Impaired F-actin signaling. (A) Experimental design for HSCT into lethally irradiated 3-month-old wild-type mice (time point at the end of experiment: 13 months, 13R). Age-matched control group (13A) without HSCT. Analysis of the BM niche in 13-month-old mice (13A and 13R), and Y control group (3 months). RNA-seq analysis of sorted MSPCs (B) with subsequent cultivation of the MSPCs until p4 for analysis with IF assay. (C) Volcano plot showing differential protein expression of autophagy genes, with adjusted P values (false discovery rate [FDR]) plotted against log₂ fold change (FC). 13A, n = 10 and 13R, n = 7. The dark dots show proteins that meet the FDR threshold for statistical significance (FDR < 0.05) and are considered differentially expressed. (D) Analysis of the DEGs using STRING (Search Tool for Retrieval of Interacting Genes/Proteins) for visualizing an interaction network. (E) Graph shows the protein content of CDC42-GTP in cultured MSPCs (p4). (F) Left: representative IF images of MSPCs (p4). IF staining of F-actin (green) counterstained with DAPI (blue). Right: evaluation of the orientation of F-actin fibers stained with phalloidin. Percentage of all cells showing fibers (bar in dark green), intermediate oriented fibers (bar in bright green), or no stress fibers (white bar). The Mann-Whitney test was applied here between the 3 groups examined for each of the 3 stress fiber orientations (fibers, intermediate, and no fibers). (G) Experimental design for human HSCT and myeloablation via chemotherapeutics (n = 5) or irradiation (n = 2) and analysis from 6 months up to 24 months after HSCT in human BM samples. Healthy donor samples as controls (n = 6). (H) Graphs show the protein content of CDC42-GTP measured by ImageJ software from healthy age-matched controls (n = 6) and allo-HSCT recipient (n = 7; left). (I) Left: representative IF staining of F-actin (green; right) counterstained with DAPI (blue) of a patient sample after allo-HSCT. Right: evaluation of the orientation of F-actin fibers stained with phalloidin. Percentage of all cells showing fibers (bar in dark green), intermediate oriented fibers (bar in bright green pattern), or no fibers (white bar). The Mann-Whitney test was applied here between the 3 groups examined for each of the 3 fiber orientations (fibers, intermediate, and no fibers). The analysis represents 2-3 independent experiments except for the RNA-seq analysis, which was performed once with a high number of samples (Y, n = 9; 13A, n = 10; and 13R, n = 7). Scale bars, 10 μm. ∗P < .05 (Kruskal-Wallis test, panel E,H; F-actin fibers/intermediate/no fibers were either present [value = 1], or not [value = 0]). Only the results for the presence of elongated F-actin fibers are presented in panels F and I and analyzed with the nonparametric Mann-Whitney test. Data are represented as mean ± SD.