Journal: Open Biology

Article Title: Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

doi: 10.1098/rsob.160091

Figure Lengend Snippet: ATF3 regulates injury-related expression of neuropeptides and other genes. ( a–i ) Unlesioned and injured FN of Atf3 +/+ and Atf3 −/− mice were subjected to qPCR analysis after 3 days of injury to quantify mRNA abundance of primers indicated. In wt mice (grey bars), facial nerve injury resulted in induction of Sprr2j ( a ), Vip ( b ), Ngf ( c ), Wnt2b ( d ), Galanin ( e ), Grp ( f ), Timp1 ( g ) and the known ATF3 target gene Hsp27 ( h ). In contrast to wt mice, induction of Sprr2j, Vip, Ngf, Wnt2b, Galanin, Grp and Hsp27 mRNA abundance was reduced upon facial nerve lesion in Atf3 mutant mice (white bars). Timp1 mRNA induction was more pronounced upon ATF3 loss-of-function ( g ). The Vip2 receptor ( Vipr2 ) was downregulated by facial nerve injury in wt and ATF3-deficient mice ( i ). Numbers in bars in ( b ) reflect independent biological replicates for experiments in ( a–i ). ( j–l ) Confirmation of reduced galanin expression in ATF3-deficient mice upon facial nerve injury. Deafferented wt ( j ) and ATF3-deficient ( k ) FN were stained with anti-galanin directed antibodies. In wt mice ( j ), galanin localized in secretory vesicle-like structures (see inset) of FMNs (some labelled with an arrow). The number of galanin immunoreactive FMNs was reduced in Atf3 mutant mice ( k ). ( l ) Quantification of galanin positive neurons without and 3 and 12 days after lesion. Data are presented as mean ± s.d. ** p ≤ 0.01. Scale bar ( j,k ) = 50 µm; inset = 5 µm.

Article Snippet: Camptothecin was added at 2 μM for 24 h. Recombinant peptides for mouse VIP (Tocris; no.1911), human gastrin releasing peptide (GRP) (Tocris; no.1789), mouse galanin (Tocris; no.2696) and mouse Wnt2b (R&D systems; no.3900-WN-025) were added at 1 nM (VIP, GRP, galanin) and 1 μg ml −1 (Wn2tb) for 24 h to the cultures.

Techniques: Expressing, Mutagenesis, Staining

Journal: Open Biology

Article Title: Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

doi: 10.1098/rsob.160091

Figure Lengend Snippet: ATF3 mediates neuropeptide and Wnt2b expression in primary PNS neurons. ( a–d ) Adult wt or ATF3-deficient mouse DRG neurons were infected with adenoviral (AV) particles resulting in GFP (control) or ATF3 expression. mRNA levels of Atf3 ( a ), Wnt2b ( b ), Galanin ( c ) and Grp ( d ) were analysed by qPCR. Viral infection strongly enhanced Atf3 mRNA abundance in wt and Atf3 mutant neurons ( a ). Wnt2b ( b ), Galanin ( c ) and Grp ( d ) mRNA levels were augmented upon viral ATF3 overexpression in wt and Atf3 mutant DRG neurons. ( e,f ) Primary wt neurons overexpressing GFP or ATF3 were subjected to ChIP analysis with anti-ATF3 or IgG (control) antibodies. ATF3 occupancy at potential ATF3 binding sites of the Galanin ( e ) and Grp ( f ) promoter was tested with qPCR. ATF3 promoter occupancy was observed in ATF3-overexpressing samples only in the presence of anti-ATF3 but not IgG antibodies suggesting ATF3 binding at the Galanin ( e ) and Grp ( f ) promoter. Numbers in bars reflect independent numbers of experiments. Data are presented as mean ± s.d. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Camptothecin was added at 2 μM for 24 h. Recombinant peptides for mouse VIP (Tocris; no.1911), human gastrin releasing peptide (GRP) (Tocris; no.1789), mouse galanin (Tocris; no.2696) and mouse Wnt2b (R&D systems; no.3900-WN-025) were added at 1 nM (VIP, GRP, galanin) and 1 μg ml −1 (Wn2tb) for 24 h to the cultures.

Techniques: Expressing, Infection, Control, Mutagenesis, Over Expression, Binding Assay

Journal: PloS one

Article Title: Differential effects of β-catenin and NF-κB interplay in the regulation of cell proliferation, inflammation and tumorigenesis in response to bacterial infection.

doi: 10.1371/journal.pone.0079432

Figure Lengend Snippet: Figure 4. Evidence of b-catenin and NF-kB interplay in vitro. A. Measurement of Wnt2b and Wnt5a expression in YAMC (Young Adult Mouse Colon) cells in vitro. YAMC cells (56105) were either uninfected (N) or infected with CR at 90:1 MOI for 3 hr. Cells were washed thoroughly to remove bacteria and incubated in fresh medium containing antibiotics for indicated period of time. Total RNA was examined for the expression of Wnt2b and Wnt5a via RT-PCR. GAPDH was used as loading control. B. Effect of Wnt2b knockdown on reporter activity. YAMC cells were transiently transfected with TOPflash plasmid and with siRNA specific to Wnt2b and Wnt5a, respectively. After 24 h, cells were infected with CR at 90:1 MOI for 3 h, washed to remove bacteria followed by measurement of reporter activity at 48 h using Renilla luciferase as internal control (w, p,0.05 vs. N; ww, p,0.05 vs. CR; *, p,0.05 vs. CR; n = 3 independent experiments). C. Effect of Wnt2b and Wnt5a addition on reporter activity. YAMC cells, following transient transfection for 24 h with TOPflash plasmid and with siRNA specific to Wnt2b and Wnt5a, respectively. After 24 hr, cells were incubated with purified Wnt2b or Wnt5a, infected with CR at 90:1 MOI for 3 h, washed to remove bacteria followed by measurement of reporter activity at 48 h using Renilla

Article Snippet: YAMC cells (56105) were transfected with either 100 nmol/L of scrambled siRNA or siRNAs specific for Wnt2b (sc-155356) and Wnt5a (sc-41113) using 2–8 ml of transfection reagent (sc-29528; Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 24 h. CR strain DBS 100 (ATCC cat.# 51459TM) was grown under aerophilic conditions on Luria-Bertani (LB) agar plates for 24 h at 37uC and cultured in LB broth O/N at 37uC.

Techniques: In Vitro, Expressing, Infection, Bacteria, Incubation, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Purification