-related proteins in SKOV3 T cells. B. H-RAS V12 , p53 and apoptosis-related proteins in SKOV3 T /V12 cells. C. H-RAS S35 , H-RAS E38 , H-RAS C40 , p53 and apoptosis-related protein expression in SKOV3 T /S35, SKOV3 T /E38, and SKOV3 T /C40 cells. D. Different RAS mutations stimulate disparate RAS signaling cascades. E - F. p53 and H-RAS synergistically modulate cell colony formation. Representative images ( E ) and quantitative analysis of colony formation ( F ). The values are expressed as the mean ± standard deviation (n = 3 wells). *: P < 0.05 vs. the control. **: P < 0.01 vs. the control. G - H. RAS signaling alterations induced by the ERK inhibitor SCH772984 (2 μM; 8 h) ( G ) and by the AKT inhibitor GSK2110183) (10 nM; 8 h) ( H ), showing that ERK and AKT signaling are mutually suppressive. Protein markers are properly labeled in relative panels.

Article Snippet: The lung cancer cell lines A549 (p53 Wt /KRAS Mt ), H358(p53 Null /KRAS Mt ), H23 (p53 Mt /KRAS Mt ) and H1299 (p53 Null /KRAS Wt ) , ; the retroviral packaging cells (Phoenix amphotropic cells); and the lentiviral packaging cells (293 T cells) were also purchased from ATCC and were maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone).

Techniques: Expressing, Standard Deviation, Control, Labeling

Journal: Theranostics

Article Title: Interaction between p53 and Ras signaling controls cisplatin resistance via HDAC4- and HIF-1α-mediated regulation of apoptosis and autophagy

doi: 10.7150/thno.29673

Figure Lengend Snippet: p53 and RAS participate in autophagy regulation. A. Expression of autophagy-associated proteins in cells transfected with p53 and various H-RAS mutants. B. Quantitative analysis of autophagosomes. The number of autophagosomes is indicated as the average number of autophagosomes per cell based on analysis of 100 cells. C. LC3B distribution detected by immunofluorescence. D. Examination of LC3B in cells treated with chloroquine. E. Quantitative analysis of autophagosomes in cells treated with chloroquine. F - G. Alterations in autophagy-related proteins induced by ERK inhibition ( F ) or AKT inhibition ( G ). The values are expressed as the mean ± standard deviation (n = 3). *: P < 0.05 vs. control. **: P < 0.01 vs. control. Protein markers are properly labeled in relative panels.

Article Snippet: The lung cancer cell lines A549 (p53 Wt /KRAS Mt ), H358(p53 Null /KRAS Mt ), H23 (p53 Mt /KRAS Mt ) and H1299 (p53 Null /KRAS Wt ) , ; the retroviral packaging cells (Phoenix amphotropic cells); and the lentiviral packaging cells (293 T cells) were also purchased from ATCC and were maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone).

Techniques: Expressing, Transfection, Immunofluorescence, Inhibition, Standard Deviation, Control, Labeling

Journal: Theranostics

Article Title: Interaction between p53 and Ras signaling controls cisplatin resistance via HDAC4- and HIF-1α-mediated regulation of apoptosis and autophagy

doi: 10.7150/thno.29673

Figure Lengend Snippet: p53 and RAS regulate cell resistance to cisplatin. A - B. IC50 values of cells determined by treatment with different cisplatin concentrations for 48 hours. C. Apoptosis analysis via flow cytometry after treatment with DOX, cisplatin, or DOX+cisplatin. D. Quantitative analysis of the cell apoptosis percentage. E. Tumor growth following subcutaneous inoculation with SKOV3 T , SKOV3 T /V12, SKOV3 T /S35, SKOV3 T /E38, or SKOV3 T /C40 cells, followed by administration of placebo, DOX, cisplatin, or DOX+cisplatin. The data are expressed as the mean ± SEM of three independent experiments. The bars represent the mean ± SEM; n = 3; * refers to P < 0.05; ** refers to P < 0.01.

Article Snippet: The lung cancer cell lines A549 (p53 Wt /KRAS Mt ), H358(p53 Null /KRAS Mt ), H23 (p53 Mt /KRAS Mt ) and H1299 (p53 Null /KRAS Wt ) , ; the retroviral packaging cells (Phoenix amphotropic cells); and the lentiviral packaging cells (293 T cells) were also purchased from ATCC and were maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone).

Techniques: Flow Cytometry

Journal: Theranostics

Article Title: Interaction between p53 and Ras signaling controls cisplatin resistance via HDAC4- and HIF-1α-mediated regulation of apoptosis and autophagy

doi: 10.7150/thno.29673

Figure Lengend Snippet: HDAC4 and HIF-1α are key molecules in p53 and RAS signaling networks. A. Gene expression profiles of p53 and RAS signaling cascades. Red and green represent upregulated and downregulated genes, respectively. B - C. HDAC4 ( B ) and HIF-1α ( C ) mRNA levels determined by q-PCR in SKOV3 T and SKOV3 T /V12 cells. D. Effects of p53 and RAS mutants on the expression and acetylation ability of HDAC4 and pHDAC4. E. Expression of HIF-1α under normoxia and hypoxia in the presence or absence of p53 and/or RAS mutants. F-G. Altered expression of HDAC4, pHDAC4 and HIF-1α induced by inhibition of ERK ( F ) and AKT ( G ). Protein markers are properly labeled in relative panels.

Article Snippet: The lung cancer cell lines A549 (p53 Wt /KRAS Mt ), H358(p53 Null /KRAS Mt ), H23 (p53 Mt /KRAS Mt ) and H1299 (p53 Null /KRAS Wt ) , ; the retroviral packaging cells (Phoenix amphotropic cells); and the lentiviral packaging cells (293 T cells) were also purchased from ATCC and were maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone).

Techniques: Gene Expression, Expressing, Inhibition, Labeling

Journal: Theranostics

Article Title: Interaction between p53 and Ras signaling controls cisplatin resistance via HDAC4- and HIF-1α-mediated regulation of apoptosis and autophagy

doi: 10.7150/thno.29673

Figure Lengend Snippet: p53 and RAS mutants control the expression and intracellular localization of HDAC4 and HIF-1α. A. Selected images showing cellular co-localization of HDAC4 with HIF-1α. B. Selected images showing pHDAC4 was increased by p53 induction and ERK active RAS transfection. C. Detection of HIF-1α, HDAC4 and p-HDAC4 in the nucleus and cytoplasm of SKOV3 T cells. β-Actin and histone 3 were used as loading controls for the cytoplasmic and nuclear extractions, respectively. D. Direct binding of HDAC4/p-HDAC4 with HIF-1α detected by co-immunoprecipitation in SKOV3 T and SKOV3 T /V12 cells. Rabbit or mouse IgG served as a negative control for the co-IP experiment. E-F. Detection of HDAC4, pHDAC4, HIF-1α, p53, RAS in additional ovarian cancer cell lines ( E ) and lung cancer cell lines ( F ) with either or both p53 or/and RAS mutations, showing that HDAC4, pHDAC4 and HIF-1α are regulated by p53 and RAS status. Protein markers are properly labeled in relative panels.

Article Snippet: The lung cancer cell lines A549 (p53 Wt /KRAS Mt ), H358(p53 Null /KRAS Mt ), H23 (p53 Mt /KRAS Mt ) and H1299 (p53 Null /KRAS Wt ) , ; the retroviral packaging cells (Phoenix amphotropic cells); and the lentiviral packaging cells (293 T cells) were also purchased from ATCC and were maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone).

Techniques: Control, Expressing, Transfection, Binding Assay, Immunoprecipitation, Negative Control, Co-Immunoprecipitation Assay, Labeling

Journal: Theranostics

Article Title: Interaction between p53 and Ras signaling controls cisplatin resistance via HDAC4- and HIF-1α-mediated regulation of apoptosis and autophagy

doi: 10.7150/thno.29673

Figure Lengend Snippet: HDAC4 modulated by p53 and HIF-1α regulates autophagy by deacetylating CREBZF. A . HDAC4 overexpression leads to increased HIF-1α stability and HDAC4 phosphorylation (upper panel), whereas knockdown of HIF-1α stimulates HDAC4 phosphorylation. B. Association of HDAC4 with autophagy and apoptosis. C. Association of HIF-1α with autophagy and apoptosis. D . Acetylation of CREBZF in SKOV3 T and SKOV3 T /V12 cells. E. CREBZF expression with HDAC4 knockdown in SKOV3 T and SKOV3 T /V12 cells. F. Atg3, Atg12 and LC3B expression with CREBZF knockdown. G . Construction of ATG3 promoter luciferase plasmids with the CREBZF binding/mutation sites. H . SKOV3 T and SKOV3 T /V12 cells were transfected with the plasmids of the ATG3 promoter-driven luciferase for 48 h followed by a dual luciferase assay. A high luciferase activity was observed in SKOV3 T /V12 cells transfected with the wild type of Atg3 promoter, whereas the transfection of the same cell line with the binding site mutation promoter highly reduced the luciferase acitivity. **, P < 0.01 . I . A schematic diagram showing that HDAC4 and HIF-1α are key mediators between p53 and RAS signaling networks although ERK and AKT inversely regulate HDAC4 and HIF-1α through phosphorylation, translocation and protein degradation. HDAC4 and HIF-1α collaboratively inhibit cellular apoptosis, but inversely control autophagy, both confer ovarian cancer cisplatin resistance. Protein markers are properly labeled in relative panels.

Article Snippet: The lung cancer cell lines A549 (p53 Wt /KRAS Mt ), H358(p53 Null /KRAS Mt ), H23 (p53 Mt /KRAS Mt ) and H1299 (p53 Null /KRAS Wt ) , ; the retroviral packaging cells (Phoenix amphotropic cells); and the lentiviral packaging cells (293 T cells) were also purchased from ATCC and were maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone).

Techniques: Over Expression, Phospho-proteomics, Knockdown, Expressing, Luciferase, Binding Assay, Mutagenesis, Transfection, Activity Assay, Translocation Assay, Control, Labeling

Journal: Theranostics

Article Title: Interaction between p53 and Ras signaling controls cisplatin resistance via HDAC4- and HIF-1α-mediated regulation of apoptosis and autophagy

doi: 10.7150/thno.29673

Figure Lengend Snippet: HDAC4 and HIF-1α promote cisplatin resistance, and predict patient survival. A. Quantitative analysis of cisplatin IC50 in response to HDAC4 or HIF-1α expression. B. Quantitative analysis of IC50 in cells with HDAC4 or HIF-1α silencing and DOX treatment. C-D. Growth of tumors derived from cells with different HDAC4 ( C )and HIF-1α ( D ) expression levels, followed by treatment with placebo, DOX, cisplatin, or DOX+cisplatin. E-F. High HDAC4 expression was significantly correlated with a shorter overall survival ( E ) and progression-free survival ( F ) in the Kaplan-Meier plotter cohort. G-H. High CREBZF expression was significantly correlated with shorter overall survival ( G ) and progression-free survival. ( H ) I-J. High HIF-1α expression indicated poor or good OS with mutant p53 ( I ) or wild-type 53, respectively ( J ). K-L. HDAC4 was positively correlated with HIF-1α ( K ) and CREBZF ( L ) in TCGA database. The bars represent the mean ± SEM; n = 3; * refers to P < 0.05; ** refers to P < 0.01.

Article Snippet: The lung cancer cell lines A549 (p53 Wt /KRAS Mt ), H358(p53 Null /KRAS Mt ), H23 (p53 Mt /KRAS Mt ) and H1299 (p53 Null /KRAS Wt ) , ; the retroviral packaging cells (Phoenix amphotropic cells); and the lentiviral packaging cells (293 T cells) were also purchased from ATCC and were maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone).

Techniques: Expressing, Derivative Assay, Mutagenesis

Journal: Nature Communications

Article Title: ROCK1 mechano-signaling dependency of human malignancies driven by TEAD/YAP activation

doi: 10.1038/s41467-022-28319-3

Figure Lengend Snippet: a TEAD reporter activity of MCF10A cells stably expressing empty vector (EV) control, YAP WT, p53 R248Q, p53 R248W, p53 R273H, p53 R273C, p53 RS241F, p53 R175H, or p53 G245S. b TEAD reporter activity of MCF10A cells stably expressing EV control, YAP WT or p53 R273H in the presence or in the absence of dnTEAD4 stable expression. c Representative image from n = 2 biologically independent replicates showing anchorage-independent growth in soft agar of MCF10A cells stably expressing EV control, YAP WT or p53 R273H in the presence or in the absence of dnTEAD4 stable expression. d TEAD reporter activity of human tumor lines containing hot spot p53 DNA contact (in blue), conformational (in red), or null (in gray) mutations as specified in Supplementary Table . 293T (wt p53) and H2052 cells (wt p53 and NF2; LATS2 LOFs) served as negative and positive controls, respectively. e TEAD reporter activity of indicated tumor lines stably expressing empty vector control (Ctr), dominant negative TEAD4 (dnTEAD4) or p53 shRNA (shp53). f Representative plates from n = 2 biologically independent replicates showing 2D colony formation by indicated tumor lines stably expressing empty vector control (Ctr), dnTEAD4 or shp53 for 14 days. g mRNA expression levels of MVA pathway genes, HMGCR and SQLE, by real time PCR in MCF10A cells stably expressing EV control, YAP WT, p53 R273H or p53 R175H. h mRNA expression of Mevalonate (MVA) pathway genes, HMGCR and SQLE, by real time PCR in the indicated cell lines stably expressing Scramble shRNA (shScr) or shp53. i ChIP analysis on HMGCR promoter in MCF10A cells stably overexpressing either p53 R273H or p53 R175H. ChIP was performed with p53 or control immunoglobulin G (IgG) antibody and quantified by real-time PCR on the indicated promoters. Acetyl choline receptor (AchR) promoter was used as a negative control. p values were derived using two tailed t -tests from means± SD of n = 3 biologically independent replicates.* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data with actual calculated p values are provided as Source data file.

Article Snippet: The following antibodies were utilized: p53 (1801; Mount Sinai School of Medicine Hybridoma Center, New York, NY, USA, 1:1000 dilution), YAP, CTGF, MYC, MLC2, RhoA, pan-Rac, Cdc42, ROCK1, ROCK2, GAPDH (Santa Cruz, Dallas, TX, USA, 1:1000 dilution), p-YAP, p-Cofilin, p-MLC2, Cofilin, pPAK1/2, pPAK4/5, PAK2, PAK4, (Cell Signaling, Danvers, MA, USA, 1:1000 dilution), TEAD4, (Thermo Scientific, Waltham, MA, USA, 1:1000 dilution), mouse anti-α-tubulin (1:10,000 dilution), mouse anti-β-actin (Sigma, Saint Louis, MO, USA, 1:10,000 dilution).

Techniques: Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Dominant Negative Mutation, shRNA, Real-time Polymerase Chain Reaction, Negative Control, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: ROCK1 mechano-signaling dependency of human malignancies driven by TEAD/YAP activation

doi: 10.1038/s41467-022-28319-3

Figure Lengend Snippet: a , b TEAD reporter activity ( a ) and immunoblots of whole-cell lysates ( b ) from MCF10A cells transfected with EV control untreated or treated with MVA 0.5 mM for 24 h or transfected with RhoA, Rac1, or Cdc42. Lysates probed with indicated antibodies; β-actin as loading control. c Immunoblots of F-actin and G-actin fractions extracted from MCF10A cells transfected with EV control, RhoA, Rac1, or Cdc42. F/ G-actin ratio representative of n = 2 biologically independent replicates. d Immunoblots of whole-cell lysates from MCF10A cells transfected with RhoA or stably expressing EV control, p53 R273H, or p53 R175H probed with the indicated antibodies. e Immunoblots of F-actin and G-actin fractions extracted from MCF10A cells transfected with RhoA or stably expressing EV control, p53 R273H, or p53 R175H. F/G-actin ratio shown representative of n = 2 biologically independent replicates. f – h TEAD reporter activity of p53 R273H expressing MCF10A cells treated with increasing concentrations of H-1152 ( f ), Latrunculin B ( g ) or Blebbistatin ( h ) for 24 h. i Immunoblots of F-actin and G-actin fractions extracted from p53 R273H MCF10A cells either untreated (Ctr) or treated with indicated concentrations of H-1152, or 1 μM Latrunculin B or 50 μM Blebbistatin for 24 h. F/G-actin ratio shown representative of n = 2 biologically independent replicates. j Representative images from n = 2 biologically independent replicates showing anchorage-independent growth of MCF10A EV control, p53 R273H or p53 cells; treated with DMSO or 1 μM H-1152. k TEAD reporter activity of hot spot p53 DNA contact mutation harboring tumor lines treated with increasing concentrations of H-1152 for 24 h. l Representative plates from n = 2 biologically independent replicates showing 2D colony formation. Cell lines treated with DMSO or increasing concentrations of H-1152. m In vitro migration measured by wound healing assay. Cell lines treated with DMSO or 1 μM H-1152 for 48 h. p values were derived using two tailed t -tests from means± SD of n = 3 biologically independent replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data with actual calculated p values are provided as Source data file.

Article Snippet: The following antibodies were utilized: p53 (1801; Mount Sinai School of Medicine Hybridoma Center, New York, NY, USA, 1:1000 dilution), YAP, CTGF, MYC, MLC2, RhoA, pan-Rac, Cdc42, ROCK1, ROCK2, GAPDH (Santa Cruz, Dallas, TX, USA, 1:1000 dilution), p-YAP, p-Cofilin, p-MLC2, Cofilin, pPAK1/2, pPAK4/5, PAK2, PAK4, (Cell Signaling, Danvers, MA, USA, 1:1000 dilution), TEAD4, (Thermo Scientific, Waltham, MA, USA, 1:1000 dilution), mouse anti-α-tubulin (1:10,000 dilution), mouse anti-β-actin (Sigma, Saint Louis, MO, USA, 1:10,000 dilution).

Techniques: Activity Assay, Western Blot, Transfection, Control, Stable Transfection, Expressing, Mutagenesis, In Vitro, Migration, Wound Healing Assay, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: ROCK1 mechano-signaling dependency of human malignancies driven by TEAD/YAP activation

doi: 10.1038/s41467-022-28319-3

Figure Lengend Snippet: a TEAD reporter activity of MCF10A cells expressing shScr, p53 R273H, YAP WT, NF2 shRNA (shNF2), or LATS1 and LATS2 shRNAs (shLATS1/2) either untreated (Ctr) or treated with 1 μM H-1152 for 24 h. b Western blot analysis of whole-cell lysates from MCF10A cells stably expressing EV control, p53 R273H, YAP WT, shNF2 or shLATS1/2 untreated or treated with 1 μM H-1152 for 24 h and probed with the indicated antibodies. β-actin was used as a loading control. c Western blot analyses of Triton-insoluble (F-actin) and Triton-soluble (G-actin) fractions extracted from the same cells as in ( a ). Fractions were probed with β-actin antibody. Ratio between F- and G-actin is shown representative of n = 2 biologically independent replicates. d Western blot analyses of Triton-insoluble (F-actin) and Triton-soluble (G-actin) fractions extracted from same cells as in ( a ) untreated or treated with 1 μM H-1152 for 24 h. Fractions were probed with β-actin antibody. Ratio between F- and G-actin is shown representative of n = 2 biologically independent replicates. e In vitro migration measured by wound healing assay of the indicated cell lines untreated or treated with 1 μM H-1152 for 48 h. f Representative plates from n = 2 biologically independent replicates showing anchorage-independent growth in agar of the same cells as in ( a ) untreated or treated with 1 μM H-1152 every 4 days for 21 days. g TEAD reporter activity of the indicated tumor lines in response to increasing concentrations of H-1152 for 24 h. h Representative plates from n = 2 biologically independent replicates showing 2D colony formation by the indicated cell lines treated with DMSO or 1 μM H-1152 every 2 days for 14 days. I In vitro migration measured by wound healing assay of the indicated cell lines treated with DMSO or 1 μM H-1152 for 48 h. p values were derived using two-tailed t -tests from means± SD of n = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data with actual calculated p values are provided as Source data file.

Article Snippet: The following antibodies were utilized: p53 (1801; Mount Sinai School of Medicine Hybridoma Center, New York, NY, USA, 1:1000 dilution), YAP, CTGF, MYC, MLC2, RhoA, pan-Rac, Cdc42, ROCK1, ROCK2, GAPDH (Santa Cruz, Dallas, TX, USA, 1:1000 dilution), p-YAP, p-Cofilin, p-MLC2, Cofilin, pPAK1/2, pPAK4/5, PAK2, PAK4, (Cell Signaling, Danvers, MA, USA, 1:1000 dilution), TEAD4, (Thermo Scientific, Waltham, MA, USA, 1:1000 dilution), mouse anti-α-tubulin (1:10,000 dilution), mouse anti-β-actin (Sigma, Saint Louis, MO, USA, 1:10,000 dilution).

Techniques: Activity Assay, Expressing, shRNA, Western Blot, Stable Transfection, Control, In Vitro, Migration, Wound Healing Assay, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: ROCK1 mechano-signaling dependency of human malignancies driven by TEAD/YAP activation

doi: 10.1038/s41467-022-28319-3

Figure Lengend Snippet: a – c Tumor cell growth following orthotopic inoculation in immunocompromised mice of MDA-MB-468, MDA-MB-231 ( a ) or HCC1395 cells ( b ) followed by daily dosing of H-1152 (25 mg/kg) after tumors reached a volume of 100 mm 3 . n = 5 mice per group were used. p values are indicated in the figure calculated using two tailed t -test for n = 5 biologically independent mice per group. Body weight of each mouse at the beginning and at the end of the treatment is also shown ( c ). d Immunostaining of tumor sections from ( a , b ) with antibodies directed against pCofilin as described in the Methods. Scale bar: 100 µM. n = 5 biologically independent mice per group. e Model depicting inhibition of TEAD/YAP transcription in physiological conditions, activated by p53 DNA contact mutations or Hippo pathway alterations by ROCK inhibitiors.* p < 0.05, ** p < 0.01. Source data with actual calculated p values are provided as Source data file.

Article Snippet: The following antibodies were utilized: p53 (1801; Mount Sinai School of Medicine Hybridoma Center, New York, NY, USA, 1:1000 dilution), YAP, CTGF, MYC, MLC2, RhoA, pan-Rac, Cdc42, ROCK1, ROCK2, GAPDH (Santa Cruz, Dallas, TX, USA, 1:1000 dilution), p-YAP, p-Cofilin, p-MLC2, Cofilin, pPAK1/2, pPAK4/5, PAK2, PAK4, (Cell Signaling, Danvers, MA, USA, 1:1000 dilution), TEAD4, (Thermo Scientific, Waltham, MA, USA, 1:1000 dilution), mouse anti-α-tubulin (1:10,000 dilution), mouse anti-β-actin (Sigma, Saint Louis, MO, USA, 1:10,000 dilution).

Techniques: Two Tailed Test, Immunostaining, Inhibition

Journal:

Article Title: Lentiviral delivery of HIV-1 Vpr protein induces apoptosis in transformed cells

doi:

Figure Lengend Snippet: Cell cycle arrest and apoptosis mediated by HIV-1 Vpr in transformed cells

Article Snippet: Similarly, introduction of Vpr into tumor cell lines defective in different DNA repair mechanisms, such as mismatch repair (hMLH1) ( 25 ) and nucleotide excision repair (xeroderma pigmentosum, complementation group A) ( 26 ), resulted in G 2 arrest and apoptosis. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Cell name Cancer origin Phenotype Virus G 1 /G 2 ¶ Annexin V ‖ HeLa Cervical carcinoma p53 null * HR′Thy (Vpr−) 1.7 2.4 HR’Thy (Vpr+) 0.3 14.3 SW480 Colon adenocarcinoma p53 mt † HR’Thy (Vpr−) 1.0 28 HR’Thy (Vpr+) 0.35 40.6 HT1080.ATCC Fibrosarcoma p53 wt ‡ HR’Thy (Vpr−) 1.95 4.3 HR’Thy (Vpr+) 0.75 20 HT1080.6TG Fibrosarcoma p53 mt ‡ HR’Thy (Vpr−) 1.18 8.8 HR’Thy (Vpr+) 0.61 25.4 SupT1 T cell lymphoma Unknown HR’Thy (Vpr−) 1.67 4.0 HR’Thy (Vpr+) 0.41 8.9 LNCap Prostate carcinoma p53 wt § HR’Thy (Vpr−) 8.4 13.6 HR’Thy (Vpr+) 2.64 28.3 HCT116 Colon carcinoma hMLH− ‡ Mock 1.3 9.2 HR’Thy (Vpr+) 0.3 18.0 XP12BESV Xeroderma pigmentosum, XPA− ‡ HR’Thy (Vpr−) 1.54 7.3 complementation group A HR’Thy (Vpr+) 0.89 28 Open in a separate window Cells were either mock-infected or infected with HR’Thy (Vpr−) or HR’Thy (Vpr+).

Techniques: Transformation Assay, Virus