Fig 1: Alix deletion in bone marrow‐derived macrophages (BMDMs) results in decreased EV production. (a) BMDMs were differentiated from whole bone marrow from Alix fl/fl × LysM‐CreTg/+ (Alix Mye‐KO) mice and Alix fl/fl × LysM‐Cre+/+ (Alix Mye‐WT). At 24 h before the start of the assays, BMDMs were re‐seeded at equal cell densities. At the start of incubation, the culture medium was switched to Opti‐MEM. Cells were lysed and culture medium was collected for analyses 8 h after switching to Opti‐MEM medium. (b) Deflox PCR on several myeloid cell‐containing organs and BMDMs derived from AlixMye‐KO mice. The presence of the deletion band (473 bp) shows effective cre‐mediated ‘defloxing’. The flox band (368 bp) is merely absent in BMDMs while still present in the organs. (c) Alix expression in BMDMs analysed with RT‐qPCR, relative to AlixMye‐WT, showing efficient KO of Alix on RNA level. (d) ALIX western blot, visualizing the absence of ALIX (96 kDa) in BMDMs from AlixMye‐KO BMDM lysates. Red bands represent ACTIN protein (42 kDa) expression. (e–n) ExoView quantification of CD9+ EVs in BMDM culture medium (e–i) and plasma (j–n) from AlixMye‐WT (n = 3–4) and AlixMye‐KO (n = 3–4) mice, showing the total (e, j), CD81+ (f, k), CD63+ (g, l), CD9+ (h, m) and label‐free scatter+ (50–200 nm) (i, n) EVs captured on the CD9 spot. (O–S) Expression level of Smpd1 (o), Smpd2 (p), Smpd3 (q), Tsg101 (r) and Stam1 (s) in BMDMs from AlixMye‐WT (n = 6) versus AlixMye‐KO mice (n = 5). Results are represented relative to AlixMye‐WT. Data are represented as means ± SEM. Statistical analyses were performed by unpaired t‐testing or Mann–Whitney testing (*p < 0.05, ***p < 0.001, ns, not significant). Alix, apoptosis linked gene 2 interacting protein X; BMDM, bone marrow‐derived macrophage; EV, extracellular vesicle; Smpd, sphingomyelin phosphodiesterase.
Fig 2: RAB27B knockdown or overexpression regulates exosome secretion. (A) The expression of Alix, CD9, TSG101 and Calnexin in cell and exosome explored by western blotting. (B) Image of transmission electron microscopy of our exosome sample (magnification: ×150,000, high voltage: 80 kV). (C) Nanoparticle tracking analysis result of our exosome sample. (D) Protein level of RAB27B in 6 pancreatic cancer cell lines. (E) Efficiency of RAB27B knockdown in MIA PaCa-2 cells confirmed by western blotting. (F) Efficiency of RAB27B overexpression in T3M4 cells confirmed by western blotting. (G) Protein concentration detected in the same amount of cell supernatant and cell numbers of different exosome samples. Significant results were observed at **, P<0.01; and *, P<0.05, respectively. NC, negative control.
Fig 3: Delivery of miR-503-5p to HCAECs and HCASMCs by macrophage-derived EVs. (A) Structure and diameter of EVs observed by TEM (×100000). (B) Diameter and number of EVs measured by NTA. (C) Expression of EV marker proteins Alix, CD63, and CD9 determined by Western blot analysis; *p < 0.05 compared with EVs. HCAECs and HCASMCs were co-cultured with EVs derived from RAW264.7 cells with or without ox-LDL treatment. (D) Expression of miR-503-5p (normalized to U6), TGF-β1, smad7, smurf1, and smurf2 (all normalized to GAPDH) in HCAECs and HCASMCs determined RT-qPCR; *p < 0.05 compared with EVs derived from RAW264.7 cells without ox-LDL treatment. (E) EVs were phagocytosed by HCAECs and HCASMCs, observed under laser confocal microscope. PKH67-labeled EVs was green, DAPI-stained nuclei was blue, while cy3-miR-503-5p-labeled EVs was red (×400). (F) Protein expression of TGF-β1, smad7, smurf1, and smurf2 (normalized to GAPDH) in HCAECs determined by Western blot analysis. Values obtained from three independent experiments in triplicate were analyzed by unpaired t test between two groups and by one-way ANOVA followed by Tukey's post hoc test among three or more groups. *p < 0.05 compared with miR-inhibitor NC-treated HCAECs co-cultured with EVs in the absence of ox-LDL; # p < 0.05 compared with miR-503-5p inhibitor treated HCAECs co-cultured with EVs in the absence of ox-LDL.
Fig 4: Smpd3 deletion in bone marrow‐derived macrophages (BMDMs) does not affect the amount of EV release. (a) BMDMs were differentiated from whole bone marrow from Smpd3 fl/fl × LysM‐CreTg/+ (Smpd3 Mye‐KO) mice and Smpd3 fl/fl × LysM‐Cre+/+ (Smpd3 Mye‐WT). Twenty four hours before the start of the assays, BMDMs were re‐seeded at equal cell densities. At the start of incubation, the culture medium was switched to Opti‐MEM. Cells were lysed and culture medium was collected for analyses 8 h after switching to Opti‐MEM medium. (b) Deflox PCR on several myeloid cell‐containing organs and BMDMs derived from Smpd3Mye‐KO mice. The presence of the deletion band (402 bp) shows effective Cre‐mediated ‘defloxing’. The flox band (2121 bp) is absent in BMDMs while still present in the organs. (c) Smpd3 expression in BMDMs analysed with RT‐qPCR, relative to Smpd3Mye‐WT, showing efficient KO of Smpd3 on RNA level. (d) nSMase activity assay illustrating reduced nSMase enzyme activity in Smpd3Mye‐KO mice. (e–n) ExoView quantification of CD9+ EVs in BMDM culture medium (e–i) and plasma (j–n) from Smpd3Mye‐WT (n = 3–7) and Smpd3Mye‐KO (n = 3–7) mice, showing the total (e, j), CD81+ (f, k), CD63+ (g, l), CD9+ (h, m) and label‐free scatter+ (50–200 nm) (i, n) EVs captured on the CD9 spot. (O–S) Expression level of Smpd1 (o), Smpd2 (p), Alix (p), Tsg101 (r) and Stam1 (s) in BMDMs from Smpd3Mye‐WT (n = 4–5) versus Smpd3Mye‐KO mice (n = 5–6). Results are represented relative to Smpd3Mye‐WT. Data are represented as means ± SEM. Statistical analyses were performed by unpaired t‐testing (*p < 0.05, ****p < 0.0001; ns, not significant). Alix, apoptosis linked gene 2 interacting protein X; BMDM, bone marrow‐derived macrophage; EV, extracellular vesicle; Smpd, sphingomyelin phosphodiesterase.
Fig 5: Smpd3 deletion in mixed cortical cell (MCC) cultures does not affect the level of EV production. (a) Schematic overview of the experimental set‐up for Smpd3 fl/fl MCC culture, TAT‐CRE mediated Smpd3 deletion and EV collection. (b) Deflox PCR on lysed MCC cells. The presence of the deletion band (402 bp) shows effective TAT‐CRE mediated ‘defloxing’. The flox band (2121 bp) is nearly absent in TAT‐CRE induced Smpd3 KO MCCs (Smpd3 KO) while still present in the Smpd3 WT MCCs (Smpd3 WT). (c) Smpd3 expression in Smpd3 KO MCCs analysed with RT‐qPCR, relative to Smpd3 WT MCCs. (d–h) ExoView quantification of CD9+ EVs in MCC culture medium from Smpd3 WT (n = 3) and Smpd3 KO (n = 3) MCC cultures, showing the total (d), CD81+ (e), CD63+ (f), CD9+ (g) and label‐free scatter+ (50–200 nm) (h) EVs on the CD9 capture spot. (I–M) Expression level of Smpd1 (i), Smpd2 (j), Alix (k), Tsg101 (l) and Stam1 (m) in MCCs from Smpd3 WT (n = 4) versus Smpd3 KO mice (n = 3). Results are represented relative to Smpd3 WT MCC expression values. Data are represented as means ± SEM. Statistical comparison of two groups was performed by unpaired t‐testing or a Mann–Whitney test (**p < 0.01; ns, not significant). Alix, apoptosis linked gene 2 interacting protein X; EV, extracellular vesicle; MCC, mixed cortical cell; Smpd, sphingomyelin phosphodiesterase.
Supplier Page from Abcam for Anti-ALIX antibody