Pharmacological treatment rescues changes in the microenvironment and mitophagy in MSPCs in vitro. (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). MSPC culture and CASIN (blue filled symbols) or vehicle (dimethyl sulfoxide, gray-filled symbols) treatment in vitro at p4. (B) Number of colony-forming mesenchymal stem cells (CFU-F) of 300 plated cultured MSPCs (p4). (C) Average proportion of β-gal positive–stained compact bone–derived MSPCs (p4, left). Representative FACS plots for β-gal–positive 13R with or without CASIN MSPCs. (D) Protein content of CDC42-GTP measured with ImageJ software. (E) Left: representative F-actin staining in cultured 13R + CASIN MSPCs (phalloidin, green). Evaluation of the orientation of F-actin fibers stained with phalloidin. Right graph: percentage of all cells showing fibers (bar in dark green), intermediate oriented fibers (bar in bright green), or no fibers (white bar). The Mann-Whitney test was applied here between all groups examined for each of the 3 fiber orientations (fibers, intermediate, and no fibers). For a better overview, not all significant values are shown. Some of them (without/no CASIN treatment) are already shown in Figure 3F. In addition, the following groups are significant: ∗Y vs 13A+; Y vs 13R+; 13A+ vs 13R+. (F) Representative confocal microscopy images stained for F-actin (green) and TOMM20 (red) counterstained with DAPI (blue) of 13R MSPCs (p4) with and without CASIN treatment. Image section of mitochondria embedded in actin structure. (G) Representative confocal microscopy images for F-actin (stained in green) and MYO6 (yellow) in MSPCs of 13R mice (p4) with and without CASIN treatment. (H) Left: representative confocal microscopy images for RHOT1 (stained in green) and LAMP1 (red) in MSPCs of 13R mice (p4) with and without CASIN treatment. Colocalization is shown in white. Right graph: colocalization pixel of RHOT1 and LAMP1 measured by ImageJ software. The analysis represents 2-3 independent experiments. Scale bars (mitochondria), 0.2μm; Scale bars (nucleus), 10μm. ∗P < .05 (Kruskal-Wallis test: panels B,C,D,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 panel E and analyzed with the nonparametric Mann-Whitney test. Data are represented as mean ± SD. Y, young; A+, aged 13 months with CASIN (=13A+); 13R+, regenerated 13 months with CASIN.