Journal: Nature Communications

Article Title: Leukemic stem cells activate lineage inappropriate signalling pathways to promote their growth

doi: 10.1038/s41467-024-45691-4

Figure Lengend Snippet: A Schematic showing how dnFOS was induced in Kasumi-1 cells B DNase1 was performed with and without dnFOS induced by doxycycline in the Kasumi-1 cell line, ranked by fold change of the tag count at distal peaks and represented as density plots (+/1 kb of the summit). The red bar indicates dnFOS specific sites and the green bar control specific sites where the normalised tag-count of specific sites is at least two-fold different. ChIP data from FOS, CEBPA, RUNX1, RUNX1::ETO, PU.1 and GATA2 with and without dnFOS were plotted on the same axis across the same window C Specific sites were calculated for the ChIPs shown in ( A ) where the normalised tag-count is at least two-fold different in a pairwise comparison of dnFOS against control. The normalised tag count was measured in a peak union generated from control or dnFOS specific sites from all ChIPs and the Spearman correlation calculated which is plotted as a heatmap with hierarchical clustering. D A motif enrichment score was calculated based on motif frequency in the specific gained (dnFOS) and lost (CTRL) sites calculated in ( B ) and plotted as a heatmap with hierarchical clustering. E Heatmap with hierarchical clustering showing the log 2 fold change between the normalised average peak height of ChIP-seq in Kasumi-1 with dnFOS vs controls at LSC-specific and blast-specific ATAC sites. F Average profiles were generated from the CPM normalised tag counts of ChIP for H3K27me3 (+/− 10 kb from the transcription start site (TSS)) and H3K4me3 (+/− 2 kb from the TSS), at the promoters of t(8;21) LSC or blast specific genes with or without induction of dnFOS in Kasumi-1 cells, poised LSC genes are those with both H3K27me3 and H3K4me3 at their promoter. G Density plots showing the signal at each of the sites used in ( F ), with active, silenced and poised genes indicated.

Article Snippet: Dynabeads protein G were pre-incubated with antibodies against FOS (Cat# MA5-15055 ThermoFisher), CEBPA (Cat# sc-61X Santa Cruz), RUNX1 (Cat# ab23980 Abcam), RUNX1::ETO (Cat# C15310197 Diagenode), PU.1 (Cat# sc-352 Santa Cruz), GATA2 (Cat# AF2046 R & D Systems), H3K27ac (Cat# ab4729 Abcam), H3K9acS10P (Cat# ab12181 Abcam) or H3K4me3 (Cat# 04-745 Millipore) for 2 h at 4°, then added to the chromatin.

Techniques: Control, Comparison, Generated, ChIP-sequencing

Journal: Genes & Development

Article Title: miRNAs cooperate in apoptosis regulation during C. elegans development

doi: 10.1101/gad.288555.116

Figure Lengend Snippet: The 3′ UTR of egl-1 is a target of miR-35 and miR-58 family miRNAs in vivo. ( A ) The 3′ UTR of egl-1 is illustrated in blue, with a reported C. elegans Argonaut ALG-1-binding site from a recent study indicated above . Sequences are given for predicted miR-35 family-binding sites and miR-58 family - binding sites, and conserved bases within each site are highlighted in gray across four Caenorhabditis species. The mutated sequences corresponding to egl-1 mut mir-35 (red) and egl-1 mut mir-58 (purple) are shown (for complete mutated sequences, see the Materials and Methods). ( B ) A schematic representation of the 3′ UTR reporters constructed for this study. ( C ) Analysis of single-copy 3′ UTR reporter expression during embryogenesis. The genetic background and transgene under investigation are indicated at the left of each image sequence, which shows representative embryos from three developmental stages. For each embryo, a DIC image ( left ) and GFP image ( right ) are shown. The following alleles were used: bcSi25 [P mai-2 gfp::h2b::mai-2 3′ UTR ], bcSi26 [P mai-2 gfp::h2b::egl-1 wt 3 ′ UTR], bcSi27 [P mai-2 gfp::h2b::egl-1 mut mir-35 3′ UTR ], bcSi46 [P mai-2 gfp::h2b::egl-1 mut mir-35 3′ UTR ], and bcSi47 [P mai-2 gfp::h2b::egl-1 mut mir-35 mir-58 3′ UTR ]. Asterisks indicate autofluorescence. Transgenic strains were homozygous for unc-119(ed3) and the cb-unc-119(+) selection marker. Bars, 10 µm.

Article Snippet: Second, this P mai-2 fragment was fused to the 1287-bp coding sequence of gfp::h2b as amplified from the plasmid pCM1.35 (a gift from G. Seydoux; Addgene plasmid no. 17248) using the primers 5′-CTAATTCACTCAATTTTCAGAATGAGTAAAGGAGAAGAACTTTT-3′ and 5′-GCTCGCGTTCTTGTACTGCAAATTACTTGCTGGAAGTGTACTTG-3′.

Techniques: In Vivo, Binding Assay, Construct, Expressing, Sequencing, Transgenic Assay, Selection, Marker