![Attenuation of elevated CDC42 activity during auto/mitophagy. (A) IP injection of 5-FU in O5AΔ/Δ mice at day 0. Additional in vivo IP injection of vehicle ([−], n = 5) or CASIN ([+], n = 4) at day 5 through day 8. Rescue and analysis of autophagy relevant mechanisms in compact bone-derived MSPCs, isolated at day 8 and cultured until passage 4. (A-G) Shown are the results of O5AΔ/Δ mice with vehicle (−) and CASIN (+) treatment. CASIN-treated mice show the same phenotype as the control groups 5Afl/fl and O5A+/+ (Figure 3). (B) Fluorescent microscopy images of LC3 (red) and DAPI (blue) staining. Graph shows the pixel intensity as measured by ImageJ software. (C) Fluorescent microscopy image of CDC42-GTP (green), LC3 (red), and DAPI (blue) staining. The graph shows the percentage of MSPCs with colocalization measured by ImageJ software (plugin colocalization) and visualized in white. (D) Fluorescent microscopy images of F-actin (phalloidin/green) and LC3 (red) in MSPCs. Colocalization was measured by ImageJ software (plug-in colocalization) and visualized in white. The graph shows colocalization counted with ImageJ software. (E) Fluorescent microscopy images of LAMP1 (green) and DAPI (blue) staining. The pixel intensity (left graph) and Feret’s diameter (right graph) were measured by ImageJ software. (F) Fluorescent microscopy images of LAMP1 (green), LC3 (red), and DAPI (blue) staining. Yellow arrows show colocalized vesicle staining, red arrows depict LC3+ vesicles that did not colocalize with green LAMP1+ vesicles. Perinuclear colocalization of LAMP1 and LC3 measured by ImageJ software (plugin colocalization, depicted in white). (G) Representative FACS plots and quantification (graph) of Cyto-ID dye levels (DMFI: Cyto-ID dye level chloroquine treated, Cyto-ID dye level w/o treatment). (H) Fluorescent microscopy images of TOMM20 (green), LC3 (red), and DAPI (blue) staining. Colocalization was measured by ImageJ software (plugin colocalization) and visualized in the bottom row in white. Scale bars, 10 μm. ∗P < .05 (2-sided parametric Student’s t test; B-H). The results represent 2 independent experiments. Data are represented as mean ± SEM. Symbol legend shown in Figure 7A.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/142/8/10.1182_blood.2023021108/2/blood_bld-2023-021108-gr3.jpeg?Expires=1724223379&Signature=ssjuy6yo~Tx6pmQ00P9YiG-8HDzFSTcChLTylQ0ujfWGsO3he-~dmPB4r0D1XSVuk3RfaAxli7Qa8wR0~8AT1-~ABcEnpcOeIAmEu1y~BJXBVf7gA1uWuMy4kGBedern1pBTEh0sdodaZJTVXX5FkgHu4Pea9iZMP8RyxM5X9NZkPqM7SqRL~vJjyHvdbaCaHSflPo1Rwj9YA02YcVj5hWwEPpeeHoeTgc9ZAZD1DFGFfaWB8CgK28WavUg51I3tvl9zRoYabaDBnfPjVMJbj2nRo~nuyu~GO3WlmB714nisy25e1oH6zMFg6djaVkl2pVjrs~4w7W-~BJrjEY5FOA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Attenuation of elevated CDC42 activity during auto/mitophagy. (A) IP injection of 5-FU in O5AΔ/Δ mice at day 0. Additional in vivo IP injection of vehicle ([−], n = 5) or CASIN ([+], n = 4) at day 5 through day 8. Rescue and analysis of autophagy relevant mechanisms in compact bone-derived MSPCs, isolated at day 8 and cultured until passage 4. (A-G) Shown are the results of O5AΔ/Δ mice with vehicle (−) and CASIN (+) treatment. CASIN-treated mice show the same phenotype as the control groups 5Afl/fl and O5A+/+ (Figure 3). (B) Fluorescent microscopy images of LC3 (red) and DAPI (blue) staining. Graph shows the pixel intensity as measured by ImageJ software. (C) Fluorescent microscopy image of CDC42-GTP (green), LC3 (red), and DAPI (blue) staining. The graph shows the percentage of MSPCs with colocalization measured by ImageJ software (plugin colocalization) and visualized in white. (D) Fluorescent microscopy images of F-actin (phalloidin/green) and LC3 (red) in MSPCs. Colocalization was measured by ImageJ software (plug-in colocalization) and visualized in white. The graph shows colocalization counted with ImageJ software. (E) Fluorescent microscopy images of LAMP1 (green) and DAPI (blue) staining. The pixel intensity (left graph) and Feret’s diameter (right graph) were measured by ImageJ software. (F) Fluorescent microscopy images of LAMP1 (green), LC3 (red), and DAPI (blue) staining. Yellow arrows show colocalized vesicle staining, red arrows depict LC3+ vesicles that did not colocalize with green LAMP1+ vesicles. Perinuclear colocalization of LAMP1 and LC3 measured by ImageJ software (plugin colocalization, depicted in white). (G) Representative FACS plots and quantification (graph) of Cyto-ID dye levels (DMFI: Cyto-ID dye level chloroquine treated, Cyto-ID dye level w/o treatment). (H) Fluorescent microscopy images of TOMM20 (green), LC3 (red), and DAPI (blue) staining. Colocalization was measured by ImageJ software (plugin colocalization) and visualized in the bottom row in white. Scale bars, 10 μm. ∗P < .05 (2-sided parametric Student’s t test; B-H). The results represent 2 independent experiments. Data are represented as mean ± SEM. Symbol legend shown in Figure 7A.
