TLNRD1 silencing does not impact CCM2 localization. (A and B) HUVECs expressing various CCM2-GFP constructs were stained for DAPI and PECAM and imaged using a spinning disk confocal microscope. (A) SUM projections are displayed. Scale bar: 10 µm. (B) For each condition, the CCM2 nuclear-cytoplasmic ratio was quantified (three biological repeats, n > 110 cells per condition). (C–E) TLNRD1 expression was silenced in HUVECs using two independent siRNA. (C and D) Cells were then transfected to express CCM2-GFP and allowed to form a monolayer. Cells were fixed and stained for DAPI and PECAM and imaged using a spinning disk confocal microscope. (C) SUM projections are displayed. Scale bar: 10 µm. (D) For each condition, the CCM2 nuclear-cytoplasmic ratio was quantified (three biological repeats, n > 60 cells per condition). (E) HUVECs were then allowed to form a monolayer without flow stimulation. KLF4 expression levels were measured by qPCR. (B and D) The results are displayed as Tukey boxplots. The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. For all panels, the P values were determined using a randomization test. NS indicates no statistical difference between the mean values of the highlighted condition and the control.