Fig. 1: Increasing NE tension activates RhoA in HSCs.

a, Representative HSC gating strategy and experimental setup for the confinement experiments. b, Two representative nuclei are depicted to illustrate the strategy to measure the NHA and diameter. NHA is calculated as the sum of n measurements of the nucleus height in the y−z axis, divided by the n number of measurements. NHA and diameter are represented against the different confinement conditions used in the experiments, and a correlation plot between both is shown from three independent experiments (total: unconfined n = 25, 8 µm n = 30, 5 µm n = 16, 3 µm n = 26). Data on graphic bars show mean ± s.e.m., and they were analyzed using Mann−Whitney two-tailed tests, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data on the correlation plot were analyzed by simple linear regression. Pearsonʼs r coefficient and P value are shown, ****P < 0.0001. c, Representative images of 3D immunofluorescence reconstruction from x−y and y−z axes of single HSCs under different levels of confinement of three independent experiments (total: unconfined n = 25, 8 µm n = 30, 5 µm n = 16, 3 µm n = 26). Antibody anti-RhoA-GTP was used to stain active RhoA (red). The nuclei are stained by DAPI (gray). White arrows indicate nucleoplasm-containing blebs. Scale bars, 1 µm. Videos of these confocal acquisitions are available in Supplementary Videos 1–4. Quantification of RhoA-GTP (volume of positive signal by intensity) under different confinements: unconfined, 8 µm, 5 µm and 3 µm. Data on graphic bars show mean ± s.e.m., analyzed by Mann−Whitney test, two-tailed, *P < 0.05, **P < 0.01, ***P < 0.001. Correlation plot in between the RhoA-GTP and the diameter is shown. Data on correlation were analyzed by simple linear regression. Pearsonʼs r coefficient and P value are shown, *P < 0.05. d, Representative images of 3D confocal reconstruction showing LT-HSCs from Rhoa^fl/fl and Rhoa^−/− mice. Cells were treated in vitro with 4-hydoxy tamoxifen overnight and then confined under 5 µm for 2 hours. Cells were stained for RhoA-GTP (red) and DAPI (gray). Graph shows volume in µm³ of RhoA-GTP signal (total: Rhoa^fl/fl n = 20; RhoaKO n = 12). Data on graphic bars are from three independent experiments showing mean ± s.e.m., analyzed by Mann−Whitney test, two-tailed, ****P < 0.0001. e, Representative images of 3D confocal reconstruction showing RhoA-GTP signal (red) and DAPI (gray) of HSCs treated with NaB. Graph shows volume in µm³ of RhoA-GTP signal. Data on graphic bars are from three independent experiments showing mean ± s.e.m. (total: −NaB n = 31; +NaB n = 40), Mann−Whitney test, two-tailed, ****P < 0.0001. f, Representative images of 3D confocal reconstruction showing DAPI staining of nuclei from young control LT-HSCs and young LT-HSCs treated with NaB. Graph shows the maximum diameter at x−y in µm at each condition. Data on graphic bars are from three independent experiments showing mean ± s.e.m. (total: −NaB n = 22; +NaB n = 63), 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 compartmentalized PcPLA2 signal within the nucleus and at the NE. We assigned light pink to NE PcPLA2 and magenta to nuclear PcPLA2. NE PcPLA2 only is also shown. Right: zoomed-in image of the NE localization of PcPLA2, which was reconstructed in light pink. Graph showing the volume in µm³ of PcPLA2 signal at the NE. Videos of representative confocal acquisitions are available in Supplementary Videos 5 and 6. Data on graphic bars are from three independent experiments showing mean ± s.e.m. (total: −NaB n = 38; +NaB n = 25), Mann−Whitney, two-tailed test, ****P < 0.0001. h, Representative images of 3D confocal reconstruction showing HSCs after 16-hour incubation on hydrogels of 1-kPa or 40-kPa stiffness, stained with RhoA-GTP (red) and DAPI (gray). Graph shows volume in µm³ of RhoA-GTP signal from two independent experiments (total: after 1 kPa = 13; after 40 kPa n = 20). Data on graphic bars show mean ± s.e.m., Mann−Whitney test, two-tailed. i, Graphs showing the number of colonies and the number of cells on CFU assays of wild-type and RhoaKO HSCs after incubation on hydrogels of different stiffness (number of independent experiments per condition: wt 1 kPa n = 4; KO 1 kPa n = 8; wt 40 kPa n = 5; KO 40 kPa n = 8). Graphs show mean ± s.e.m., Mann−Whitney test, two-tailed. Scale bars, 1 µm. j,k, Graphs showing hematopoietic progenitor cells (c-Kit⁺) (j) and myeloid cells (Mac1⁺Gr1⁺ and Mac1⁺) (k) in CFU assays of wild-type and RhoaKO HSCs after incubation on hydrogels at 1 kPa or 40 kPa (number of independent experiments per condition: wt 1 kPa n = 4; KO 1 kPa n = 8; wt 40 kPa n = 5; KO 40 kPa n = 8). Graphs show mean ± s.e.m., Mann−Whitney test, two-tailed, *P < 0.05. l, Graphics depict RhoA-GTP and PcPLA2 under normal conditions and nuclear stretching, either by confinement or NaB treatment. FITC, fluorescein isothiocyanate; LT, long-term; NS, not significant; ST, short-term; LMPP, lymphoid multi-potent progenitor; wk, weeks; wt, wild-type. Panel a and l created with BioRender.

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