Journal: Development (Cambridge, England)

Article Title: Nelfb promotes dermal white adipose tissue formation through RNA polymerase II-mediated adipogenic gene regulation

doi: 10.1242/dev.204976

Figure Lengend Snippet: Nelfb is necessary for adipocyte differentiation by promoting the expression of adipocyte transcription factors. (A) Oil Red O staining of control (CTL: Nelfb fl/wt Pdgfra-Cre + or Nelfb wt/wt Pdgfra-Cre + ) and Nelfb −/− ( Nelfb fl/fl Pdgfra-Cre + ) cells cultured in adipocyte differentiation medium for 14 days ( N =4). (B) Staining of CTL and Nelfb −/− cells with antibodies against perilipin 1 (Plin1; mature adipocyte marker: green) and Hoechst 33342 (nuclei stain: blue) ( N =4). (C) RNA-seq analysis of CTL and Nelfb −/− cells cultured in adipocyte differentiation medium for 14 days. Volcano plot shows 762 genes increased (red) and 358 genes decreased (blue) on a log 2 scale upon Nelfb depletion. N =3. (D) Gene Ontology (GO) terms of the 358 decreased and 762 increased genes using Enrichr. (E) Heat map of the 43 genes (≥2-fold change) found in the top five GO terms of decreased genes in D. (F) CUT&RUN on CTL and Nelfb −/− cells cultured in adipocyte differentiation medium for 14 days using an anti-Nelfb and anti-IgG antibody. qPCR analysis was conducted on transcription start site (TSS) regions of Pparg , Cebpa , Cebpb , Stat3 and Krox20. Signal was calculated as a percent of total input DNA ( N =3). (G,H) CUT&RUN using an anti-RNA Polymerase II (RNA Pol II) antibody and anti-IgG antibody ( N =3). qPCR analysis was performed on the TSS (G) or gene body (H) regions of Pparg , Cebpa , Stat3 and Krox20 . (I,J) CUT&RUN using an anti-RNA Polymerase II Ser2 phosphorylation (Pol II Ser2 Phospho) antibody and anti-IgG antibody ( N =3). qPCR analysis was performed on the TSS (I) or gene body (J) regions of Pparg , Cebpa , Stat3 and Krox20 . (K) CUT&RUN using active chromatin mark anti-H3K4me3 and anti-IgG antibody. qPCR analysis on TSS regions of Pparg , Cebpa , Stat3 and Krox20 . Signal was calculated as a percent of total input DNA ( N =3). Data are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparison tests). ns, not significant. Scale bars: 150 μm.

Article Snippet: Quantities used were: Nelfb: 5 μl/reaction (Cell Signaling Technology, #14894S); RNA Pol II: 3 μl/reaction (Active Motif, #39097); RNA Pol II Ser2 Phosphorylation: 2 μl/reaction (Cell Signaling Technology, #13499S); Nelfe: 5 μl/reaction (Proteintech, #10705-1-AP); IgG: 5 μl/reaction (Cell Signaling Technology, #66362); H3K4me3: 5 μl/reaction (Cell Signaling Technology, #9751S); H3K27me3: 2 μl/reaction (Cell Signaling Technology, #9733S); H3K9me3: 2 μl/reaction (Cell Signaling Technology, #13969S); H3K27Ac: 1 μl/reaction (Cell Signaling Technology, #8173S).

Techniques: Expressing, Staining, Control, Cell Culture, Marker, RNA Sequencing, Phospho-proteomics, Comparison