Fig 2: Inhibition of neutral SMases disrupts repair of damaged lysosomes.a Time-course plotting LysoTracker-positive puncta in HeLa cells pre-treated with siRNA targeting GFP (72 h) and GW4869 (10 µM) or DMSO (0.5%, 30 min) and pulse-treated with GPN. Data are means ± SD. n = 52 cells for DMSO and 39 cells for GW4869 over three independent experiments. b Time-course plotting LysoTracker-positive puncta in HeLa cells pre-treated with siRNAs targeting ALIX/TSG101 and GW4869 or DMSO and pulse-treated with GPN. Data are means ± SD. n = 46 cells for DMSO and 44 cells for GW4869 over three independent experiments. c Survival rate of HeLa cells pre-treated with siRNA targeting GFP after 5 h of exposure to LLOMe at indicated concentrations in the presence of GW4869 or DMSO. Data are means ± SD. n = 3 independent experiments. d Survival rate of HeLa cells pre-treated with siRNA targeting ALIX/TSG101 after 5 h exposure to LLOMe at indicated concentrations in the presence of GW4869 or DMSO. Data are means ± SD. n = 3 independent experiments. e Time-course plotting LysoTracker-positive puncta in HeLa cells pre-treated with siRNAs targeting ALIX/TSG101 and GFP or nSMase-1 and pulse-treated with GPN. Data are means ± SD. n = 28 cells for siGFP and 28 cells for sinSMase-1 over three independent experiments. f Time-course plotting LysoTracker-positive puncta in HeLa cells pre-treated with siRNAs targeting ALIX/TSG101 and GFP or nSMase-2 and pulse-treated with GPN. Data are means ± SD. n = 28 cells for siGFP and 40 cells for sinSMase-2 over three independent experiments. g Time-course plotting LysoTracker-positive puncta in WT or nSMase-2 KO HeLa cells pulse-treated with GPN. Data are means ± SD. n = 58 cells for WT and 66 cells for nSMase-2 KO over three independent experiments. h Survival rate of wild-type (WT) or nSMase-2 KO HeLa cells after 5 h exposure to LLOMe at the indicated concentration. Data are means ± SD. n = 4 independent experiments. P values were calculated by paired (c, d, h) or unpaired two-tailed t test (a, b, e, f, g). i Model illustrating how Ca2+-activated SM scrambling and turnover may promote restoration of damaged lysosomes.

Fig 3: Characterization of CML SP-derived EVs.(a) CD45.1 and CD45.2 expression on the BM cells and splenocytes in CML-bearing mice was determined 4 weeks after BMT therapy and is shown in the left panels. As a control, cells derived from CD45.1+ and CD45.2+ mice were mixed immediately before staining with molecule-specific Abs. Representative immunofluorescence images for CD45.2 on the sorted CD45.1+CD45.2+ cells (inside square gates) from three independent experiments are shown in the right panels. Expression pattern of CD45.2 on the cells derived from CD45.2+ mice are shown as a control. (b) Protein concentration of SP-derived EVs. Data represent the mean ± SD from three independent experiments. **P < 0.01 using Tukey-Kramer test. (c) EV surface marker proteins (CD9 and CD81), an EV cytosolic marker protein (Alix), and a non-EV protein (GAPDH) were detected using western blot. Each lane represents an individual sample (n = 3). (d) Size distribution of control EVs, non-rad CML EVs, and rad CML EVs were analyzed using NTA. Representative results from three independent experiments are shown here.

Fig 4: In vitro transfer of functional Cre-recombinase through RBC-extracellular vesicles (EVs) and functional effects.(A) Schematic representation of isolation of EVs into 12 fractions, from mouse cell-free plasma using C-DGUC. (B) Detection of EV markers Alix and CD9 in the 12 fractions from C-DGUC through Western Blot. Molecular weight markers are indicated. The exposure time to detect Alix and CD9 was 200 s. (C) Flow cytometry scatterplots of mTmG HEK293 cells treated with EpoR-Cre RBC-EVs for 36 h. Shown are Td on the y-axis and GFP in the x-axis and divided in four quadrants (Q4 is background: Q1 is mTd+[red], Q3 is mGFP+ [green] and Q2 is mTd+/mGFP+ [double-positive]); n = 3 independent replicates for each condition. (D) Box and whisker plot quantifying the frequency of recombination (mGFP+ and mTd+/mGFP+ combined, expressed as percentage of total fluorescent living cells) of mTmG HEK293 cells treated with EpoR-Cre RBC EVs (4.54% ± 1.51%, n = 9, six-well cell culture dish representing one individual event) (n = 9 biological replicates) Controls were mTmG HEK293 cells that were untreated with no recombination. (E) Ingenuity Pathway Analysis of enriched pathways for transcriptomic data for flow-sorted mGFP+ HEK293 cells and mTd+ HEK293 cells after treatment of mT/mG HEK293 cells with EpoR-Cre EVs and mTmG EVs for 36 h (n = 3 each). A total of 143 genes that were expressed with at least 25 reads per million (rpm) and a twofold change between mGFP+ HEK293 and mTd+ HEK293 cells were included for pathway analysis. Data are presented as median ± SEM.

Fig 5: Distributions of protein markers, chromosomal DNA, and mitochondrial DNA in the pentapartite fractions of oral fluids (OFs) from 6 healthy volunteers. (a) Western blots of proteins markers for canonical exosomes (CD81, CD9, Alix, CD63 and HSP70), salivary gland (AQP5), mitochondria (ATP5A), chromatin (Histone H2B), and miRNA-associated protein (Ago2). Numbers on the right indicate molecular weight × 10–3. Black arrow shows the expected size of ATP5A. Asterisks in the 160K fractions of Ago2 are discussed in the text. Note that Sup was diluted 35-fold. The copy numbers of chromosomal DNA (b), and mitochondrial DNA (c), were determined by quantitative PCR. Target genes were SLCO2B1 and SERPINA1 for chromosomal DNA and ND1 and ND3 for mitochondrial DNA. The OFs were obtained from n = 6 healthy donors. Data are expressed as the mean ± standard deviation (SD). Dots indicate the mean value of the three measurements for each subject, while the mean values obtained from six subjects are represented as solid horizontal lines. Note that Sup was diluted 35-fold. The data obtained from HC45 are shown by red filled circles (see “Results” section).