Journal: Science Advances
Article Title: Modulating immune cell fate and inflammation through CRISPR-mediated DNA methylation editing
doi: 10.1126/sciadv.adt1644
Figure Lengend Snippet: ( A ) Clustering of the DEGs during transdifferentiation between dCas9-DNMT3A CTRL and IL1RN edited cells. ( n = 2 biologically independent replicates, FDR < 0.05). TC1 to TC4, transdifferentiation clusters. Representative genes for each cluster are annotated. ( B ) Gene Ontology Biological Processes (GO:BP) enrichment analysis for the genes associated with clusters in (A). The top five most significant terms for each cluster are plotted. Significant terms ( q value < 0.05) are highlighted with a black stroke. ( C ) Mean fluorescence intensity (MFI) of macrophage-lineage surface markers in CTRL and edited iMacs. ( D ) Representative histograms showing signal intensity for selected cell surface markers in CTRL and edited iMacs. ( E ) Phagocytic capacity evaluation [by fluorescence-activated cell sorting (FACS)] for CTRL and edited iMacs. Left, histogram showing the uptake of blue fluorescent beads. Right, blue beads MFI quantification. Unpaired two-tailed Student’s t test, n = 3, means ± SEM, (*** P < 0.001). ( F ) Up, schematic of the IL1RN shRNAs experiments during transdifferentiation. Bottom, MFI quantification of myeloid markers in shLuc and shIL1RN iMacs. Two-way ANOVA with Dunnett’s post hoc test, n = 3, means ± SEM, (**** P < 0.0001). ( G ) Phagocytic capacity evaluation (by FACS) for shLuc and shIL1RN iMacs. Left, histogram showing the uptake of blue fluorescent beads. Right, blue beads MFI quantification. Two-way ANOVA with Dunnett’s post hoc test, n = 3, means ± SEM, (**** P < 0.0001). ( H ) Left, schematic of the colony-forming assay performed in shLuc and shIL1RN bone marrow–derived human CD34 + cells; CFU, colony forming unit. Right, total number of CFUs obtained from shLuc and shIL1RN_1/_2 CD34 + cells. n = 3 biologically independent samples per group, means ± SEM. ( I ) Comparison of different types of CFUs obtained from shLuc and shIL1RN_1/_2 CD34 + cells. Two-way ANOVA with Dunnett’s post hoc test, n = 3, means ± SEM,(* P < 0.05; ** P < 0.01; **** P < 0.0001).
Article Snippet: Primary human bone-marrow CD34 + cells (STEMCELL Technologies, catalog no. 70002.3) were cultured in StemSpan SFEM (STEMCELL Technologies, catalog no. 9600) supplemented with StemSpan TM CC100 (STEMCELL Technologies, catalog no. 2690). shLuc or IL1RN shRNA_1/2–expressing viruses were centrifuged onto RetroNectin-coated plates prepared according to manufacturer’s instructions.
Techniques: Fluorescence, FACS, Two Tailed Test, Derivative Assay, Comparison
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![( A ) Clustering of the DEGs during transdifferentiation between dCas9-DNMT3A CTRL and IL1RN edited cells. ( n = 2 biologically independent replicates, FDR < 0.05). TC1 to TC4, transdifferentiation clusters. Representative genes for each cluster are annotated. ( B ) Gene Ontology Biological Processes (GO:BP) enrichment analysis for the genes associated with clusters in (A). The top five most significant terms for each cluster are plotted. Significant terms ( q value < 0.05) are highlighted with a black stroke. ( C ) Mean fluorescence intensity (MFI) of macrophage-lineage surface markers in CTRL and edited iMacs. ( D ) Representative histograms showing signal intensity for selected cell surface markers in CTRL and edited iMacs. ( E ) Phagocytic capacity evaluation [by fluorescence-activated cell sorting (FACS)] for CTRL and edited iMacs. Left, histogram showing the uptake of blue fluorescent beads. Right, blue beads MFI quantification. Unpaired two-tailed Student’s t test, n = 3, means ± SEM, (*** P < 0.001). ( F ) Up, schematic of the IL1RN shRNAs experiments during transdifferentiation. Bottom, MFI quantification of myeloid markers in shLuc and shIL1RN iMacs. Two-way ANOVA with Dunnett’s post hoc test, n = 3, means ± SEM, (**** P < 0.0001). ( G ) Phagocytic capacity evaluation (by FACS) for shLuc and shIL1RN iMacs. Left, histogram showing the uptake of blue fluorescent beads. Right, blue beads MFI quantification. Two-way ANOVA with Dunnett’s post hoc test, n = 3, means ± SEM, (**** P < 0.0001). ( H ) Left, schematic of the colony-forming assay performed in shLuc and shIL1RN bone marrow–derived human <t>CD34</t> + cells; CFU, <t>colony</t> <t>forming</t> <t>unit.</t> Right, total number of CFUs obtained from shLuc and shIL1RN_1/_2 CD34 + cells. n = 3 biologically independent samples per group, means ± SEM. ( I ) Comparison of different types of CFUs obtained from shLuc and shIL1RN_1/_2 CD34 + cells. Two-way ANOVA with Dunnett’s post hoc test, n = 3, means ± SEM,(* P < 0.05; ** P < 0.01; **** P < 0.0001).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6127/pmc12266127/pmc12266127__sciadv.adt1644-f4.jpg)