active rhoa detection kit  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Targeting RhoA activity rejuvenates aged hematopoietic stem cells

doi: 10.1101/2025.04.23.647902

Figure Lengend Snippet: A, Representative HSC gating strategy and experimental set-up for the confinement experiments. B. Two representative nuclei are depicted to illustrate the strategy to measure the Nuclear Height Average (NHA) and diameter. NHA is calculated as the sum of n measurements of the nucleus height in the YZ axis, divided by the n number of measurements. Nuclear Height Average (NHA) and Diameter are represented against the different confinement conditions used in the experiments and a correlation plot between both is shown from 3 independent experiments. Data on graphic bars have been analyzed using Mann-Whitney two-tailed tests **p<0.01; ***p<0.001, ****p<0.0001. Data on correlation plot have been analyzed by simple linear regression. Pearson r coefficient and P value are shown. ****P<0.0001 C. Representative images of 3D immunofluorescence reconstruction from XY and YZ axes of single HSCs under different levels of confinement of 3 independent experiments. Antibody anti-RhoAGTP was used to stain active RhoA (red). The nuclei are stained by DAPI (gray). Scale bars=1 µm. Movies of these confocal acquisitions are available in Video S1-4 . Quantification of RhoAGTP (volume of positive signal by intensity) under different confinements: unconfined, 8 µm, 5 µm and 3 µm. Mann-Whitney-test, two-tailed n =3* p<0.05; **p<0.01, ***p<0.001 . Correlation plot in between the RhoAGTP and the diameter is shown. Data on correlation have been analyzed by simple linear regression. Data is obtained from 3 independent experiments. Pearson r coefficient and P value are shown. * p<0.05 . D, Representative images of 3D confocal reconstruction showing LT-HSC from RhoA fl/fl and RhoA -/- mice. Cells were treated in vitro with 4OH-tamoxifen o/n and then confined under 5 µm for 2 hours. Cells were stained for RhoAGTP (red) and DAPI (gray). Graph shows volume in µm 3 of RhoAGTP signal. n=3 Mann-Whitney two-tailed, ****p<0.0001 E, Representative images of 3D confocal reconstruction showing RhoAGTP signal (red) and DAPI (gray) of HSCs treated with NaB. Graph shows volume in µm 3 of RhoAGTP signal. n=3, Mann-Whitney two-tailed **** p <0.0001. F, Representative images of 3D confocal reconstruction showing DAPI staining of nuclei from young control LT-HSC and young treated with NaB. Graph shows the maximum diameter at XY in µm at each condition. n=3, Mann-whitney test one-tailed * p <0,05. G, Representative images of 3D confocal reconstruction showing phospho-cPLA2 (PcPLA2) (pink) and DAPI (gray) in young control and young NaB treated LT-HSCs. By using image analysis software (Imaris and Volocity) we have compartmentalize PcPLA2 signal within the nucleus and at the nuclear envelope (NE). We have assigned light pink to NE PcPLA2 and magenta to nuclear PcPLA2. NE PcPLA2 only is also shown. The zoom inset shows membrane localization of PcPLA2 which has been reconstructed in light pink in the last panel. Graph showing the volume in µm 3 of PcPLA2 signal at the NE. Movies of representative confocal acquisitions are available in Video S5-6 . n=3, Mann-Whitney, two-tailed **** p <0.0001. H, Representative images of 3D confocal reconstruction showing HSC after 16h incubation on hydrogels of 1 KPa or 40 KPa stiffness, stained with RhoAGTP (red) and DAPI (gray). Graph shows volume in µm 3 of RhoAGTP signal. n=2; Mann-Whitney, two-tailed. I, Graphs showing the number of colonies and the number of cells on CFU assays of wt and RhoAKO HSCs after incubation on hydrogels of different stiffness. n=4, Mann-Whitney,two-tailed. Scale bars=1µm. J and K, Graphs showing haematopoietic progenitor cells (c-Kit+) (J) and myeloid cells (mac1+gr1+ and mac1+) (K) in CFU assays of Rhoa fl/fl and RhoA -/- LT-HSC after incubation on hydrogels at 1KPa or 40KPa. n=4, Mann-Whitney, two-tailed, * p <0.05. L, Graphics depicting RhoAGTP and PcPLA2 under normal conditions and nuclear stretching, either by confinement or NaB treatment.

Article Snippet: To isolate the active isoform of RhoA, GTP-bound RhoA, Active RhoA Detection Kit (Cell Signaling, 8820) was used following manufacturer instructions.

Techniques: MANN-WHITNEY, Two Tailed Test, Immunofluorescence, Staining, In Vitro, Control, One-tailed Test, Software, Membrane, Incubation