Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) Inducible clonal T47D cells with mdm2 shRNA or control vector were treated with or without 4μg/ml doxycycline (dox) for 3 days, followed by 10nM estrogen for 5 days in the presence or absence of dox. A representative image of western blot analysis of MDM2, phospho Rb, E2F1, p53 and Actin protein levels from 50μg whole cell protein extract is shown. (B) ImageJ analysis was performed for MDM2, phospho Rb and E2F1 protein levels normalized to Actin. The graph represents an average of three independent experiments with standard deviation in inducible clonal T47D cells with mdm2 shRNA or control vector. (C) A constitutive pool of T47D cells with mdm2 shRNA or control vector were grown with or without 10nM estrogen for 5 days. A representative image of western blot analysis of MDM2, phospho Rb, E2F1, total Rb and Actin protein levels from 50μg whole cell protein extract is shown. (D) ImageJ analysis was performed for MDM2, phospho Rb and E2F1 protein levels normalized to Actin. Graph represents average of three independent experiments with standard deviation in constitutive pool of T47D cells with mdm2 shRNA or control vector. * represents a p-value ≤ 0.05, ** represents a p-value ≤ 0.01, *** represents a p-value ≤ 0.001. The p-value was determined by 2-tailed Student t-test.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: shRNA, Control, Plasmid Preparation, Western Blot, Standard Deviation

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) T47D.sh mdm2 cells were treated with 10nM estrogen (lane 1) and either had MDM2 knockdown (lane 2) or 10μM fulvestrant treatment (lane 3), or both (lane 4) for 5 days. A representative western blot analysis of MDM2, phosphoRb and Actin protein levels from 50μg whole cell protein extract is shown. Dot plot diagram shows quantified ImageJ values of phospho Rb protein levels normalized to Actin from three independent experiments. (B) MTT assay was performed in T47D.control vector and inducible T47D.sh mdm2 clonal cell lines after treatments. Percentage mitochondrial activity represents an average of 2 independent experiments. (A) & (B) * represents a p-value ≤ 0.05, *** represents a p-value ≤ 0.001, **** represents a p-value ≤ 0.0001. The p-value was determined by 2-tailed Student t-test. (C) A representative live cell image by confocal microscopy with 20X objective of T47D.vector and inducible clonal T47D.sh mdm2 clonal cell lines after treatments. Red fluorescence represents staining with propidium iodide. Blue fluorescence represents staining of nuclear DNA. (D) MDM2 drives phosphorylation of Rb in ER+ breast cancer cells. In vitro kinase assay was performed to detect phosphorylation of Rb with or without overnight estrogen treatment in either presence or absence of bacterially expressed and purified MDM2 (1μl or 2μl). A representative image of Western blot analysis of MDM2, phospho Rb and total Rb protein level from nuclear extract of MCF7 (left) and T47D (right) cells are shown. Dot plot diagram shows quantified ImageJ values of phospho Rb protein levels normalized to lamin A from three independent experiments, when 1μl of purified MDM2 was added to the nuclear extracts of MCF7 (left) and T47D (right) cells. The p-value for MCF7 cells with overnight estrogen treatment and addition of purified MDM2 (compare lane 4 to lane 5) was statistically significant. The p-value was determined by 2-tailed Student t-test.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Western Blot, MTT Assay, Control, Plasmid Preparation, Activity Assay, Confocal Microscopy, Fluorescence, Staining, Phospho-proteomics, In Vitro, Kinase Assay, Purification

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) Number of large colonies (50μm or larger) determined by counting the colonies of MCF7 cells when grown in soft agar in the presence of estrogen and in the presence or absence of shRNA induction (viewed by inverted fluorescence microscope). Average of three independent experiments are shown. The number of colonies for 3 independent experiments were as follows: control vector –shRNA induction (160, 158, 175), control vector + shRNA induction (153, 151, 197), mdm2 shRNA –shRNA induction (140, 137, 200) and mdm2 shRNA + shRNA induction (17, 11, 8). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.002. (B) Number of large colonies (100μm or larger) determined by counting the colonies of T47D cells when grown in soft agar in the presence of estrogen and in the presence or absence of shRNA (viewed by inverted fluorescence microscope). Average of three independent experiments are shown. The number of colonies for 3 independent experiments were as follows: control vector –shRNA induction (220, 191, 70), control vector + shRNA induction (202, 195, 33), mdm2 shRNA –shRNA induction (166, 175, 69) and mdm2 shRNA + shRNA induction (27,20, 9). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.02. (C) Representative images of colonies that T47D cells formed in soft agar in the presence or absence of MDM2 knockdown. (D) T47D cells grown in matrigel for 3 weeks in presence of estrogen and in presence or absence of 4μg/ml doxycycline, were fixed and stained with propidium iodide. Colony masses were categorized in 5 different groups and the number of masses in each group were counted and presented as percentages in the total population. This is an average of two independent experiments. The total number of masses scored for 2 independent experiments were control vector –shRNA induction (20, 15), control vector + shRNA induction (25, 27), mdm2 shRNA –shRNA induction (93, 95) and mdm2 shRNA + shRNA induction (86, 83). The p-value was determined by 2-tailed Student t-test. The p-value for large and small colonies for comparisons with and without MDM2 knockdown were p-value=0.03 and p-value=0.05 respectively. Two independent scorers counted the numbers of colonies for each independent experiment.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: shRNA, Fluorescence, Microscopy, Control, Plasmid Preparation, Knockdown, Staining

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) T47D cells grown in matrigel for 3 weeks in presence of estrogen and in the presence or absence of 4 μg/ml dox, were fixed, stained with F-Actin and mounted with DAPI containing mounting media. Confocal z-stack images were acquired. Masses with lumen were counted and presented as percent of total number of masses grown in 3D matrigel. An average of two independent experiments are shown. The number of masses counted for 2 independent experiments were control vector -shRNA induction (21, 41), control vector +shRNA induction (21, 47), mdm2 shRNA -shRNA induction (31, 46) and mdm2 shRNA +shRNA induction (31, 62). The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.01. Two independent scorers counted the numbers of masses for each independent experiment. (B) A representative image from confocal immunofluorescence microscopy showing a single slice from z-stack of DAPI, GFP and F-Actin of estrogen treated inducible clonal T47D.sh mdm2 cells grown in 3D-matrigel in the presence or absence of 4μg/ml doxycycline (dox) for 3 weeks. The top and middle rows show hollow lumen and ductal lumen respectively in the presence of shRNA expression to mdm2 ; the GFP (green) indicates shRNA induction to mdm2 . The third row shows mass structure (disruption of normal mammary glandular architecture) in the absence of shRNA expression to mdm2 .

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Staining, Control, Plasmid Preparation, shRNA, Knockdown, Immunofluorescence, Microscopy, Expressing, Disruption

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) & (B) T47D cells ((A) inducible sh mdm2 clonal and vector pool; (B) constitutive sh mdm2 and vector pool) grown in the presence of estrogen in 3D matrigel for 3.5 weeks. The cells were fixed, permeabilized, blocked, stained with phospho-histoneH3 antibody and mounted with DAPI containing mounting media. Images were taken with confocal microscope. Quantitative analysis of phospho-histone H3 positive cells were performed by capturing optical Z-stack sections of masses and dividing the number of positive phospho-histone H3 cells by the total number of masses. (A) The number of masses counted in each group for 2 independent experiments were control vector -shRNA induction (29, 30), control vector +shRNA induction (30, 30), mdm2 shRNA -shRNA induction (29, 30) and mdm2 shRNA +shRNA induction (30, 30). (B) The number of masses counted in each group for 2 independent experiments were control vector (49, 60) and sh mdm2 (48, 60). Average of two independent experiments are shown for each knockdown method. The p-value determined by 2-tailed Student t-test comparing with and without MDM2 knockdown was p-value=0.001 (A) and p-value=0.009 (B). Two independent scorers counted the numbers of masses for each independent experiment. (C) Representative confocal Z-stack image (single slice) showing DAPI, phospho-histone H3 and GFP in estrogen-treated inducible clonal T47D.sh mdm2 cells in the presence and absence of 4μg/ml doxycycline. (D) Cell cycle analysis by FACS (Fluorescence Activated Cell Sorting). T47D cells were harvested, fixed and stained with propidium iodide and subjected to cell cycle analysis by FACS. Data are presented as percent of cells in S phase in a total population of 10,000 cells and analyzed by FACS in each group. Average of 4 independent experiments are shown. The p-value was determined by 2-tailed Student t-test and * represents a p-value ≤ 0.05

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Plasmid Preparation, Staining, Microscopy, Control, shRNA, Knockdown, Cell Cycle Assay, Fluorescence, FACS

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: (A) Inducible clonal MCF7 cells with mdm2 shRNA or control vector were treated with and without 2μg/ml doxycycline(dox) for 3 days to induce shRNA expression, followed by 10nM estrogen for 5 days in the presence and absence of dox. A representative image of western blot analysis of phospho Rb, Total Rb, E2F1, MDM2 and Actin protein levels from 50μg whole cell protein extract is shown. (B) ImageJ analysis was performed for phospho Rb, E2F1 and MDM2 protein levels normalized to Actin. Graph represents average of four independent experiments with standard deviation in inducible clonal of MCF7 cells with mdm2 shRNA or control vector. * represents a p-value ≤ 0.05, ** represents a p-value ≤ 0.01. The p-value was determined by 2-tailed Student t-test.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: shRNA, Control, Plasmid Preparation, Expressing, Western Blot, Standard Deviation

Journal: Oncotarget

Article Title: Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

doi: 10.18632/oncotarget.18147

Figure Lengend Snippet: Model showing that MDM2 is a central hub in estrogen signaling and works through an Rb-E2F1 pathway to promote proliferation. Breast cancer cells harboring SNP309 have increased binding of the transcription factor Sp1, which causes elevated MDM2 protein levels. Fulvestrant blocks the MDM2 pathway and the Rb-E2F1 pathway.

Article Snippet: 2D culture: Human breast cancer cells T47D ( mdm2 SNP309 G/G, mutant p53 L194F), MCF7 ( mdm2 SNP309 T/G, wild-type p53), and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Binding Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 1 MKRN1 is highly expressed and associated with poor prognosis in patients with CRC. A The expression distribution of mRNA in different tumour cell lines. B The distribution of MKRN1 expression in tumour and normal tissues. C MKRN1 expression in CRC tumour and adjacent non-tumour tissues was verified by WB analysis. D Immunohistochemistry detection of MKRN1 expression in tissue sections of patients with CRC and colitis showing typical photographs (scale bars are 100 and 50 µm, respectively). E The Kaplan–Meier Plotter in the R2 Genomics Analysis Platform was used to draw the overall survival curve. F WB analysis of MKRN1 expression levels in CRC cells (HT29, HCT116, HCT15, and RKO) and normal human colonic fibroblasts (CCD-18Co). ** P < 0.01, *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: Expressing, Immunohistochemistry

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 2 MKRN1 has a role in CRC cell proliferation, migration, and invasion. A–C WB analysis of MKRN1 transfection rates in HCT116, HT29, and HCT15 cells. D, E CCK-8 assay for CRC cell viability. F, G Colony formation assay to detect CRC cell proliferation. H, I Migration ability of CRC cells with different MKRN1 expression levels detected using a wound healing assay (Scale bar: 100 µm). G-K Transwell assays for migration and invasion of MKRN1 knockdown and overexpressing cells (Scale bar: 50 µm). L, M Microscopic observation of the morphology of the cell lines HCT116 (Control, sh1-MKRN1) and HCT15 (Vector, OE-MKRN1) (Scale bar: 100 µm). N, O Comparison of epithelial and mesenchymal marker expression following knockdown and MKRN1 overexpression. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: Migration, Transfection, CCK-8 Assay, Colony Assay, Expressing, Wound Healing Assay, Knockdown, Control, Plasmid Preparation, Comparison, Marker, Over Expression

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 3 MKRN1 and SNIP1 interaction. A Prediction of protein interaction with MKRN1 using the STRING database. B Prediction of protein interaction with MKRN1 using the IntAct database. C The experimental process of MKRN1 quantitative proteomics and ubiquitination modification omics. D Expression distribution of SNIP1 (upper) and TRA2A (lower) in colorectal tumour and normal tissues. E Univariate and multifactorial Cox analyses of P-values, hazard rate, and confidence intervals for gene expression and clinical characteristics. F, G WB analysis showing that MKRN1 expression level affects SNIP1 protein expression. H Confocal microscopy showing MKRN1 and SNIP1 localisation (Scale bar: 20 µm). I, J Forward and reverse validation of MKRN1 interactions with SNIP1 in HCT116 and HCT15 cells using co-immunoprecipitation (Co-IP) and WB. K Validation of exogenous MKRN1 interaction with SNIP1 in HCT15 cells using Co-IP and WB. *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: Quantitative Proteomics, Ubiquitin Proteomics, Modification, Expressing, Gene Expression, Confocal Microscopy, Biomarker Discovery, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 5 MKRN1 induces EMT in CRC cells by degrading SNIP1 protein. A, B WB was used to determine the transfection rate of SNIP1 in HCT116 and HCT15 cells. C, D WB was used to measure expression levels of epithelial and mesenchymal markers following SNIP1 knockdown and overexpression. E, F Transwell assays for migration and invasion of SNIP1 knockdown and overexpressing cells (Scale bar: 50 µm). G WB was used to determine the level of major EMT proteins in HCT116 cells co-transfected with control, sh1-MKRN1, and sh-SNIP1. H WB analysis of the level of major EMT proteins in HCT15 cells co-transfected with vector, OE-MKRN1, and OE-SNIP1. I Transwell assay of the migration ability of HCT116 cells after co-transfection with control, sh1-MKRN1, and sh-SNIP1 (Scale bar: 50 µm). J Transwell assay of HCT15 cells co-transfected with vector, OE-MKRN1, and OE-SNIP1 for cell migration (scale bar: 50 µm). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: Transfection, Expressing, Knockdown, Over Expression, Migration, Control, Plasmid Preparation, Transwell Assay, Cotransfection

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 7 MKRN1 promotes tumour proliferation and metastasis in vivo. A Comparative graph showing the number of intestinal lesions in the MKRN1 [+ / +] and MKRN1 [f/f] groups. B Haematoxylin–eosin (H&E) staining of the intestine of both groups of mice (scale bar: 100 μm). C H&E staining of the liver in the two groups of mice (scale bar: 100 µm; scale bar: 20 µm). D IHC staining for E-cadherin, MKRN1, SNIP1, and TGF-β1 in the intestinal tissues of the two groups of mice (scale bar: 100 µm). E Western blotting analysis of E-cadherin, MKRN1, SNIP1, and TGF-β1 protein expression in intestinal tissues of the two groups of mice. F MKRN1 facilitates the TGF-β signalling via ubiquitination and degradation of SNIP1, thereby promoting EMT in CRC cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: In Vivo, Staining, Immunohistochemistry, Western Blot, Expressing, Ubiquitin Proteomics

Journal:

Article Title: Loss of Oncogenic H-ras-Induced Cell Cycle Arrest and p38 Mitogen-Activated Protein Kinase Activation by Disruption of Gadd45a

doi: 10.1128/MCB.23.11.3859-3871.2003

Figure Lengend Snippet: Disruption of Gadd45a abolishes p38 activation after H-ras overexpression. (A) wt and Gadd45a−/− MEF were infected with H-ras-expressing retrovirus, and activities of ERK, JNK, and p38 were analyzed, with MBP, GST-Jun, and GST-ATF2 as substrates, respectively. puro, puromycin. (B) At the same time that the protein extracts were obtained, the levels of p16/Ink4a, p53, and p21/Waf1 were determined. (C) MEF were infected with either puromycin- or H-ras-expressing retroviruses, and 5 days after selection with puromycin, mRNA was purified and the levels of p53-inducible genes (those encoding p21/Waf1, XPC, and ATF2) were analyzed by using a quantitative filter hybridization procedure (29). The relative induction ratio was obtained after dividing the relative level of mRNA after infection with H-ras retrovirus by the level of mRNA after infection with puromycin vector alone. (D) The levels of Gadd45a and p19/ARF mRNA in wt and Gadd45a−/− MEF on day 5 after retroviral infection were measured by Northern blotting. GAPD was included as a loading control.

Article Snippet: Analysis of protein levels was carried out by using polyclonal antibodies (Abs) against p16/ink4a (M-156; Santa Cruz) and p53 (AB7; Oncogene), monoclonal Abs against p21/Waf1 (AB4; Oncogene) and actin (AB1; Oncogene), anti-Flag monoclonal Ab M2 (Sigma) or anti-Flag monoclonal Ab M2 conjugated with horseradish peroxidase (Sigma), anti-HA polyclonal Ab (Covance Research Products), anti-Myc-tag monoclonal Ab 9E10 (Santa Cruz), anti-Gadd45a polyclonal Ab H-165 (Santa Cruz), anti-p38 polyclonal Ab C-20 (Santa Cruz), anti-JNK polyclonal Ab C-17 (Santa Cruz), and anti-ERK2 polyclonal Ab C-14 (Santa Cruz).

Techniques: Activation Assay, Over Expression, Infection, Expressing, Selection, Purification, Hybridization, Plasmid Preparation, Northern Blot

Journal:

Article Title: Loss of Oncogenic H-ras-Induced Cell Cycle Arrest and p38 Mitogen-Activated Protein Kinase Activation by Disruption of Gadd45a

doi: 10.1128/MCB.23.11.3859-3871.2003

Figure Lengend Snippet: Gadd45a and p38 are required for p53 activation after H-ras overexpression. (A) wt and Gadd45a−/− MEF were incubated in the presence of a MEK1 (50 μM PD98059) or p38 (10 μM SB202190) inhibitor, and protein extracts were obtained on day 5 after selection (see Materials and Methods). The levels of p16/Ink4a, p21/Waf1, and p53 proteins were analyzed. DMSO, dimethyl sulfoxide; puro, puromycin. (B) wt and Gadd45a−/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or H-ras. Some cells were additionally transfected with a dominant-negative p38α vector (p38DN). Four days later, cells were treated with either a MEK1 (PD90859) or p38 (SB202190) inhibitor, and CAT assays were carried out 12 h later. (C) wt and Gadd45a−/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or MKK6(E). Four days later, CAT activity was analyzed, and representative results are shown. Relative induction, as measured by increased CAT activity, was consistently twofold or greater in wt MEF compared to that in Gadd45a−/− MEF.

Article Snippet: Analysis of protein levels was carried out by using polyclonal antibodies (Abs) against p16/ink4a (M-156; Santa Cruz) and p53 (AB7; Oncogene), monoclonal Abs against p21/Waf1 (AB4; Oncogene) and actin (AB1; Oncogene), anti-Flag monoclonal Ab M2 (Sigma) or anti-Flag monoclonal Ab M2 conjugated with horseradish peroxidase (Sigma), anti-HA polyclonal Ab (Covance Research Products), anti-Myc-tag monoclonal Ab 9E10 (Santa Cruz), anti-Gadd45a polyclonal Ab H-165 (Santa Cruz), anti-p38 polyclonal Ab C-20 (Santa Cruz), anti-JNK polyclonal Ab C-17 (Santa Cruz), and anti-ERK2 polyclonal Ab C-14 (Santa Cruz).

Techniques: Activation Assay, Over Expression, Incubation, Selection, Plasmid Preparation, Expressing, Transfection, Dominant Negative Mutation, Activity Assay

Journal: Cell death and differentiation

Article Title: Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

doi: 10.1038/s41418-023-01167-4

Figure Lengend Snippet: Fig. 2 CuET alters the nucleolar morphology. A Representative IF images of A549 nuclei treated with CuET or ActDL for the indicated time points. Fibrillarin (FBL) or nucleophosmin (NPM1) were used as nucleolar markers. Scale bar: 5 μM. B Representative IF images of nucleolin and 5.8S rRNA levels in A549 cells treated with CuET or ActDL for the indicated time points. Scale bar: 50 µm. C AgNOR staining of U2OS or A549 cells following a four-hour treatment of increasing CuET doses. Scale bar: 10 μm. D Ethylene uridine (EU) levels were calculated following IF and high content imaging of U2OS treated with 1 μM CuET for the indicated time points. 750–1500 cells were analyzed per experiment (data are shown as mean ± SD, n = 3 biological replicates, **p < 0.01). Scale bar: 50 µm. E Detection of NPL4 protein levels with immunocytochemistry in samples from patients with triple-negative breast cancer (TNBC) or ovarian carcinoma. Regions in red squares are presented magnified in the bottom panel. Scale bar: 100 μM. F Representative IF images of nucleolar structure in U2OS treated with ActDL or BMH-21 as nucleolar stress inducers. Fibrillarin (FBL) was used as a nucleolar marker. Insets depict magnifications of the regions designated in squares. Scale bar: 2 μM.

Article Snippet: U2OS osteosarcoma, A549 lung epithelial carcinoma, MDA-MB-231 breast carcinoma, and RPE1 human normal retina epithelial cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Staining, Imaging, Immunocytochemistry, Marker

Journal: Cell death and differentiation

Article Title: Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

doi: 10.1038/s41418-023-01167-4

Figure Lengend Snippet: Fig. 3 CuET triggers p53 entrapment in NPL4-rich aggregates. A Immunoblotting of p53 protein levels following increasing concentrations of CuET in U2OS or A549 cells. B IF-based quantitation of p53 levels following CuET treatment of A549 cells. Data are shown as mean ± SD, n = 3 biological replicates, ****p < 0.001. Scale bar: 10 μM. C NPL4, p53, MDM2, and CHK2 protein levels following CuET treatment and fractionation of A549 cells. Lamin B and α-tubulin were used as markers for the soluble (S/N) and insoluble (pellet) fractions, respectively. D Representative IF images of GFP-tagged NPL4 and p53 protein levels following treatment of NPL4-GFP U2OS cells with CuET. Scale bar: 10 μM. E NPL4 and p53 protein levels in U2OS cells ectopically expressing NPL4mut. Lamin B and α-tubulin were used as markers for the soluble (S/N) and insoluble (pellet) fractions, respectively. F Immunoblotting of various p53 post-translational modifications (PTM) in A549 cells treated with CuET+/−(6 h) Etoposide (last 2 h). Etoposide was used as a positive control known to induce activating p53 PTMs. G CDKN1A mRNA levels following treatment with CuET (6 h) +/−Etoposide (last 2 h). Two different cell models were used: A549 transfected with siRNA against TP53 or control siRNA and U2OS cells compared to U2OS cells carrying a dominant negative p53 mutant (ddp53) (data are shown as mean ± SD, n = 3 biological replicates, ****p < 0.001, ***p = 0.01, **p < 0.01, ns non-significant).

Article Snippet: U2OS osteosarcoma, A549 lung epithelial carcinoma, MDA-MB-231 breast carcinoma, and RPE1 human normal retina epithelial cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Quantitation Assay, Fractionation, Expressing, Positive Control, Transfection, Control, Dominant Negative Mutation, Mutagenesis

Journal: Cell death and differentiation

Article Title: Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

doi: 10.1038/s41418-023-01167-4

Figure Lengend Snippet: Fig. 5 RiBi and autophagy inhibition potentiate the cytotoxic effect of CuET. A Survival analysis of U2OS cells treated with the synergistic pairs of CuET, BMH-21, or their combination (data are shown as mean ± SD, n = 3 biological replicates, ***p = 0.01, *p < 0.05). B Survival analysis of U2OS cells treated with the synergistic pairs of CuET, CX-5461, or their combination (data are shown as mean ± SD, n = 3 biological replicates, ***p = 0.01,**p < 0.01). C, D Survival analysis of U2OS (C) or RPE1 (D) cells treated with the synergistic pairs of CuET, chloroquine (CQ), or their combination (data are shown as mean ± SD, n = 3 biological replicates, ****p < 0.001, ***p = 0.01). E Comparative plot showing the Bliss synergy scores of the dose pairs tested among combinations of CuET/AQ (amodiaquine) and CuET/CQ. F Differential survival analysis between three cancer cell lines (A549, U2OS, MDA-MB-231) and RPE1 treated with CuET+/−AQ (data are shown as mean ± SD, n = 3 biological replicates, ****p < 0.001). G Quantification of the transplanted fluorescent tumor area in zebrafish xenografts treated with DMSO vehicle, AQ, CuET, or their combination for 48 h. The end-point signal (2 days) is normalized to the initial one (0 days) for each sample tested (Δtumor area). H Representative IF images of xenografts treated with DMSO or the combination of AQ/CuET. Tumors composed of transplanted MDA-MB-231 cells are shown in red.

Article Snippet: U2OS osteosarcoma, A549 lung epithelial carcinoma, MDA-MB-231 breast carcinoma, and RPE1 human normal retina epithelial cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Succinate Accumulation Is Associated with a Shift of Mitochondrial Respiratory Control and HIF-1α Upregulation in PTEN Negative Prostate Cancer Cells

doi: 10.3390/ijms19072129

Figure Lengend Snippet: Loss of PTEN results in increased ROUTINE respiration in human and mouse cell lines ( A ) Schematic overview of the electron transfer through the mitochondrial complexes (CI–CII–CIV and CII–CIII–CIV) as part of the electron transfer system. NADH, the substrate for CI, is oxidized to NAD + (N-pathway). Succinate, the substrate for CII, is oxidized to fumarate leading to the reduction of FAD and formation of FADH 2 (S-pathway). ( B , C ) ROUTINE respiration expressed as O 2 flow per cell was determined by high-resolution respirometry in intact human and mouse cell lines. Androgen receptor (AR) and PTEN expression was validated by western blotting. Glycerinaldehyd-3-phosphate dehydrogenase (GAPDH) was used as internal loading control. Representative blots from three independent experiments are shown. ( D ) ROUTINE respiration expressed as O 2 flow per cell was determined in intact JP11066 Pten KO cells after 24 h treatment with 25 µM PI3K inhibitor LY29004 compared to mock control (DMSO). Data were expressed as mean and SEM of at least three independent experiments. Statistical differences were calculated with t -test and indicated with asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: The following primary antibodies were used: rabbit anti AR PG-21 (androgen receptor, dilution 1:500, Merck-Millipore, Burlington, CA, USA), rabbit anti PTEN (phosphatase and tensin homologe, 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), mouse anti GAPDH (glycerinaldehyd-3-phosphat-dehydrogenase, dilution: 1:50,000, Merck-Millipore), and rabbit anti NaDC3 (dilution: 1:250, Proteintech).

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Succinate Accumulation Is Associated with a Shift of Mitochondrial Respiratory Control and HIF-1α Upregulation in PTEN Negative Prostate Cancer Cells

doi: 10.3390/ijms19072129

Figure Lengend Snippet: Loss of phosphatase and tensin-homolog (PTEN) is associated with increased capacity for mitochondrial complex II respiration and elevated intracellular succinate levels. Capacities of mitochondrial pathways assessed in permeabilized cells: ( A ) FN(GM) OXPHOS capacity: activation of fatty acid oxidation (F) and NADH linked pathway (N) after addition of glutamate (G) and malate (M), FN(PGM): respiratory capacity after subsequent addition of pyruvate (P), FNS(PGM) OXPHOS capacity after addition of succinate (S). ( B ) N-pathway (complex I, CI) and S-pathway (complex II, CII) respiration. Succinate was added before maximum ETS capacity was reached by addition of uncoupler. Rotenone was added to inhibit CI and assessment of CII-mediated respiration. ( C ) FNS(PGM) OXPHOS capacity determined in LNCaP 3D spheroids and compared to that of LNCaP cells grown in standard 2D culture. Representative images are shown below the graph (magnification 100× (left), 40× (right)) ( D ) Intracellular levels of succinate and fumarate ( E ) were assessed by GC-MS and values expressed as µg per million cells. Data were expressed as mean and SEM. Statistical differences are indicated (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: The following primary antibodies were used: rabbit anti AR PG-21 (androgen receptor, dilution 1:500, Merck-Millipore, Burlington, CA, USA), rabbit anti PTEN (phosphatase and tensin homologe, 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), mouse anti GAPDH (glycerinaldehyd-3-phosphat-dehydrogenase, dilution: 1:50,000, Merck-Millipore), and rabbit anti NaDC3 (dilution: 1:250, Proteintech).

Techniques: Activation Assay, Gas Chromatography-Mass Spectrometry

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 knockdown in MDA-MB-231 orthotopic transplants reduces CTCs. MDA-MB-231 cells with constitutive sh mdm2 , sh mdmx , or mir30 shRNA-expressing vector were implanted into the mammary fat pads of 6-week-old female NSG mice. a Western blot analysis of MDM2, MDMX, and mtp53 protein levels from 50 μg of whole-cell lysates from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells (lanes 1, 2, and 3, respectively) prior to mammary fat pad implantation. Actin is shown as a loading control. b Box-and-whisker plot represents the numbers of CTCs per milliliter from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells engrafted into animals. The number of CTCs was determined by flow cytometry, and the total events were counted (gates were set by the GFP signal intensity and cell size). The number of CTCs per milliliter was obtained by dividing the number of positive events by blood volume from individual animals. The adjusted p value was obtained with two-tailed, two-sample t tests using a permutation test. c Representative fluorescence-activated cell sorting plots showing GFP-positive events in different mouse groups

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, shRNA, Expressing, Plasmid Preparation, Western Blot, Control, Whisker Assay, Flow Cytometry, Two Tailed Test, Fluorescence, FACS

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 knockdown in MDA-MB-231 orthotopic transplants does not significantly reduce primary tumor growth. a Primary tumor volumes of 231.mir30.vector ( n = 6), 231.sh mdm2 ( n = 7), and 231.sh mdmx (n = 7) engrafted animals were measured using calipers over 36 days. b The endpoint tumor volumes were determined on dissected masses at the time of necropsy. c mRNA levels of mdm2 and mdmx normalized to gapdh in primary tumors were determined by real-time qRT-PCR. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001, NS = nonsignificant. The p value was calculated using two-tailed unpaired t tests. d Protein expression of MDM2, MDMX, and mtp53 from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx engrafted primary tumors were determined by Western blot analysis. Three tumors per group were used, and actin is shown as a loading control

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test, Expressing, Western Blot, Control

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 provoke in vitro MDA-MB-231 cell migration without altering cell proliferation. a Representative Western blot demonstrating the levels of MDM2, MDMX, and mtp53 in 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells (lanes 1, 2, and 3, respectively). Fifty micrograms of lysate was loaded per lane. Actin was used as a loading control. b The number of cells was determined by hemocytometer cell counting. Cells ( n = 50,000) were seeded in triplicate, and cell counting was performed at 2, 4, 5, and 6 days. Dots represent mean values, and error bars represent SD. Experiments were carried out with three biological replicates. c Wound closure was observed by phase-contrast microscopy and photographed at 0 and 12 h. One representative image from each group at 0 and 12 h is shown. d The wound area was measured by using NIS-Elements software (Nikon Instruments, Melville, NY, USA). The percentage of wound closure was quantified from four independent biological experiments. The p value was obtained by two-tailed unpaired t test

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: In Vitro, Migration, Western Blot, Plasmid Preparation, Control, Cell Counting, Microscopy, Software, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX knockdown in primary tumors blocks the transcription of CXCR4 and PTGS2 . The 231.mir30.vector-, 231.sh mdm2 -, and 231.sh mdmx -derived primary tumors were lysed and used for total RNA extraction and complementary DNA synthesis. a Microarray analysis revealed selected tumor metastasis-related genes that were either up- or downregulated in 231.sh mdm2 and 231.sh mdmx compared with 231.mir30 vector. Fold changes were gated either > 2 or < 0.5. Two tumor samples per group were used for the analysis. b From the respective cells derived from all the primary tumors, the total CXCR4 and PTGS2 levels were determined by real-time qRT-PCR, and these were compared with those of the parental cells grown in culture. The bars represent mean values, and error bars represent SD. The p values were obtained by two-tailed unpaired t test

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Derivative Assay, RNA Extraction, DNA Synthesis, Microarray, Quantitative RT-PCR, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDM2 knockdown in ERα + T47D orthotopic transplant reduces tumor volume. a T47D cells with inducible sh mdm2 or mir30 shRNA-expressing control vector were treated with 4 μg/ml doxycycline (Dox) for 10 days to induce and maintain shRNA expression. Western blot shows the levels of MDM2, MDMX, E-cadherin, and mtp53 with Dox treatment (lanes 1 and 2) prior to mammary fat pat implantation. Actin was used as a loading control. b Animals were provided with 2 mg/ml Dox and 8 μg/ml E 2 in their drinking water during the entire experiment. Primary tumor growth was measured over a period of 60 days using calipers *** p < 0.001 calculated by two-tailed unpaired t test. c The experimental endpoint tumor volume was determined at the time of necropsy. d mdm2 mRNA expression in primary tumors was determined by real-time qRT-PCR. The p value was determined by two-tailed unpaired t test. e E-cadherin, MDM2, MDMX, and mtp53 protein levels from primary tumors were determined by Western blot analysis. Actin was used as the loading control. f Representative H&E staining images of T47D.vector and T47D.sh mdm2 under 200× and 1000× magnification. T represents Tumor; nm represents normal mammary fat pad; M represents muscle; arrowhead depicts tumor cells infiltrating into muscle layer

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, shRNA, Expressing, Control, Plasmid Preparation, Western Blot, Two Tailed Test, Quantitative RT-PCR, Staining

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: Comparative levels of CXCR4 in ERα + T47D and TNBC MDA-MB-231 tumors. a CXCR4 RNA expression normalized to gapdh with MDM2 knockdown was determined using real-time qRT-PCR. 231.mir30.vector ( n = 6), T47D.mir30.vector ( n = 10), and T47D.sh mdm2 ( n = 9) tumor samples were analyzed. The RNA level of CXCR4 was set as 1 for the 231.mir30.vector group, and T47D samples were expressed relative to 231.mir30.vector values. b Protein expression of CXCR4 was compared from parental cell lines and two tumors from each group. Representative Western blot demonstrating protein levels of CXCR4 and actin in MDA-MB-231 and T47D groups shown using two gels (lanes 1–9 and 10–16, with tumor 2 for 231.sh mdmx used in lane 10 as a common reference)

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: RNA Expression, Knockdown, Quantitative RT-PCR, Plasmid Preparation, Expressing, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 in TNBC promote metastasis, and in ERα + breast cancer MDM2 promotes proliferation. In TNBC, MDMX promotes expression of CXCR4 and PTGS2 with associated release of CTCs but no increase in cell proliferation. In ERα + breast cancer, estrogen stimulates MDM2 expression with no influence on CXCR4 and causes an increase in cell proliferation without correlated metastasis

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Expressing