Journal: Carcinogenesis

Article Title: miR-106b downregulates adenomatous polyposis coli and promotes cell proliferation in human hepatocellular carcinoma.

doi: 10.1093/carcin/bgs320

Figure Lengend Snippet: Fig. 4. miR-106b activates the Wnt/β-catenin pathway. (A) Left panel, western blotting analysis of expression of cyclin D1, phosphorylated pRb (p-pRb) and total pRb protein in indicated HCC cells. α-Tubulin served as the loading control. Right panel: Real-time PCR analysis of expression of cyclin D1 in indicated HCC cells. GAPDH was used as a loading control. (B) Cyclin D1-luciferase reporter gene assays with wild-type promoter and TCF-binding site-mutated promoter were performed in indicated cells. (C) TOP/FOP luciferase ratio reported Wnt/β-catenin pathway activity in the indicated cells. (D) Relative mRNA expression of Wnt/β-catenin-regulated genes in the indicated cells was assessed by real-time PCR. GAPDH was used as a loading control. (E) Western blotting analysis of β-catenin expression in the cytoplasm (C) and nucleus (N) of the indicated cells. Nuclear protein p84 was used as a nuclear protein marker and EF-1α as a loading control. (F) Flow cytometric analysis of the indicated HCC cells transfected with miR-106b or miR-106 combined with TCF4-dn. Each bar represents the mean of three independent experiments. *P < 0.05.

Article Snippet: Cyclin D1 promoter-Wt (plasmid 32727; Addgene) and cyclin D1 promoter-Mut (plasmid 32733; Addgene) were used to examine the Wnt/β-catenin-dependent regulation of cyclin D1 expression.

Techniques: Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction, Luciferase, Binding Assay, Activity Assay, Marker, Transfection

Journal: bioRxiv

Article Title: Hotspot ESR1 mutations are multimodal and contextual drivers of breast cancer metastasis

doi: 10.1101/2021.02.10.430701

Figure Lengend Snippet: A & B. Representative images (A) and quantification (B) of wound scratch assay of T47D WT and ESR1 mutant cells performed using IncuCyte living imaging system over 72 hours. The migratory region normalized to T0 are labelled in blue. Images were taken under 10x magnification. Cell migration rates were quantified based on relative wound densities with 8 biological replicates. Representative experiment from 11 independent repeats is shown. Pairwise two-way ANOVA between WT and each mutant was performed. (** p<0.01) C. Representative magnified images of the migratory edge of each group in wound scratch assays in A. D & E. Representative images (D) and quantification (E) of spheroid collective migration assays in T47D mutant cells. T47D cells were initially seeded into round bottom ULA plates to form spheroids, which were then transferred onto collagen I coated plates. Collective migration was measured after 4 days. The migratory edge of each spheroid is circled with a white line. Migratory distances were calculated based on the mean radius of each spheroid normalized to corresponding original areas. Representative experiment from three independent repeats is shown. Dunnett’s test was used for statistical analysis. (** p<0.01) F. Dot plots representing the enrichment distribution of the 50 MSigDB curated Hallmark gene sets in T47D-Y537S and T47D-D538G models normalized to WT cells.Significantly enriched gene sets (FDR<0.25) are highlighted in red, with names labeled in the venn diagram plot on the right panel. Gene sets enriched in Y537S and D538G cell models are in green and blue circles respectively. G. Immunoblot detection of ß-catenin, phospho-GSK3ß (Ser9), phospho-GSK3α (Ser21) total GSK3ß and total GSK3α levels in T47D WT and mutant cells after hormone deprivation. Tubulin was blotted as a loading control. Representative blots from three independent repeats is displayed for each protein. H. Quantification of IncuCyte wound scratch assay with or without 5μM LGK974 treatment for 72 hours. The migratory region normalized to T0 are labelled in blue. Images were taken under 10x magnification. Cell migration rates were quantified based on relative wound densities with eight biological replicates. Representative experiment from three independent repeats is shown. Pairwise two-way ANOVA between WT and each mutant was performed. (** p<0.01) I. IncuCyte migration assay with combination treatment of four different doses of LGK974 and Fulvestrant in T47D-D538G cells. Inhibition rates were calculated using the wound density at 48 hours normalized to vehicle control with values labelled using color scales in the heatmap. Positive Bliss scores are considered a synergistic combination. Representative experiment from three independent repeats is shown. J. Dot plot representing the fold changes of all Wnt signaling component genes in both T47D ESR1 mutant cell models normalized to WT cells. The blue dotted frame highlights the unique T47D-D538G enriched genes as well as genes that are enriched in both mutants, but with a larger magnitude of enrichment in the T47D-D538G cells. K & L. Immunoblot validation of Fulvestrant-induced ER degradation (K) and FOXA1 knockdown (L). Cell lysates were subjected to ER and FOXA1 detection. Tubulin was blotted as a loading control. These validation experiments were performed once. M & N. Wound scratch assay in T47D-D538G and WT cells with 1μM of Fulvestrant treatment (M) or knockdown of FOXA1 (N) for 72 hours. Cell migration rates were quantified based on wound closure density. For fulvestrant treatment, data were merged from 3 (WT) or 6 (D538G) independent experiments. For FOXA1 knockdown, representative result from three independent repeats is displayed. Pairwise two-way ANOVA between siScramble/siFOXA1 or vehicle/Fulvestrant conditions in each cell type was performed. (* p<0.05, ** p<0.01) O. PCA plot showing the FOXA1 peak distribution of T47D WT, WT+E2, T47D-Y537S and T47D-D538G groups. P. Heatmaps representing the comparison of FOXA1 binding intensities in T47D-D538G mutants to FOXA1 binding in WT cells. Displayed in a horizontal window of ± 2kb from the peak center. The pairwise comparison between WT and mutant samples was performed to calculate the fold change (FC) of intensities. Binding sites were sub-classified into sites with increased intensity (FC>2), decreased intensity (FC<-2), and non-changed intensity (−22, FDR<0.005 in D538G vs WT excluding genes with fold change|>1.5, FDR<0.01 in WT+E2 vs WT groups). Intersected genes are indicated in the right panel. S. Wound scratch assay in T47D-WT and T47D-D538G cells with or without prior transfection of a dominant negative TCF4 plasmid for 72 hours. Pairwise two-way ANOVA between vehicle and treatment conditions was performed. Data from one representative experiment of three independent experiments (each with six biological repeats) is shown. (** p<0.01)

Article Snippet: For the dominant negative TCF4 overexpression experiment, Myc-tagged DN TCF4 plasmids (Addgene, #32729) were transiently transfected into targeted cells for a total of 24 hours before being subjected to the wound scratch assay.

Techniques: Wound Healing Assay, Mutagenesis, Imaging, Migration, Labeling, Western Blot, Inhibition, Binding Assay, RNA Sequencing Assay, Transfection, Dominant Negative Mutation, Plasmid Preparation

Journal: bioRxiv

Article Title: Hotspot ESR1 mutations are multimodal and contextual drivers of breast cancer metastasis

doi: 10.1101/2021.02.10.430701

Figure Lengend Snippet: Mutated ER triggers differential gene regulatory reprogramming through 1) ligandin-dependent transcriptional gene activation or repression, 2) secondary transcriptional regulation and 3) FOXA1-driven epigenetic remodeling. Ligand independent transcription constitutively induces or represses canonical ER regulated sites (e.g. TIMP3 ). Secondary transactivation induces gene expression indirectly via activation of an intermediate regulator (e.g. GJA1 ). Novel epigenetic remodeling includes de novo FOXA1 redistribution and increased chromatin accessibility at specific gene loci (e.g. TCF4 ). Consequently, increased desmosome and gap junction expression, TIMP3 -MMP axis alteration and hyperactivation of the Wnt pathway results in enhanced cell-cell adhesion, collagen invasion, migration and decreased cell-ECM adhesion, ultimately facilitating metastases of ESR1 mutant cells. Corresponding therapeutic vulnerabilities can be efficiently targeted by carbenoxolone, marimatsat and LGK974. These mechanisms are highly context dependent with phenotypes labeled for specific cell line models.

Article Snippet: For the dominant negative TCF4 overexpression experiment, Myc-tagged DN TCF4 plasmids (Addgene, #32729) were transiently transfected into targeted cells for a total of 24 hours before being subjected to the wound scratch assay.

Techniques: Activation Assay, Expressing, Migration, Mutagenesis, Labeling

Journal: Nature Communications

Article Title: Concurrent BMP7 and FGF9 signalling governs AP-1 function to promote self-renewal of nephron progenitor cells

doi: 10.1038/ncomms10027

Figure Lengend Snippet: ( a , b ) CCNE1 (G1-phase) and PCNA (S-phase) immunostaining of E14.5 and E17.5 NPCs treated for 24 h with BMP7, TAK1 and JNK inhibitors. Graphs show the percentage of G1, G1-S and S-phase cells per condition. Three biological replicates analysed per condition ( n =3). Error bars represent s.d. ** P <0.005 and ** P <0.001, Student's t -test. ( c , d ) RT–qPCR of JUN and MYC targets in E14.5 and E17.5 NPCs treated with vehicle, BMP7, TAK1 and JNK inhibitors for 2 h. Error bars indicate s.d. Three biological replicates analysed per condition ( n =3). ( e , f ) RT–qPCR of JUN and MYC targets on NPCs isolated from E14.5 Jun het , Jun NP C and E17.5 Tak1 het , Tak1 NPC kidneys. Error bars represent s.d. Two biological replicates analysed per genotype ( n =2). ( g , h ) CCND1 and CCNE1 immunostaining in E14.5 Jun het , Jun NPC and E17.5 Tak1 C-WT , Tak1 C-NPC kidneys. Scale bars, 100 and 150 μM. Quantitation of CCND1+ and CCNE1+ cells per cap mesenchyme (yellow arrows) was calculated by scoring at least 30 random cap mesenchymes per kidney section per genotype for a total of 5 sections from each experimental group. Error bars indicate mean (s.d.). ** P <0.005 and ** P <0.001, Student's t -test. ( i ) Model for BMP7-TAK1-JNK-JUN pathway regulation of NPC self-renewal in early (E14.5) and later (E17.5) phases of nephrogenesis. CD, collecting duct; CM, cap mesenchyme.

Article Snippet: 3 × AP1-pGL3 was a gift from Dr Alexander Dent (Addgene # 40342) . pGL3Basic-962 CCND1 promoter luciferase), pGL3Basic-962 CCND1 promoter AP-1 site mutant (Addgene #32727 and # 32728) were gifts from Dr Frank McCormick . pRL-CMV (Renilla-Luciferase) was obtained from Promega.

Techniques: Immunostaining, Quantitative RT-PCR, Isolation, Quantitation Assay

Journal: Nature Communications

Article Title: Concurrent BMP7 and FGF9 signalling governs AP-1 function to promote self-renewal of nephron progenitor cells

doi: 10.1038/ncomms10027

Figure Lengend Snippet: ( a ) Immunostaining of EdU (S-phase) and pHH3 (M-phase), ( b ) CCNE1 (G1-phase) and PCNA (S-phase) in E17.5 NPCs stimulated with vehicle, BMP7, FGF9 or BMP7+FGF9 for 24 h. Scale bars, 50 μM. Graphs show the percentage of EdU+,pHH3+ cells and G1, G1-S and S-phase cells in each condition. Error bars represent mean (s.d.). ** P <0.005 and P <0.001 (Student's t -test). Two to three biological replicates analysed per condition ( n =2 in a ) and ( n =3 in b ) ( c , d ) RT–qPCR of cell cycle genes ( Ccnd1 and Myc ), Jun and Fos in NPCs stimulated with vehicle, BMP7, FGF9 or BMP7+FGF9 for 2 h. Error bars represent s.d. Three biological replicates analysed per condition, n =3. ( e , f ) pJUN and pFOS immunoblot of NPCs stimulated with vehicle, BMP7, FGF9 or BMP7+FGF9 for 20 min. Graph shows the relative density of pJUN and pFOS normalized to β-tubulin in each condition . ( g – i ) Bars in the graphs represent the average fold change in luciferase activity of 3xAP1-Luc, CCND1-Luc and CCND1 ΔAP-1 -Luc in NPCs stimulated with BMP7 and FGF9 relative to vehicle treatment for 24 h. Three biological replicates analysed per condition ( n =3). Error bars represent s.d. ** P <0.005, NS, not significant P >0.05, Student's t -test.

Article Snippet: 3 × AP1-pGL3 was a gift from Dr Alexander Dent (Addgene # 40342) . pGL3Basic-962 CCND1 promoter luciferase), pGL3Basic-962 CCND1 promoter AP-1 site mutant (Addgene #32727 and # 32728) were gifts from Dr Frank McCormick . pRL-CMV (Renilla-Luciferase) was obtained from Promega.

Techniques: Immunostaining, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) Effect of CBDL on protein levels of the c-Myc, Mnt, Max, p53, and cyclin D1 in liver tissues. Protein samples from liver tissues of CBDL mice from 0 to 28 days were analyzed by Western Blot analysis. To ensure equal loading, membranes were stripped and re-probed with anti-actin antibodies. Total protein (15 ug/lane) was loaded for protein expression analysis. Representative blots from at least 3 mice for each time point are shown and densitometric values are shown in supplemental figure 1A. B) Effects of CBDL on mRNA levels of c-myc, Mnt, Max, p53, and cyclin D1 in liver tissues. RNA was isolated from liver tissues of CBDL mice from day 0 to day 28 for Northern blot analysis (15 μg/lane). Membranes were stripped and re-probed with β-actin to ensure equal loading. Representative blots from at least 3 mice for each time point are shown and densitometric values are shown in supplemental figure 1B. C) Effects of CBDL on mRNA levels of c-myc, Mnt, Max, p53, and cyclin D1 in hepatocytes isolated from CBDL mice from day 0 to 28 post operation. Representative blots from at least 3 mice for each time point are shown and densitometric values are shown in supplemental figure 1C.

Article Snippet: The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Western Blot, Expressing, Isolation, Northern Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) E-box sequences of mouse and human cyclin D1 and p53. The E-box sequence of mouse and human cyclin D1 is completely conserved and the E-box sequence of mouse p53 has only one mismatch as compared to the human p53. B) EMSA analysis using the mouse p53 E-box element showed the top band (Mnt) was decreased and the bottom band (c-Myc) was increased from day 0 to day 14 after CBDL. C) Supershift analysis using the mouse p53 E-box element identified bound proteins to include c-Myc and Mnt. The c-Myc antibody generated a strong supershift band derived from lower band; while the Mnt antibody supershifted the top band. Similar results were obtained using the mouse cyclin D1 E-box element (not shown).

Article Snippet: The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Sequencing, Generated, Derivative Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) Western blot analysis of c-Myc, Mnt, Max, p53, and cyclin D1 in LCA (100 μM) treated HuH-7 cells from 1 to 48 hours. To ensure equal loading, membranes were stripped and re-probed with anti-actin antibodies. Total protein (15 μg/lane) was used for assay. Representative blots from at least 3 independent experiments for each time point are shown and densitometric values are shown in supplemental figure 2A. B) Effects of LCA on protein levels of c-Myc, Mnt, p53 and cyclin D1 in primary human hepatocytes treated for varying time points. Total protein (15 μg/lane) was subjected to Western Blot analysis. Membranes were stripped and re-probed with actin for housekeeping control. Representative blots from at least 3 independent experiments for each time point are shown and densitometric values are shown in supplemental figure 2B. C) Dose-response of LCA on apoptosis in HuH-7 cells. HuH-7 cells were treated with LCA (0 to 125 μM) for 24 hours and apoptosis was assessed as described in Methods. Results represent mean±SEM from 3 experiments. *p<0.05, **p<0.005 vs. 0. D) Dose-response of LCA on p53, cyclin D1, c-Myc, and Mnt protein levels in HuH-7 cells. HuH-7 cells were treated with LCA (0 to 100 μM) for 24 hours and protein levels were determined by Western blot analysis as described above. E) Effect of LCA on HuH-7 and primary human hepatocyte apoptosis. After LCA treatment (100 μM), apoptosis increased from 24 hours to 48 hours in HuH-7 and from 12 hours to 36 hours in primary hepatocytes. Results represent mean±SEM from 3 independent experiments. *p<0.05 vs. respective controls for the different time points.

Article Snippet: The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Western Blot, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) Efficiency of c-myc, p53 and cyclin D1 knockdown in HuH-7 cells. Cells were treated with siRNA for 36 hours and mRNA levels were determined by Northern blot analysis. Scrambled siRNA had no effect (not shown). B) Effect of siRNA against c-myc, p53, and cyclin D1 on protein levels of c-Myc, p53 and cyclin D1 in LCA-treated HuH-7 cells. HuH-7 cells were transfected with siRNA for these genes for 0, 8, 16, 24 or 48 hours and then treated with LCA for 8 hours for evaluation of c-Myc, p53 and cyclin D1 knockdown efficiency. Numbers below each blot refer to densitometric values as % of 0 h (LCA treatment alone). C) Effect of c-myc, p53 and cyclin D1 siRNA on LCA- mediated HuH-7 apoptosis. HuH-7 cells were transfected with siRNA for 24 hours followed by LCA treatment for another 24 hours. *p<0.05 vs. respective controls, †p<0.05 vs. LCA+scrambled siRNA. D) Effect of c-myc siRNA on Mnt and Myc nuclear binding activity to E box of p53 promoter in HuH-7 cells. HuH-7 cells were treated with c-myc siRNA for 48 hours and EMSA for p53 promoter E-box binding was performed as described in Methods. For specificity, 50X cold probe was added. E) Effect of Mnt knockdown on baseline and LCA-mediated nuclear binding to E-box. HuH-7 cells were treated with Mnt siRNA for 48 hours followed by LCA (100 μM) for another 8 hours. EMSA for p53 promoter E-box binding activity was performed as above. F) Effect of Mnt knockdown on baseline and LCA-mediated induction of p53 and cyclin D1 protein levels. HuH-7 cells were treated with Mnt siRNA for 48 hours followed by LCA (100 μM) for another 8 hours and Western blot analysis was performed as described in Methods.

Article Snippet: The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Knockdown, Northern Blot, Transfection, Binding Assay, Activity Assay, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: Effects of LCA and c-myc siRNA on promoter activity of p53 and cyclin D1. A) HuH-7 cells were treated with c-myc siRNA or scrambled siRNA for 24 hours, then transfected with either the p53 or cyclin D1 promoter for another 10 hours. LCA (100 μM) was added during the last 8 hours of the p53 or cyclin D1 promoter transfection. Results represent mean±SEM from 3 independent experiments done in triplicates. *p<0.05 vs. control or scrambled siRNA. †p<0.05 vs. LCA. B) The protocol used was the same as in A) except the promoter constructs consisted of only the E-box elements of these promoters in native or mutated sequences as described in Methods. Results represent mean±SEM from 3 independent experiments done in triplicates. *p<0.05 vs. respective control.

Article Snippet: The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Activity Assay, Transfection, Control, Construct

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) Northern blot analysis of c-myc, p53 and cyclin D1 in livers of CBDL and sham-operated mice treated with lentivirus harboring c-myc siRNA or empty vector injection. See supplemental figure 3A for densitometric analysis. B) Northern blot analysis of c-myc, p53 and cyclin D1 in left lobes and right lobes from LHBDL mice treated with lentivirus harboring c-myc siRNA or empty vector injection. See supplemental figure 3B for densitometric analysis. C) Effect of c-myc siRNA on Myc and Mnt nuclear binding activity in LHBDL mice. Mice were treated with c-myc siRNA or empty vector followed by LHBDL as described in Methods. EMSA for E-box binding of p53 was performed using nuclear protein extracts from the left and right lobes 3 days later.

Article Snippet: The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Northern Blot, Plasmid Preparation, Injection, Binding Assay, Activity Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) Effect of CBDL on protein levels of the c-Myc, Mnt, Max, p53, and cyclin D1 in liver tissues. Protein samples from liver tissues of CBDL mice from 0 to 28 days were analyzed by Western Blot analysis. To ensure equal loading, membranes were stripped and re-probed with anti-actin antibodies. Total protein (15 ug/lane) was loaded for protein expression analysis. Representative blots from at least 3 mice for each time point are shown and densitometric values are shown in supplemental figure 1A. B) Effects of CBDL on mRNA levels of c-myc, Mnt, Max, p53, and cyclin D1 in liver tissues. RNA was isolated from liver tissues of CBDL mice from day 0 to day 28 for Northern blot analysis (15 μg/lane). Membranes were stripped and re-probed with β-actin to ensure equal loading. Representative blots from at least 3 mice for each time point are shown and densitometric values are shown in supplemental figure 1B. C) Effects of CBDL on mRNA levels of c-myc, Mnt, Max, p53, and cyclin D1 in hepatocytes isolated from CBDL mice from day 0 to 28 post operation. Representative blots from at least 3 mice for each time point are shown and densitometric values are shown in supplemental figure 1C.

Article Snippet: P53 and cyclin D1 promoter and E-box mutational analysis The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Western Blot, Expressing, Isolation, Northern Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) E-box sequences of mouse and human cyclin D1 and p53. The E-box sequence of mouse and human cyclin D1 is completely conserved and the E-box sequence of mouse p53 has only one mismatch as compared to the human p53. B) EMSA analysis using the mouse p53 E-box element showed the top band (Mnt) was decreased and the bottom band (c-Myc) was increased from day 0 to day 14 after CBDL. C) Supershift analysis using the mouse p53 E-box element identified bound proteins to include c-Myc and Mnt. The c-Myc antibody generated a strong supershift band derived from lower band; while the Mnt antibody supershifted the top band. Similar results were obtained using the mouse cyclin D1 E-box element (not shown).

Article Snippet: P53 and cyclin D1 promoter and E-box mutational analysis The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Sequencing, Generated, Derivative Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) Western blot analysis of c-Myc, Mnt, Max, p53, and cyclin D1 in LCA (100 μM) treated HuH-7 cells from 1 to 48 hours. To ensure equal loading, membranes were stripped and re-probed with anti-actin antibodies. Total protein (15 μg/lane) was used for assay. Representative blots from at least 3 independent experiments for each time point are shown and densitometric values are shown in supplemental figure 2A. B) Effects of LCA on protein levels of c-Myc, Mnt, p53 and cyclin D1 in primary human hepatocytes treated for varying time points. Total protein (15 μg/lane) was subjected to Western Blot analysis. Membranes were stripped and re-probed with actin for housekeeping control. Representative blots from at least 3 independent experiments for each time point are shown and densitometric values are shown in supplemental figure 2B. C) Dose-response of LCA on apoptosis in HuH-7 cells. HuH-7 cells were treated with LCA (0 to 125 μM) for 24 hours and apoptosis was assessed as described in Methods. Results represent mean±SEM from 3 experiments. *p<0.05, **p<0.005 vs. 0. D) Dose-response of LCA on p53, cyclin D1, c-Myc, and Mnt protein levels in HuH-7 cells. HuH-7 cells were treated with LCA (0 to 100 μM) for 24 hours and protein levels were determined by Western blot analysis as described above. E) Effect of LCA on HuH-7 and primary human hepatocyte apoptosis. After LCA treatment (100 μM), apoptosis increased from 24 hours to 48 hours in HuH-7 and from 12 hours to 36 hours in primary hepatocytes. Results represent mean±SEM from 3 independent experiments. *p<0.05 vs. respective controls for the different time points.

Article Snippet: P53 and cyclin D1 promoter and E-box mutational analysis The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Western Blot, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) Efficiency of c-myc, p53 and cyclin D1 knockdown in HuH-7 cells. Cells were treated with siRNA for 36 hours and mRNA levels were determined by Northern blot analysis. Scrambled siRNA had no effect (not shown). B) Effect of siRNA against c-myc, p53, and cyclin D1 on protein levels of c-Myc, p53 and cyclin D1 in LCA-treated HuH-7 cells. HuH-7 cells were transfected with siRNA for these genes for 0, 8, 16, 24 or 48 hours and then treated with LCA for 8 hours for evaluation of c-Myc, p53 and cyclin D1 knockdown efficiency. Numbers below each blot refer to densitometric values as % of 0 h (LCA treatment alone). C) Effect of c-myc, p53 and cyclin D1 siRNA on LCA- mediated HuH-7 apoptosis. HuH-7 cells were transfected with siRNA for 24 hours followed by LCA treatment for another 24 hours. *p<0.05 vs. respective controls, †p<0.05 vs. LCA+scrambled siRNA. D) Effect of c-myc siRNA on Mnt and Myc nuclear binding activity to E box of p53 promoter in HuH-7 cells. HuH-7 cells were treated with c-myc siRNA for 48 hours and EMSA for p53 promoter E-box binding was performed as described in Methods. For specificity, 50X cold probe was added. E) Effect of Mnt knockdown on baseline and LCA-mediated nuclear binding to E-box. HuH-7 cells were treated with Mnt siRNA for 48 hours followed by LCA (100 μM) for another 8 hours. EMSA for p53 promoter E-box binding activity was performed as above. F) Effect of Mnt knockdown on baseline and LCA-mediated induction of p53 and cyclin D1 protein levels. HuH-7 cells were treated with Mnt siRNA for 48 hours followed by LCA (100 μM) for another 8 hours and Western blot analysis was performed as described in Methods.

Article Snippet: P53 and cyclin D1 promoter and E-box mutational analysis The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Knockdown, Northern Blot, Transfection, Binding Assay, Activity Assay, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: Effects of LCA and c-myc siRNA on promoter activity of p53 and cyclin D1. A) HuH-7 cells were treated with c-myc siRNA or scrambled siRNA for 24 hours, then transfected with either the p53 or cyclin D1 promoter for another 10 hours. LCA (100 μM) was added during the last 8 hours of the p53 or cyclin D1 promoter transfection. Results represent mean±SEM from 3 independent experiments done in triplicates. *p<0.05 vs. control or scrambled siRNA. †p<0.05 vs. LCA. B) The protocol used was the same as in A) except the promoter constructs consisted of only the E-box elements of these promoters in native or mutated sequences as described in Methods. Results represent mean±SEM from 3 independent experiments done in triplicates. *p<0.05 vs. respective control.

Article Snippet: P53 and cyclin D1 promoter and E-box mutational analysis The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Activity Assay, Transfection, Control, Construct

Journal: Hepatology (Baltimore, Md.)

Article Title: Switch from Mnt-Max to Myc-Max Induces p53 and cyclin D1 Expression and Apoptosis During Cholestasis in Mice and Human Hepatocytes

doi: 10.1002/hep.22720

Figure Lengend Snippet: A) Northern blot analysis of c-myc, p53 and cyclin D1 in livers of CBDL and sham-operated mice treated with lentivirus harboring c-myc siRNA or empty vector injection. See supplemental figure 3A for densitometric analysis. B) Northern blot analysis of c-myc, p53 and cyclin D1 in left lobes and right lobes from LHBDL mice treated with lentivirus harboring c-myc siRNA or empty vector injection. See supplemental figure 3B for densitometric analysis. C) Effect of c-myc siRNA on Myc and Mnt nuclear binding activity in LHBDL mice. Mice were treated with c-myc siRNA or empty vector followed by LHBDL as described in Methods. EMSA for E-box binding of p53 was performed using nuclear protein extracts from the left and right lobes 3 days later.

Article Snippet: P53 and cyclin D1 promoter and E-box mutational analysis The p53 promoter and the 3.3 kb human cyclin D1 promoter were gifts from Dr. S. Sukumar (Johns Hopkins Oncology Center, Baltimore, Maryland) and Dr. RG.

Techniques: Northern Blot, Plasmid Preparation, Injection, Binding Assay, Activity Assay