Calnexin Antibody - #AF5362

Fig. 3 CD45 shed from WBCs could be transferred via EVs to tumor cells. a GFP+ Caco2 cells co-cultured with Jurkat cells at a ratio of 1:4 for different time (0, 1, 2, 4, 8, 16 h), then GFP+ Caco2 cells were collected and stained with anti-CD45-APC to dynamically analyze the MFI shifting of CD45 among GFP+ Caco2 cells using flow cytometry. b Flow cytometric analysis of CD45 transfer from Jurkat cells to Caco2 cells, or from Jurkat cells/ THP1 cells/ WBCs to DLD1 cells following 16 h of indirect co-culture. c, d Bar graph showing the MFI of CD45-PE of Caco2 cells or DLD1 cells after 16 h of indirect co-culture with Jurkat cells/ THP1 cells/ WBCs. e, f Immunoblot analysis showing the makers of EVs isolated from supernatant of THP1 and Jurkat cells. e Density gradient fractions of EVs from Jurkat cells. f Positive (TSG101 and CD63) and negative (Calnexin) markers of EVs. WCL: whole cell lysis. Each ladder was loaded with protein 30 μg. g Nanoparticle tracking analysis of Jurkat cell-derived EVs. h Transmission electron microscope imaging of Jurkat cell-derived EVs. Scale bar = 50 nm. i Plasma EVs charactering by immunoblotting. Jurkat-EVs was used as positive control. P1- and P2-EVs were examples of two CRC cancer patients, HD1 and HD2 were examples of two healthy donors. j Plasma EVs charactering by nano-flow cytometry analysis. k, l CD45 ELISA assay for the measurement of EVs-derived CD45 from healthy donors (HD) and CRC patients (PD) with or without metastasis. m Time series of live cell imaging of DLD1-RFP cells after the addition of EVs (20 μg/mL) derived from CD45-GFP-expressing HEK293T cells for up to 8 h. Scale bar = 5 μm. n Immunofluorescence images of GFP+ Caco2 cells after incubation with PBS or EVs isolated from Jurkat cell supernatant for 12 h. Left scale bar = 20 μm, right scale bar = 5 μm. o Flow cytometric analysis for the measurement of Jurkat cell-derived EVs uptake by Caco2 cells. Caco2 cells were incubated with different concentration of PKH26-stained EVs (0, 1, 2, or 4 μg/mL) for 12 h before analysis. p Caco2 cells were incubated with PKH26-stained EVs (2 μg/mL) and chlorpromazine (CPZ, 0, 5, 10, or 20 μg/mL) for 12 h before collection for flow cytometric analysis of PKH26+ Caco2 cells. All bar graph data are presented as means ± SEM. *P 

Fig. 5 | CNP increases exosome release through HSP–p53–TASP6 signalling pathway. a, Simulated temperature changes at five selected locations. A 200 V and 10 ms pulse created a localized ‘hot spot’ in the nanochannel outlet with a power density of ~1 × 1014 W m−3 and a peak temperature up to 60 °C from room temperature. Once the pulse ended, the hot spot vanished rapidly due to the extremely small volume of the heated fluid inside the nanochannel (~1 × 10−12 cm3 ) compared with the bulk solution outside the nanochannel (~0.1 cm3 ). b, Top-down images of MEFs (green) attaching to the surface of the CNP device. Red dots show nanochannel locations and room temperature before CNP transfection (0 s). White arrows indicate locations of the nanochannels. The CNP electric pulse (CNP) sharply increases temperature at the nanochannel–cell surface interface. c, Cross-section view of nanochannels shows temperature changes in the nanochannels before (0 s), during and after (1 s) a CNP pulse. d, Temperature at the cell–nanochannel interface transiently (<1 s) increases to ~60 °C. e, Western blot of HSP90 and HSP70 from untreated (PBS) and CNP (with PBS)-stimulated (CNP) MEFs. f, DLS measurements of exosome concentrations from 108 CNP-stimulated MEFs with or without HSP inhibitors show that HSP70 and HSP90 are critical for the production of exosomes. NVP-HSP990, HSP90 inhibitor; VER155008, HSP70 inhibitor. g, Western blots show that CNP increases expression of p53 and TSAP6 protein in p53 wild-type MEFs, but does not affect p53 or TSAP6 protein expression in p53−/− MEFs. h, DLS measurements of exosome concentrations show that knockdown of p53 can partially block exosome release after CNP. i, Schematic of a proposed mechanism for CNP triggering of exosome release in CNP-transfected cells. Data are from three independent experiments and are presented as mean ± s.e.m. Two-sided Student’s t-test was used for the comparison.

Figure 2 Creation and characterization of cNPcancer cell@MVDC (A) Schematic diagram illustrating the production of cNPorganoid@MVhDC. (B) TEM analysis of cNPs before and after adsorbing PDAC patient-derived tumor organoid lysate, with curve charts showing the size changes. Results shown are representative of three independent experiments. (C) SEM analysis of human or mouse immature DC morphology after indicated treatments. White arrows indicate MV budding. (D) TEM analysis of cNPorganoid@MVhDC and cNPDT6066@MVDC, including representative images and magnified views. White dot lines represent MV’s membrane thickness. (E) Confocal microscopy examination of red PKH26 membrane fluorescent dye stained cNPorganoid@MVhDC or cNPDT6066@MVDC derived from UV-stimulated human/mouse DCs pulsed with green PKH67 fluorescently labeled cNPorganoid or cNPDT6066. Representative IF images of three independent experiments are given. (F) Curve charts show the size of cNPorganoid@MVhDC (top) and cNPDT6066@MVDC (bottom), respectively. Results shown are representative of three independent experiments. (G and H) Venn diagram analysis comparing proteomics data between different indicated groups (n = 3 independent samples). (I and J) Heatmap analysis of the proteomics data in (G) and (H). (K and L) Western blot analysis of the indicated proteins in each group. TSG101 was used as a loading control (n = 3 independent experiments). (M and N) Flow cytometry analysis of CD86, MHC-I, or MHC-II expression in each group. Results shown are representative of three independent experiments. Scale bars in (B) represent 200 nm. (C) 5 μm. (D) 0.5μm (top), 200 nm (middle), 50 nm (bottom). (E) 100 μm. See also Figure S2.

Fig. 4 The isolation and identifcation of Cal-27 exosomes. a A sketch of isolation path and identifcation of exosomes. b Western blot analyzed of exosomes for CD63, CD9 and CD81 (exosome biomarker), Calnexin (negative marker) and CMTM6. c Western blot analyzed of M0 cells and exosomes incubated M0 cells for CMTM6. d Representative fuorescent images for PKH26-labeled exosomes taken by M0 after 12 h incubation. Exosomes were stained red and M0 nuclei were stained blue by DAPI (scale bar: 25um). Cal-27 cells incubated with non-labeled exosomes (PBS was added to 25 μg exosomes instead of PKH26) and Cal-27 cells with only PKH26 (PKH26 was added to PBS and was re-isolated as stated) were used as controls

FIGURE 1Cell identification and exosome characterization. (A) Representative flow cytometry histograms of BMSCs and ADSCs show positive staining for CD29 and CD90 but not for CD34 and CD45. (B) BMSCs and ADSCs were successfully induced into osteoblasts (positively stained with Alizarin Red S) and adipocytes (positively stained with Oil Red O). The magnification is 100×. (C) Representative immunofluorescence results of CCSMCs show positive expression for α-SMA and desmin. Scale bars = 50 μm. (D) Exosomes derived from CCSMCs, BMSCs and ADSCs were observed using transmission electron microscopy, and the particle size distributions of the exosomes were measured by nanoparticle tracking analysis. Scale bars = 100 nm. (E) Representative results of Western blot analysis of exosomes derived from CCSMCs, BMSCs and ADSCs show positive expression for CD9, CD63 and TSG101 but not for calnexin. CCSMC: corpus cavernosum smooth muscle cell; BMSC: bone marrow stem cell; ADSC: adipose-derived stem cell; CCSMC-EXOs: exosomes derived from corpus cavernosum smooth muscle cells; BMSC-EXOs: exosomes derived from bone marrow stem cells; ADSC-EXOs: exosomes derived from adipose-derived stem cells; α-SMA: α-smooth muscle actin; DAPI: 4’,6-diamidino-2-phenylindole