Journal: Antioxidants

Article Title: Murine Neonatal Oxidant Lung Injury: NRF2-Dependent Predisposition to Adulthood Respiratory Viral Infection and Protection by Maternal Antioxidant

doi: 10.3390/antiox10121874

Figure Lengend Snippet: Placenta transcriptome changes by prenatal sulforaphane (SFN) and affected biological functions and molecular networks predicted by pathway analyses in wild-type ( Nrf2 +/+ ) and Nrf2 -deficient ( Nrf2 −/− ) mice at embryonic day 18.5. ( A ) Hierarchical clustering analysis generated a heap map depicting expression profiles of placenta genes altered by maternal SFN in Nrf2 +/+ mice and Nrf2 −/− mice. Color bar indicates average expression intensity ( n = 3/group) normalized to PBS- Nrf2 +/+ group. ( B ) Prenatal SFN modulated 814 placenta genes in Nrf2 +/+ mice (1.5-fold changed 708 genes). Pathway analysis of SFN-altered transcriptome predicted inhibition of perinatal death and activation of vasculature development by upstream modulators prolactin (PRL) and immunoglobulin in Nrf2 +/+ placenta. A key network of SFN-influenced Nrf2 +/+ placenta genes was connected with p42/p44 mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) and predicted to play roles in protein synthesis and cellular assembly and organization. ( C ) Prenatal SFN modulated 634 placenta genes in Nrf2 −/− mice (1.5-fold changed 367 genes). Pathway analysis of SFN-altered Nrf2 −/− transcriptome also suggested potential inhibition of perinatal death and activation of vasculature development. MicroRNA miR-21-5p was suggested as one of the upstream modulators of Nrf2 −/− placenta transcriptome changes by maternal SFN. ERK was also predicted to play central roles in crosstalk of SFN-modulated Nrf2 −/− placenta genes involved in cellular assembly and organization and organ development. Molecular color and its intensity indicate increased (red) or decreased (green) regulation degree of the genes by maternal SFN compared to maternal PBS in each genotype. Ingenuity Pathway Analysis and GeneSpring software were used for data analyses.

Article Snippet: Lung nuclear (7 μg) or pooled total placenta (50 μg) proteins were subjected to Western blotting using antibodies against mouse c-Fos (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), lamin B (Santa Cruz, Dallas, TX, USA), phosphor-p42/p44 mitogen-activated protein kinase/extracellular signal-regulated kinase ( P -p42/p44 MAPK/ P -ERK, Santa Cruz) and pan-actin (Santa Cruz).

Techniques: Generated, Expressing, Inhibition, Activation Assay, Software

Journal: Antioxidants

Article Title: Murine Neonatal Oxidant Lung Injury: NRF2-Dependent Predisposition to Adulthood Respiratory Viral Infection and Protection by Maternal Antioxidant

doi: 10.3390/antiox10121874

Figure Lengend Snippet: Validation of prenatal sulforaphane (SFN) effects on neonate lung and placenta transcriptome and signaling pathways. ( A ) Aliquots of nuclear proteins (7 μg) were subjected to colorimetric DNA-binding activity assay for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 which was predicted to be involved in Nrf2 +/+ and Nrf2 −/− neonate lung transcriptomics altered by maternal SFN after hyperoxia (O 2 ) exposure. Group mean ± S.E.M presented ( n = 4/group). ( B ) Western blotting determined nuclear protein level of c-Fos transcription factor which was proposed to play roles in SFN-altered lung transcriptomics in Nrf2 +/+ and Nrf2 −/− neonates exposed to O 2 . Lamin B level was detected as a housekeeping control for nuclear proteins. Representative images from duplicate analyses presented. Scanned band images were quantitated by densitometry and relative protein band intensities normalize to PBS/air- Nrf2 +/+ were depicted (mean ± S.E.M.). kDa = kilodalton. ( C ) qPCR was performed to determine SFN-mediated changes in DNA base lesions caused by O 2 in nuclear and mitochondrial genomes. Lesion frequencies in genomic (DNA polymerase β gene) and mitochondrial DNA were compared in Nrf2 +/+ and Nrf2 −/− neonate lungs after air or O 2 . All data were normalized to air-exposed Nrf2 +/+ and group mean ± S.E.M. presented ( n = 4/group). Background noise level (dashed lines) is set at ± 0.15. *, significantly different from genotype-matched PBS/Air controls ( p < 0.05). +, significantly different from prenatal treatment/neonatal exposure matched Nrf2 +/+ mice ( p < 0.05). #, significantly different from genotype-matched PBS/hyperoxia (O 2 ) mice ( p < 0.05). ( D ) Placenta expressions of SFN-altered genes, ubiquitin carboxy-terminal hydrolase L1 ( Uchl1 ), glutathione-s-transferase, theta 1 ( Gstt1 ) and ADAM-like, decysin 1 ( Adamdec1 ) were determined by qRT-PCR. Group mean ± S.E.M. presented ( n = 3/group). ( E ) Western blotting determined placenta protein level of activated (phosphor) p42/p44 mitogen-activated protein kinase/extracellular signal-regulated kinase ( P -ERK) which was proposed to play roles in SFN-altered placenta transcriptomics of Nrf2 +/+ and Nrf2 −/− mice. Pan-actin level was determined as a housekeeping protein control for total proteins. Representative images from duplicate analyses presented. Scanned P -ERK band images were quantitated by densitometry and relative protein band intensities normalize to PBS- Nrf2 +/+ were depicted (mean ± S.E.M.). kDa = kilodalton. #, significantly different from genotype-matched PBS controls ( p < 0.05). +, significantly different from prenatal treatment matched Nrf2 +/+ mice ( p < 0.05).

Article Snippet: Lung nuclear (7 μg) or pooled total placenta (50 μg) proteins were subjected to Western blotting using antibodies against mouse c-Fos (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), lamin B (Santa Cruz, Dallas, TX, USA), phosphor-p42/p44 mitogen-activated protein kinase/extracellular signal-regulated kinase ( P -p42/p44 MAPK/ P -ERK, Santa Cruz) and pan-actin (Santa Cruz).

Techniques: Biomarker Discovery, Protein-Protein interactions, Binding Assay, Activity Assay, Western Blot, Control, Ubiquitin Proteomics, Quantitative RT-PCR