Journal: Chemical Senses

Article Title: Commensal Bacteria Regulate Gene Expression and Differentiation in Vertebrate Olfactory Systems Through Transcription Factor REST

doi: 10.1093/chemse/bjz050

Figure Lengend Snippet: NRSF/REST-binding sites are commonly enriched at genes that are downregulated in olfactory tissue in response to microbiota. (A) TF motif enrichment (−log10P-value) using the 1000 bp upstream of the TSS of genes that are significantly more highly expressed in CV conditions in zebrafish and mouse olfactory tissue. A dashed line represents the threshold for motif enrichment. TBX5 and STAT4 motifs that are enriched in both zebrafish and mouse are marked in red and within the gray area. (B) Same as (A) for genes that were significantly higher in GF conditions. NRSF/REST and LHX2 motifs that are enriched in both zebrafish and mouse are marked in red and within the gray area (P < 0.05). (C) Gene expression levels of REST in GF zebrafish and mouse compared to CV animals (N = 4). Asterisk indicates statistically significant differences when P < 0.05. (D) Protein expression of REST in brain and olfactory organ of adult mice (lanes 1–2), juvenile mice (lanes 3–4), adult zebrafish brain and olfactory organ (lanes 5–6), and olfactory organs of CV and GF zebrafish larvae (lanes 7–8). Blots probed with an isotype control instead of anti-REST antibody produced no bands. GAPDH was used as a loading control.

Article Snippet: Samples were loaded onto a mini Protean TGX precast gel (Bio-Rad), transferred to a PVDF membrane, and incubated for 2 h at room temperature with either the primary antibody rabbit anti-human NRSF/REST (1:500, GeneTex, GTX37363), which recognizes 2 bands (1 of 120 KDa and 1 of 55 KDa) or with the primary rabbit polyclonal GAPDH antibody (1:1000, GeneTex, GTX100118).

Techniques: Binding Assay, Expressing, Produced

Journal: Chemical Senses

Article Title: Commensal Bacteria Regulate Gene Expression and Differentiation in Vertebrate Olfactory Systems Through Transcription Factor REST

doi: 10.1093/chemse/bjz050

Figure Lengend Snippet: Silencing NRSF/REST results in decreased differentiation of Odora cells in vitro. (A) REST mRNA levels in differentiated Odora cells, Odora cells treated with siNEG oligo (negative control), siGAPDH oligo (positive control), and a cocktail of 3 different siREST oligos. Gene expression levels were measured by RT-qPCR. Relative expression levels are shown as the ratio between REST expression and the housekeeping gene EF1a. ***P < 0.001. (B) OMP mRNA levels; (C) oCNGb mRNA levels; and (D) ADCY3 mRNA levels in undifferentiated Odora cells, differentiated Odora cells (Diff. positive control), Odora cells differentiated in the presence of S. aureus and the negative control silencing probe (siNEG), Odora cells differentiated in the presence of S. aureus and the GAPDH silencing probe, differentiated Odora cells and siREST probe (Diff. positive control + siREST), Odora cells differentiated in the presence of S. aureus and the siREST probe. Different letters denote statistically significant differences among treatments following 1-way ANOVA and Tukey’s post hoc test (P < 0.05).

Article Snippet: Samples were loaded onto a mini Protean TGX precast gel (Bio-Rad), transferred to a PVDF membrane, and incubated for 2 h at room temperature with either the primary antibody rabbit anti-human NRSF/REST (1:500, GeneTex, GTX37363), which recognizes 2 bands (1 of 120 KDa and 1 of 55 KDa) or with the primary rabbit polyclonal GAPDH antibody (1:1000, GeneTex, GTX100118).

Techniques: In Vitro, Negative Control, Positive Control, Expressing, Quantitative RT-PCR