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