primary adult human aortic endothelial cells haecs  (ATCC)

Journal: Nature Communications

Article Title: Conserved transcription factors promote cell fate stability and restrict reprogramming potential in differentiated cells

doi: 10.1038/s41467-023-37256-8

Figure Lengend Snippet: a Expression of AJSZ in HDFs. Immunostaining for AJSZ is shown in red and fibroblast markers Vimentin (VIM) or Transgelin (TAGLN) are shown in green. Nuclei are stained with DAPI. n = 4 per condition. b Quantification of the % of cardiac marker ACTN2-expressing cells normalized to MGT + siControl condition after 30 days of cardiac reprogramming in HDFs. n = 5 and n = 9 for siControl and siAJSZ treated, respectively. c Representative images of HDFs treated with MGT + siControl or MGT + siAJSZ and immunostained for cardiac (ACTN2, green) and fibroblast (TAGLN, red) markers. Nuclei are stained with DAPI (blue). White arrows indicate iCMs. d Quantification of the % of MAP2 (green) and TUJ1 (red)-double-positive cells, 3 days after overexpression of Ascl1, Brn2, and Mytl1 (ABM) in HDFs. n = 7 and n = 8 for siControl and siAJSZ treated, respectively. e Representative images of HDFs treated with ABM + siControl or ABM + siAJSZ and immunostained for MAP2 (green) and TUJ1 (red). Nuclei are stained with DAPI (blue, top left insets). White arrows indicate TUJ1 + MAP2 + double-positive cells = neurons. f Quantification of the % of NANOG-positive cells, 7 days after overexpression of OCT4, KLF4, SOX2, and cMYC (OKSM) in HDFs. n = 10 per condition. g Representative images of HDFs treated with OKSM + siControl or OKSM + siAJSZ and immunostained for pluripotent marker NANOG (red). Nuclei are stained with DAPI (blue, top left insets). White arrows indicate NANOG-positive cells= iPSCs. h Quantification of the % of ACTN2 + 20 days after MGT overexpression. i Representative images of HAECs treated with MGT + siControl or MGT + siAJSZ and immunostained for the endothelial marker (PECAM1, red) and the cardiac marker (ACTN2, green). n = 4 per condition. Nuclei are stained with DAPI (blue). White arrows indicate iCMs. Scale bars: 50 µm. Student’s t -test: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Groups were compared using two-tailed unpaired analysis. Data in figure are presented as mean values ± standard deviation.

Article Snippet: Human adult aortic endothelial cells (HAECs) were obtained from ATCC (PCS-100-011) and cultured in plates precoated with 0.1% gelatin in Vascular Cell Basal Medium (VCBM; ATCC, PCS-100-030) containing endothelial cell growth kit-BBE (ATCC, PCS-100-040) and 0.1% P/S.

Techniques: Expressing, Immunostaining, Staining, Marker, Over Expression, Two Tailed Test, Standard Deviation

Journal: Nature Communications

Article Title: Conserved transcription factors promote cell fate stability and restrict reprogramming potential in differentiated cells

doi: 10.1038/s41467-023-37256-8

Figure Lengend Snippet: a Schematic depicting the hypothesis that AJSZ promotes the expression of reprogramming barriers. b Volcano plot showing the screening results for siRNAs directed against top 25 percentile downregulated (MGT + siAJSZ vs MGT) and core promoter-bound genes in the iMGT-MEF CR assay. The top three candidate barrier genes are circled. n = 4 per condition. c Histogram showing validation of siNceh1 and siChst2 effect on CR. n = 8 per condition. d Representative images for siControl, siChst2, and siNceh1 conditions. Myh6-eGFP+ cells are shown in green and cell nuclei are stained blue (DAPI, top right insets). e Schematic depicting the hypothesis that AJSZ negatively regulates reprogramming agonists. f Volcano plot showing the screening results for siRNAs directed against the top 25 percentile of upregulated (MGT + siAJSZ vs MGT) and core promoter-bound genes in siAJSZ-induced iMGT-MEF assay. g Histogram showing validation of top nine siRNAs that blunt siAJSZ-induced CR without affecting cell viability. n = 4 per condition. h Representative images for siAJSZ+ siControl, siMef2c, siHspb3, or siOlfml3 conditions. i Heatmap summarizing AJSZ expression dependence of identified barriers and agonists in HAECs and HDFs, 2 days after MGT, ABM, or OKSM overexpression. j Model showing that AJSZ regulates cell fate reprogramming by controlling the expression of a conserved set reprogramming agonists. Scale bars: 50 µm. Student’s t -test. * p < 0.05, ** p < 0.01, and **** p < 0.0001. Groups were compared using two-tailed unpaired analysis. Data in the figure are presented as mean values ± standard deviation. j Schematic is modified from Cunningham, T. J. et al. Id genes are essential for early heart formation. Genes & development, 10.1101/gad.300400.117 (2017). - CC-BY 4.0.

Article Snippet: Human adult aortic endothelial cells (HAECs) were obtained from ATCC (PCS-100-011) and cultured in plates precoated with 0.1% gelatin in Vascular Cell Basal Medium (VCBM; ATCC, PCS-100-030) containing endothelial cell growth kit-BBE (ATCC, PCS-100-040) and 0.1% P/S.

Techniques: Expressing, Biomarker Discovery, Staining, Over Expression, Two Tailed Test, Standard Deviation, Modification