cells ecfcs star methods n a ipscs  (R&D Systems)

Journal: Cell reports

Article Title: Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells.

doi: 10.1016/j.celrep.2018.06.083

Figure Lengend Snippet: Figure 3. Shear Responsiveness, Cilia, and Stress Fibers in hiPSC-ECs (A–C) BC1 E6-CHIR (A), C12 E6-CHIR (B), and hiPSC-ECFCs (C) were assessed for (i) shear responsiveness (acetylated a-tubulin in red, VECad in green, and nuclei in blue) and (ii) cytoskeletal organization (F-actin in red, acetylated a-tubulin in green, and nuclei in blue). (D) Quantification of (i) nuclei alignment and (ii) aspect ratio after arterial shear exposure compared with unaligned static controls (dotted blue line). In addition, (iii) percentage of ciliated cells and (iv) number of stress fibers per cell were quantified. (E) Protein expression of cytoplasmic (C) and nuclear (N) fractions of acetylated a-tubulin with GAPDH and HDAC1 as endogenous controls, respectively. Cilia and stress fiber quantification are shown from at least three independent differentiations, using five fields of view for image quantification (BC1-E6 CHIR: n = 813 serum starved; C12 E6-CHIR: n = 1428 serum starved; hiPSC-ECFCs: n = 197 serum starved). Statistical significance and analysis were determined using one-way ANOVA with Bonferroni post hoc tests. Shear stress experiments are shown from at least biological duplicates. Aspect ratio analysis was conducted on n R 50 and nuclei alignment on n > 200 cells. All graphical data are reported as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p< 0.0001. White arrowheads show primary cilia.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER iblot transfer stack nitrocellulose regular Thermo Fisher Scientific IB23001 20X Bolt MOPS SDS Running Buffer Thermo Fisher Scientific B0001 NE-PER Nuclear and Cytoplasmic Extraction Reagents Thermo Fisher Scientific 78835 MagicMark XP Western protein standard Thermo Fisher Scientific LC5603 iBlot 2 Transfer Stacks, PVDF, regular size Thermo Fisher Scientific IB24001 Critical Commercial Assays Trizol reagent Thermo Fisher Scientific 15596018 M-MLV Reverse Transcriptase Promega M1701 RNaseOUT Thermo Fisher Scientific 10777019 RT PCR master mix Thermo Fisher Scientific 4369016 Deposited Data hiPSC-EC cDNA microarray This Study GEO: GSE115870 Experimental Models: Cell Lines HUVEC single donor Promocell C-12200 HUAEC Promocell C-12202 Endothelial Colony Forming Cells (ECFCs) STAR Methods N/A iPSCs and Derivatives STAR Methods N/A Irradiated Mouse Embryonic Fibroblasts R&D Systems PSC001 Oligonucleotides Random primers Promega C1181 TaqMan Gene Arrays, see Table S2 Thermo Fisher Scientific N/A Software and Algorithms ImageJ NIH https://imagej.nih.gov/ij/ MATLAB MathWorks https://www.mathworks.com/products/ matlab.html Imaris BITPLANE http://www.bitplane.com/imaris-for- cell-biologists GraphPad GraphPad Software https://www.graphpad.com/scientific- software/prism/ Illustrator Adobe https://www.adobe.com

Techniques: Shear, Expressing

Journal: Cell reports

Article Title: Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells.

doi: 10.1016/j.celrep.2018.06.083

Figure Lengend Snippet: Figure 6. Transcriptional Differences between hiPSC-Derived ECs (A) Cilia gene expression profile between hiPSC-derived ECs from BC1, C12, and DF19-9-11T (hiPSC-ECFCs) cell lines. N = 3 biological replicates for BC1 and hiPSC-ECFCs. Biological replicates are N = 2 for C12. Data are represented as ± SEM. (B) Immunofluorescence images of BC1-ECs, C12-ECs, and hiPSC-ECs before and after serum starvation for PLK-1 (green), acetylated-a-tubulin (red), and nuclei (blue) (yellow arrowheads denote co-localization of PLK-1 and tubulin bodies; white arrowheads show primary cilia). (C) Quantification of cilia length between the hiPSC-ECs. (D) Protein expression of cytoplasmic (C) and nuclear (N) fractions of acetylated a-tubulin with GAPDH and HDAC1 as endogenous controls, respectively. Comparison of cytoplasmic HDAC6 protein expression is shown. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER iblot transfer stack nitrocellulose regular Thermo Fisher Scientific IB23001 20X Bolt MOPS SDS Running Buffer Thermo Fisher Scientific B0001 NE-PER Nuclear and Cytoplasmic Extraction Reagents Thermo Fisher Scientific 78835 MagicMark XP Western protein standard Thermo Fisher Scientific LC5603 iBlot 2 Transfer Stacks, PVDF, regular size Thermo Fisher Scientific IB24001 Critical Commercial Assays Trizol reagent Thermo Fisher Scientific 15596018 M-MLV Reverse Transcriptase Promega M1701 RNaseOUT Thermo Fisher Scientific 10777019 RT PCR master mix Thermo Fisher Scientific 4369016 Deposited Data hiPSC-EC cDNA microarray This Study GEO: GSE115870 Experimental Models: Cell Lines HUVEC single donor Promocell C-12200 HUAEC Promocell C-12202 Endothelial Colony Forming Cells (ECFCs) STAR Methods N/A iPSCs and Derivatives STAR Methods N/A Irradiated Mouse Embryonic Fibroblasts R&D Systems PSC001 Oligonucleotides Random primers Promega C1181 TaqMan Gene Arrays, see Table S2 Thermo Fisher Scientific N/A Software and Algorithms ImageJ NIH https://imagej.nih.gov/ij/ MATLAB MathWorks https://www.mathworks.com/products/ matlab.html Imaris BITPLANE http://www.bitplane.com/imaris-for- cell-biologists GraphPad GraphPad Software https://www.graphpad.com/scientific- software/prism/ Illustrator Adobe https://www.adobe.com

Techniques: Derivative Assay, Gene Expression, Expressing, Comparison