pgl3basic 962 ccnd1 promoter ap 1 site mutant  (Addgene inc)

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