Sulfisoxazole inhibits the secretion of small extr...
Fig. 1
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SFX inhibits the secretion of…
Fig. 1
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SFX inhibits the secretion of sEV from breast cancer cells quantitatively and qualitatively.…
SFX inhibits the secretion of sEV from breast cancer cells quantitatively and qualitatively. a Screening flow chart of primary and secondary screenings with some exclusion criteria to identify an inhibitor of sEV secretion, sulfisoxazole. b The number of secreted sEV with the indicated concentrations of SFX. n = 3. c Left, electron microscopy image. Scale bar, 100 nm. Right, Quantification of the number of secreted sEV. Randomized fields were captured and counted. n = 30. d Measurement of the microvesicle concentration by nanoparticle tracking analysis (NTA). n = 3. e Measurement of soluble cytokines secreted from MDA-MB231 cells. n = 3. f qRT-PCR analysis of the indicated miRNAs in MDA-MB231 cells after treatment of 100 μM SFX. n = 3. The GEO accession number of miRNA microarray set is GSE124320. g Immunoblot of various proteins in MDA-MB231 cell-derived sEV. Equal amounts of sEV protein (10 μg) were loaded per lane. n = 3. Experiments were performed with 95% confluent cells. Significance was determined using an unpaired two-tailed Student’s t- test. ***p < 0.001, **p < 0.005, and *p < 0.05. Error bar, SD. Source data (b–g) are provided as a Source Data file
Fig. 2
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SFX suppresses breast cancer cell…
Fig. 2
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SFX suppresses breast cancer cell proliferation and metastasis in two cancer xenograft models.…
SFX suppresses breast cancer cell proliferation and metastasis in two cancer xenograft models. a Schematic illustration of the in vivo experimental designs with different treatments. b Top, Representative image of cancer cells tracked with the IVIS imaging system following the injection of mice with luciferase-expressing MDA-MB231 cells. Bottom, Tumor mass volume of in BALB/c nude female mice inoculated with MDA-MB231-luci (+) cells and then treated with an indicated drug or vehicle. n = 7 per group. c Top, in vivo images of 4T1 cancer cell-bearing mice using an IVIS imaging system. Bottom, tumor mass volume of 4T1-luci (+) cells inoculated BALB/c nude female mice treated with an indicated drug or vehicle n = 9 per group. d Measurement of the survival rates in tumor-bearing mice after different treatments. The survival curves were generated according to the Kaplan–Meier method n = 5 per group. e Quantitative representation of lung and liver nodules from mice bearing 4T1-luci (+) cells. n = 5 per group. f Left top, immunoblot of human CD63 in circulating cancer-cell-derived sEV from MDA-MB231-luci (+)-bearing mice. n = 3 per group where similar amounts of protein/lane were verified by Ponceau S staining (Bottom). Right, Analysis of the relative protein intensity of CD63 measured using a densitometer system. Significance was determined using an unpaired two-tailed Student’s t-test. ***p < 0.001, **p < 0.005, and *p < 0.05. Error bar, SD. Source data (b–f) are provided as a Source Data file
Fig. 3
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SFX impairs sEV secretion through…
Fig. 3
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SFX impairs sEV secretion through interfering with the ESCRT-dependent MVE biogenesis. a Heatmap…
SFX impairs sEV secretion through interfering with the ESCRT-dependent MVE biogenesis. a Heatmap of the selected transcriptome of SFX-treated MDA-MB231 cells compared to untreated control cells. Heatmap represents probe sets for transcripts expressed at significantly higher or lower levels after exposure to 100 μM SFX, respectively (FC = ±1.3 p value = 0.05). n = 3. b Advanced bubble chart shows enrichment of differentially expressed genes in the indicated signaling pathways. The x-axis label indicates gene ontology (GO) biological processes, and the y-axis label represents p value. c qRT-PCR analysis of various genes in 24 h SFX-treated MDA-MB231 (upper) and 6 h SFX-treated SK-MEL-28 human melanoma cells (lower). n = 3. d Immunoblot for the indicated proteins in MDA-MB231 cells. Beta-actin and Lamin B were used as loading controls for cell lysates and nuclear fraction, respectively. Right, Analysis of the relative protein intensity of proteins measured using a densitometer system. n = 3. Experiments were performed with 95% confluent cells. Significance was determined using an unpaired two-tailed Student’s t-test. ***p < 0.001, **p < 0.005 and *p < 0.05. Error bar, SD. Source data (c, d) are provided as a Source Data file
Fig. 4
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Identification of the endothelin receptor…
Fig. 4
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Identification of the endothelin receptor as the novel target of SFX. a Schematic…
Identification of the endothelin receptor as the novel target of SFX. a Schematic illustration of three acetylated SFX derivatives. N1, N1-acetylation site (blue). N4, N4-acetylation site (green). b The minimal inhibitory concentration (MIC) of SFX and its derivatives against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. c Measurement of the number of sEV and the amounts of sEV proteins. Left, the number of sEV. Right, the amounts of sEV proteins. n = 3. d Immunoblot (Top) and qRT-PCR analysis (bottom) of RAB27A in MDA-MB231 cells treated with 100 µM SFX or its structural derivatives. e Candidate proteins for binding to SFX by searching binding DB. Tanimato coefficients (Tcs) as the measure of similarity are summarized. f Detection of ETA by immunoblot. Eluted parts from the membrane fraction of MDA-MB231 cells were used for biotin-based pull-down assay. WT-SFX was used as a negative control. n = 3. g Immunoblot of various proteins in MDA-MB231 cells after transfection of different ETA siRNAs. AccuTargetTM siRNA was used for control siRNA. n = 3. h Immunoblot of Rab27a protein after transfection of each siRNA specific to AGTR1, CA13, or KMO into MDA-MB231. n = 3. i Dose response curve for the inhibition of binding of 125I-ET1 to the human ETA receptor. IC50, the half maximal inhibitory concentration. Ki the inhibitory constant, nH Hill coefficient. j Immunoblot of ETA and its ligand, ET1 and ET2 in MCF10A, MCF7, and MDA-MB231 cells. n = 3 per cell line. Experiments were performed with 95% confluent cells. Significance was determined using an unpaired two-tailed Student’s t-test. ***p < 0.001, **p < 0.005 and *p < 0.05. Error bar, SD. Source data (b–d, i–j) are provided as a Source Data file
Fig. 5
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Identification of ETA as the…
Fig. 5
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Identification of ETA as the novel regulator of sEV biogenesis. a Measurements of…
Identification of ETA as the novel regulator of sEV biogenesis. a Measurements of the number of sEV secreted (left) and the amounts of sEV proteins (right) from WT, ETA-overexpressed (ETA-OE), and ETA knockdown (K/D) MDA-MB231 cells. n = 3. b Immunoblot of various proteins in ETA-OE and ETA-K/D MDA-MB231 cells. n = 3. c Measurement of the number of sEV secreted and the amount of sEV proteins from ETA agonist- or antagonist-treated MDA-MB231 cells. n = 3. d Measurement of the number of sEV and the amounts of sEV proteins from MDA-MB231 cells treated with 10 nM ET1 or 10 nM ET2 in the presence of 100 μM SFX. n = 3. e Measurement of soluble proteins secreted from MDA-MB231 cells in the presence of PD156707 or zibotentan for 24 h. The unit of IL8, IL2, CXCL13, CCL2, TGFB1, and IL6 is pg ml−1. The unit of CX3CL1 is ng ml−1. n = 3. f Tumor mass volume of in BALB/c nude female mice inoculated with MDA-MB231 or MDA-MB231 ETA K/D. Bar, 1 cm. n = 8 per group. g Left top, immunoblot of human CD63 in circulating cancer-cell-derived sEV from MDA-MB231 or MDA-MB231 ETA K/D-bearing mice (n = 8 per group) where similar amounts of protein/lane were verified by Ponceau S staining (Bottom). Right, densitometric analysis of the relative protein intensity of CD63. Experiments were performed with 95% confluent cells. Significance was determined using an unpaired two-tailed Student’s t-test. ***p < 0.001, **p < 0.005 and *p < 0.05. Error bar, SD. Source data (a, c–g) are provided as a Source Data file
Fig. 6
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SFX induces fusion of MVE…
Fig. 6
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SFX induces fusion of MVE with lysosomes in breast cancer cells. a TEM…
SFX induces fusion of MVE with lysosomes in breast cancer cells. a TEM analysis of SFX-treated MDA-MB231 cells. Red arrows indicated unusual structures in SFX-treated MDA-MB231 and MCF7, and white arrows indicated MVEs in control cells. Scale bar, 5 μm. b Top, Images of lysosome and autolysosome structures observed in SFX-treated MDA-MB231 cells. Bottom left, the numbers of distributed lysosome and autolysosome structures were counted. n = 20. Bottom right, LAMP-1 protein expression was upregulated in SFX-treated MDA-MB231 cells. n = 3. c Confocal image of CD63-GFP (+)-MDA-MB231 in the presence or absence of SFX. Red, lysotracker. Green, CD63-GFP. Scale bar, 2 μm. d Measurement of Lysotracker (red) intensity in rapamycin-treated (positive control) or SFX-treated MDA-MB231 cells. Lysotracker intensity measurement >170 cells per group. e Left, Image of MDA-MB231 treated by 100 μM SFX. Green, CellLight-Lysosome-GFP (GFP-LAMP1). Red, CellLight-late endosome/MVE-RFP (RFP-RAB7). Scale bar, 2 μm. Right, Quantitative colocalization rates of lysosomes and MVE in SFX-treated MDA-MB231. Colocalization puncta count >60 cells per group using ImageJ program. Bafilomycin A1 (BafA1) was used as a negative control. Experiments were performed with 95% confluent cells. Significance was determined using an unpaired two-tailed Student’s t-test. ***p < 0.001, **p < 0.005 and *p < 0.05. Error bar, SD. Source data (b, d,e) are provided as a Source Data file
Fig. 7
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Antagonists against the endothelin receptor…
Fig. 7
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Antagonists against the endothelin receptor induce fusion of MVEs with lysosomes in MDA-MB231…
Antagonists against the endothelin receptor induce fusion of MVEs with lysosomes in MDA-MB231 cells. a TEM analysis of ETA-antagonist-treated MDA-MB231 cells. Scale bar, 5 μm. b Measurement of lysotracker (red) intensity in rapamycin-treated or antagonist (Zibotentan and PD156707)-treated MDA-MB231. Lysotracker intensity measurement >100 cells per group. c Top, Immunoblot of LAMP-1 protein in antagonist (Zibo, BQ-123, and PD156707)-treated MDA-MB231. Bottom, Immunoblot of LAMP-1 protein in ETA-siRNA-transfected MDA-MB231 and in ETA-K/D MDA-MB231. n = 3. d Left, Image of MDA-MB231 treated by SFX. Green, CellLight-Lysosome-GFP (GFP-LAMP1). Red, CellLight-late endosome/MVE-RFP (RFP-RAB7). Scale bar, 2 μm. Right, Quantitative colocalization rates of lysosomes and MVE in MDA-MB231 treated with Zibotentan, PD156707, or BQ123. Colocalization puncta count >65 cells per group. e Images of ETA K/D MDA-MB231 or WT MDA-MB231 cells. Different colors represent the same markers as described in d. Scale bar, 2 μm. Experiments were performed with 95% confluent cells. Significance was determined using an unpaired two-tailed Student’s t-test. ***p < 0.001, **p < 0.005 and *p < 0.05. Error bar, SD. Source data (b, d) are provided as a Source Data file
Fig. 8
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Proposed model for SFX-mediated inhibition…
Fig. 8
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Proposed model for SFX-mediated inhibition of sEV secretion in MDA-MB231 cancer cells. SFX,…
Proposed model for SFX-mediated inhibition of sEV secretion in MDA-MB231 cancer cells. SFX, through ETA binding and subsequent interference, reduced the expression of proteins involved in the sEV biogenesis and secretion, increased the fusion of MVEs with lysosomes, and finally decreased the number of secreted sEV from breast cancer cells
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