Journal: Nature Communications
Article Title: Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells
doi: 10.1038/s41467-024-54640-0
Figure Lengend Snippet: a Workflow of in vivo labeling strategy using TAM and the experimental design. b PCA indicating the variations of transcriptomes among Lin - ZsGreen + cells isolated from BF-Ctrl, BF-cKO, MSFL-Ctrl and MSFL-cKO groups. c GO and KEGG enrichment analysis of FACS-RNA-seq data. d Heatmap of replicate data for H3K4me1 and H3K27ac enrichment as detected by CUT&Tag. n = 3 from 3 biological replicates. e GO-biological process enrichment analysis of differentially enriched super-enhancers. f Heatmap of replicate data for Zfp260-V5 enrichment detected by ChIP-seq. n = 2 from 2 biological replicates. g Top enriched de novo motifs of Zfp260-V5 enriched genes. h Distribution of peaks in the genome. i GO and KEGG enrichment analysis of Zfp260-V5 enriched genes. j Screening strategy for the potential master downstream regulator. k Transcripts Per Kilobase (TPM) of Runx2 expression level from fracture and MSFL derived Lin - ZsGreen + cells. n = 3 from 3 biological replicates of RNA-seq data. l Genome browser view of peaks enriched for H3K4me1, Brd4, H3K27ac, and Zfp260-V5 over the Runx2 gene locus on chromosome 17 (left) with the magnified super-enhancer region displayed on the right. Primers 1 and 2 indicated the primer sets for the subsequent ChIP-qPCR detection. m Co-IP was performed to examine the condensates for the super-enhancer via immortalized PSCs. n = 3 from 3 biological replicates. n , o mIHC co-staining for Zfp260 (purple) with Brd4 (gold), Med1 (cyan), and P300 (gray) in the homeostatic and osteogenic states of PSCs. The yellow dotted line indicated the route for the subsequent fluorescence intensity measurements. n = 3 from 3 biological replicates. p Fluorescence intensity measurements along the route, with black triangles indicating the merged signals of the four channels. q , r ChIP-qPCR assays for H3K27ac and Brd4 binding via immortalized PSCs. n = 6 from 2 biological replicates. Two-way ANOVA. Scalebars: 5 μm. All data in this figure are represented as mean ± SD. Source data and exact p values are provided in the Source Data file.
Article Snippet: The primary antibodies used in mIHC (dilution 1:400 for all antibodies) included goat anti-mouse/human/rat Itgav (AF1219, Novus Biologicals), mouse anti-mouse/rat CD90 (NB100-65543, Novus Biologicals), mouse anti-mouse/human CD105 (NBP2-22122, Novus Biologicals), rabbit anti-human/mouse/rat CD200 (AF2724, Novus Biologicals), rabbit anti-mouse/human/rat Runx2 (ab236639, Abcam), rabbit anti-mouse/human/rat Sox9 (ab185966, Abcam), rabbit anti-mouse/human Alpl (MA5-24845, Invitrogen), rabbit anti-mouse/human/rat Zfp260 (ABE295, Merck), mouse anti-human/mouse/rat p300 (NB100-616, Novus Biologicals), rabbit anti-human/mouse MED1 (NB100-2574, Novus Biologicals), rabbit anti-human/mouse BRD4 (NBP2-76393, Novus Biologicals), mouse anti-human/mouse/rat Prkca (NB600-201, Novus Biologicals), rabbit anti-V5 tag (13202, CST), mouse anti-Collagen type I (67288-1-Ig, proteintech), rabbit anti-Collagen type II (28459-1-AP, proteintech).
Techniques: In Vivo, Labeling, Isolation, RNA Sequencing, ChIP-sequencing, Expressing, Derivative Assay, ChIP-qPCR, Co-Immunoprecipitation Assay, Staining, Fluorescence, Binding Assay
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