myc t58a transgene  (Addgene inc)

Journal: The EMBO Journal

Article Title: HAND1 level controls the specification of multipotent cardiac and extraembryonic progenitors from human pluripotent stem cells

doi: 10.1038/s44318-025-00409-0

Figure Lengend Snippet: ( A ) Western blot of HAND1-AM-Tag (Active Motif) at day 3 and 4 of differentiation in different conditions compared to undifferentiated hESCs. ( B ) Motif enrichment analysis of HAND1 ChIP-seq peaks with control (low HAND1) and SB (high HAND1) sets merged. Significance is relative to the background control. ( C ) Doxycycline-inducible HAND1-BFP transgene expressed in HAND1-null cells for cell sorting by HAND1 expression (12-hour induction) and molecular analysis by ATAC- and RNA-seq (both performed on 3 biological replicates). ( C ’) Euler plots for the ATAC-seq analysis representing the number of differentially accessible chromatin regions in HAND1+ vs HAND1-negative cells and the subset with detected HAND1 binding. ( D ) Schematic illustration of the motifs enriched in chromatin with increased or decreased accessibility as a result of HAND1 expression. ( E ) Identification of HAND1[+] (activating) and HAND1[-] (repressing) regulons. ( F ) Chord plot showing significantly enriched gene ontology terms for HAND1 target genes. The red and blue text colouring indicates terms enriched in activated targets and repressed targets, respectively. ( G ) UMAP plots showing the activity of constant and HAND1-sensitive regulons in endoderm and mesoderm of wild-type and HAND1-null cells. The activity of the HAND1[+] and HAND1[-] regulons are shown with the domains of strong activity highlighted. ( H – J ) ATAC-seq in HAND1-null and HAND1+ (12 h induction) populations, and HAND1 binding at low-HAND1 (CTRL) and high-HAND1 (SB) levels at the ( H ) FOXF1 locus, ( I ) HOXB cluster and ( J ) CDX2 locus. ( K ) Gene regulatory network model of HAND1. * Direct early target of HAND1. The dashed line indicates a link at high HAND1 levels. CTRL control/vehicle-only, DEG differentially expressed genes, EMT epithelial-mesenchymal transition, GO gene ontology, WT wild-type. See also Appendix Fig. . .

Article Snippet: A TO-MYC system was introduced by transducing the hPSCs with two lentiviruses, one carrying a MYC T58A transgene under the control of a TRE-CMV promoter (Addgene ID 19775), and the second carrying the tetracycline transactivator (Addgene ID 19780).

Techniques: Western Blot, ChIP-sequencing, Control, FACS, Expressing, RNA Sequencing, Binding Assay, Activity Assay

Journal: The EMBO Journal

Article Title: HAND1 level controls the specification of multipotent cardiac and extraembryonic progenitors from human pluripotent stem cells

doi: 10.1038/s44318-025-00409-0

Figure Lengend Snippet: ( A ) Schematic illustration of HAND1 level with primitive streak-like patterning of hESCs into cardiac and extraembryonic mesoderm. ( B ) Live images (brightfield and fluorescence) showing HAND1-Tomato in EBs at day 5 of differentiation. ( C ) UMAP plots showing the target genes AUC score (regulon activity) for MYC and N-MYC with differentiation in wild-type cells. White indicates the score is below threshold. ( D ) Target genes AUC score for MYC, N-MYC, PGC-1α (PPARGC1A) and SRF through pseudotime of mesoderm differentiation. ( E ) Scaled target gene AUC scores in bulk populations by differentiation day and impact of induction of a dox-inducible MYC transgene at day 5–6. ( F ) Expansion of HAND1-high, HAND1-low, and HAND1-neg progenitors from SB, control and DMH1-treated conditions, respectively. HAND1-high progenitors were maintained in FGF8, BMP4, and CHIR; HAND1-low progenitors were maintained in FGF8, BMP4, and WNT3A, and HAND1-negative progenitors were maintained in FGF8-only (see Methods). Flow cytometric analysis of HAND1-Tom and NKX2-5-GFP after 6 days of expansion. ( G ) Differentiation of progenitors to epicardial and endothelial cells assessed by immunostaining for WT1 and CD31, respectively, and additionally by the flow cytometric analysis of CD31. ( H ) Differentiation of progenitors to cardiomyocytes assessed by immunostaining for cTroponin T. ( I ) Quantification of differentiated cell types by population. Data in ( I ) represent mean ± SEM, n = 3 independent biological experiments. Scale bars represent 150 μm. AUC area under the curve, EB embryoid body, Epi epicardial cells, Endo endothelial cells, CM cardiomyocyte, SEM standard error of the mean. See also Appendix Fig. . .

Article Snippet: A TO-MYC system was introduced by transducing the hPSCs with two lentiviruses, one carrying a MYC T58A transgene under the control of a TRE-CMV promoter (Addgene ID 19775), and the second carrying the tetracycline transactivator (Addgene ID 19780).

Techniques: Fluorescence, Activity Assay, Control, Immunostaining