Journal: bioRxiv
Article Title: Mitofusin agonists enhances long-term engraftment and potency of HSC cultures in vivo
doi: 10.1101/2025.10.27.684807
Figure Lengend Snippet: A , Triplicate histograms illustrating OPP signal in HSCs in CD90+ HSCs expanded for 3d with DMSO or 5nM CmpB. B , Flow cytometry OPP MFI in HSCs cultured for 3d with DMSO, 5nM CmpB, or 10mM salubrinal control (n=4, *p < 0.05, one-way ANOVA). C , IF staining quantification of p-eIF2α in resorted CD90+ HSCs expanded for 3d with DMSO or 5nM CmpB; (n≥20 cells from three biological experiments, *p < 0.05, student’s t-test). D , Flow cytometry MFI of proteostat signal in CD90 + HSCs cultured for three days in either DMSO or compound B (n= 4, data points from three biological replicates, *p < 0.05, two-tailed student t-test). E , Flow cytometry Lysotracker Green MFI in HSCs cultured for 3d in DMSO or compound B (n= 16, *p < 0.05, Student’s t-test). F , Lysosensor Green MFI in HSCs treated 3d with DMSO, 5nM CmpB, or 10mM chloroquine control (n=3-7 biological replicates, *p < 0.05, one-way ANOVA, post-hoc test). G , Spot count of LAMP1 IF staining in HSCs treated for 3d with DMSO, 5nM CmpB (mean of n = 3 biological replicates, *p < 0.05, student’s t-test). H , Spot count of LC3B IF staining in HSCs treated for 3d with DMSO, 5nM CmpB or 10mM chloroquine control (n= 7 cells from 2 biological replicates, *p < 0.05, one-way ANOVA). I , Super-resolution confocal micrographs (left) and Imaris spot analysis (right) of LC3B (green) and MFN2 (red) IF staining and DAPI nuclear stain (blue) in HSCs treated for 3d with DMSO or 5nM CmpB. Scale bar is 1mm. J , SRCM image quantification of nuclear TFEB signal in HeLa cells 24h post transfection with TFEB-GFP followed by 24h treatment with DMSO, 5nM CmpB or 10mM chloroquine and 5mM rapamycin as controls. (n = 14 cells from 3 independent experiments, *p < 0.05, one-way ANOVA). K , Representative confocal micrographs of TFEB-GFP localization in HeLa cells. Nuclear surfaces derived from DAPI (blue) and phalloidin (magenta) staining are shown. Scale bar is 10μm.
Article Snippet: K562 cells (ATCC) were sub-cultured in 10% FBS/DMEM-F12 with pen/strep in 5% CO2 at 37 ° C. HeLa cells (ATCC) were sub-cultured in 10% FBS/DMEM with pen/strep in 5% CO2 at 37 ° C. For mitophagy experiments, HeLa cells were transfected with pHAGE-Mito-mKeima and pEGFP-N1-TFEB using Lipofectamine 3000 Reagent according to manufacturer protocol.
Techniques: Flow Cytometry, Cell Culture, Control, Staining, Two Tailed Test, Transfection, Derivative Assay
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