Journal: International journal of molecular sciences

Article Title: Mef2c- and Nkx2-5-Divergent Transcriptional Regulation of Chick WT1_76127 and Mouse Gm14014 lncRNAs and Their Implication in Epicardial Cell Migration.

doi: 10.3390/ijms252312904

Figure Lengend Snippet: Figure 4. Gm14014 regulates epicardial cell migration by modulating cytoskeletal proteins. Panel (A). RT-qPCR analyses of Gm14014 expression in MEC1 epicardial cells after Gm14014 ASO adminis- tration 6 h, 12 h, 24 h and 48 h after transfection. It can be observed that selective down-regulation was achieved at 6 h, 12 h and 48 h while at 24 h there was a significant up-regulation (n = 3). Panel (B). Schematic representation of wound healing scratch assay in MEC1 epicardial cells in controls, Gm14014 ASO-, siMyl9- and Myl9 ASO-treated cells, HL1 cardiomyocytes and MEVEC endocardial cells at 6 h, 8 h, 12 h and 24 h in controls and Gm14014 ASO-treated cells. Representative images of MEC1 epicardial cells at t = 0, t = 12 h and t = 24 h. Note that the migration of Gm14014-ASO treated cells was significantly decreased in MEC1 epicardial cells but not in HL1 cardiomyocytes or MEVEC endocardial cells. The migration of siMyl9 and Myl9 ASO treated cells was significantly decreased in MEC1 epicardial cells. Panel (C). Representative images of proliferation assays as revealed by phospho-histone 3 (pHH3) immunocytochemistry in control and scratched MEC1 epicardial cells corresponding to control and Gm14014 ASO conditions. Quantitative analyses are also shown, demon- strating no significant differences in cell proliferation. Panel (D). Graphical representation of lineal vs. non-linear cell migration in time lapse confocal image analyses of control and scratched MEC1 epicardial cells corresponding to control and Gm14014 ASO conditions. Panel (E). Representative images of immunocytochemical analyses of Actn1, Actn4, Myh9, Myl9, Rac1 at 6 h and of Myh9, Myl9, Rac1 at 24 h after administration of Gm14014 ASO compared to controls. It can be observed that there is significant difference in the expression of Myh9 and Myl9 at 6 h but not at 24 h. Note also that Myl9 displays both nuclear and cytoplasmic distribution in controls while in Gm14014-treated cells it is exclusively cytoplasmic. Panel (F). Quantitative analysis and representative blot of Myl9 after Gm14014 PD. Observe that Myl9 protein interacts with Gm14014. Statistical analysis: Student’s t (95% confidence interval); * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001. ns, not significant. Schemes were made with Biorender.

Article Snippet: Primary antibodies against Actn1 (ab68194, Abcam), Actn4 (ab108198, Abcam), Myh9 (ab75590, Abcam), pHH3 (CA-92590, Milipore, Burlington, MA, USA), Myl9 (sc-28329, Santa Cruz, Dallas, TX, USA), Rac1 (sc-514583, Santa Cruz) and MF20 (ATCC) were used, which were diluted (1:200) in blocking solution and applied to each culture overnight at 4 ◦C.

Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, Wound Healing Assay, Immunocytochemistry, Control

Journal: Integrative biology : quantitative biosciences from nano to macro

Article Title: Geometry sensing through POR1 regulates Rac1 activity controlling early osteoblast differentiation in response to nanofiber diameter.

doi: 10.1039/c4ib00225c

Figure Lengend Snippet: Fig. 6 Western blot demonstrating knockdown of POR1 which was found to be approximately 50% based on the ratio of POR1 to Tubulin (top). Rac1 activation as a function of fiber diameter, 24 hours after seeding, normalized by DNA (bottom). All values were normalized against the control siRNA on control surfaces. Blue bars indicate significant difference between substrates within control siRNA treatment and * represents significance between siRNA treat- ments on the same substrate, p o 0.05; n = 6, outliers removed.

Article Snippet: POR1 knockdown and active Rac1 quantification MC3T3-E1 S4 cells were transfected with POR1 siRNA in accordance with the guidelines from the supplier, Santa Cruz Biotechnology.

Techniques: Western Blot, Knockdown, Activation Assay, Control

Journal: Integrative biology : quantitative biosciences from nano to macro

Article Title: Geometry sensing through POR1 regulates Rac1 activity controlling early osteoblast differentiation in response to nanofiber diameter.

doi: 10.1039/c4ib00225c

Figure Lengend Snippet: Fig. 7 Arf1 activation as a function of fiber diameter, 24 hours after seeding, normalized by DNA (bottom). All values were normalized against the control siRNA on control surfaces. Color coded bars indicate signifi- cant differences between substrates within control and POR1 siRNA samples whereas * represents significance between siRNA treatments on the same substrate, p o 0.05; n = 6, outliers removed.

Article Snippet: POR1 knockdown and active Rac1 quantification MC3T3-E1 S4 cells were transfected with POR1 siRNA in accordance with the guidelines from the supplier, Santa Cruz Biotechnology.

Techniques: Activation Assay, Control

Journal: Integrative biology : quantitative biosciences from nano to macro

Article Title: Geometry sensing through POR1 regulates Rac1 activity controlling early osteoblast differentiation in response to nanofiber diameter.

doi: 10.1039/c4ib00225c

Figure Lengend Snippet: Fig. 9 The proposed mechanism for POR1 (arfaptin2) and Arf1 and their role in the activation of Rac1.

Article Snippet: POR1 knockdown and active Rac1 quantification MC3T3-E1 S4 cells were transfected with POR1 siRNA in accordance with the guidelines from the supplier, Santa Cruz Biotechnology.

Techniques: Activation Assay