Fig 1: Schematic and characterization of hNVs.a Schematic showing the hNVs consist of engineered SaV-C-NVs, M1-NVs, and P-NVs. b Schematic showing the hNVs efficiently interact with CTCs in the blood, accumulate in the post-surgical tumor bed, repolarize TAMs towards M1 phenotype, and block the CD47-SIRPa ‘don’t eat me’ pathway, thus promoting macrophage phagocytosis of cancer cells, as well as boosting antitumor Tcell immunity. c, d Immunofluorescence imaging c and flow cytometry analysis d of SaV expression on original and engineered B16F10 cells. Scale bar, 10 µm. e Relative mRNA expression of Cd86, Tnf, Il6, and Inos in M0 cells, M1 cells, M0-NVs and M1-NVs. f Hydrodynamic size and zeta potential of P-NVs, M1-NVs, SaV-C-NVs, and hNVs measured by DLS. g TEM images of hNVs. Scale bar, 100 nm and 50 nm in the left and right panel, respectively. The samples were negatively stained with uranyl acetate. h Immunofluorescence images of hNVs. Scale bar, 10 µm. i Western blot analysis of specific protein CD61, CD14, and Melan-A in the samples of P-NVs, M1-NVs, SaV-C-NVs, and hNVs. All data are presented as mean ± S.D. (n = 3). Statistical significance was calculated via unpaired two-tailed t test. **P < 0.01; ***P < 0.001.
Fig 2: Integrin ß3 regulates inflammatory response and permeability in vitro. PMVECs were transfected with empty or Itgb3 plasmid for 48 h, and then cells were exposed to rMMP-9 (10 ng/mL) for 1h, followed by treatment with LPS (1µg/mL) at indicated times. (A) NF-?B signaling proteins (p-IKK, IKKß, p-P65, P65, I?Ba, and p-I?Ba) were measured by Western blot. Supernatant IL-6 (C) and CXCL-1 (E) secretions were measured by ELISA. (G) The permeability of treated PMVEC monolayers grown on 0.4 mm filters was determined by the appearance of rhodamine-dextran. PMVECs were transfected with control siRNA, or Itgb3 siRNA for 48 h, and then cells were exposed to rMMP-9 for 1h, following by treatment with LPS at indicated times. (B) NF-?B signaling proteins were measured by Western blot. Supernatant IL-6 (D) and CXCL-1 (F) secretions were measured by ELISA. (H) The permeability of treated PMVECs was determined by the appearance of rhodamine-dextran. *P<0.05, **P<0.01. All the results are from at least three independent experiments; Data represent means ±SEM.
Fig 3: Characteristics of PexD and gel. A Photos of Pex and PexD. B TEM images of Pex and PexD. Scale bar (left side): 500 nm. Scale bar (right): 100 nm. C Particle size and potential of Pex and PexD. D SDS-PAGE protein analysis of Platelets, Pex, and PexD. E Western blot analyses were performed on Platelet, Pex, PexD labeled CD41, P-selectin and CD61, CD9 CD63 and TSG101 characteristic of Pex and PexD, and CD44 on B16-F10 cells. F Photos of Platelet, Pex and PexD. G DOX curve of aPD-L1-PexD-Gel in PBS at pH 6.5. Data are expressed as mean ± SD (n = 3). H Cryo-scanning electron microscopy (SEM) images of fibrin gel containing PexD nanoparticles and aPD-L1. Scale bar (left side): 40 µm. Scale bar (right): 20 µm
Fig 4: Integrin ß3/5 regulates MMP-9 expression in LPS-treated PMVECs. (A and B) Heat map of downregulated (green) and upregulated (red) mRNAs from PMVECs transfected with Itgb3 siRNA (siItgb3) or Itgb5 siRNA (siItgb5) for 48 h, and then subjected to 18 h of LPS (1µg/mL) treatment. Numbers 1, 2, and 3 refer to three independent experimental analyses of log2[(LPS+siCon)/(LPS+siItgb3/5)]. (C and D) MMP-9 gene expressions were measured in PMVECs transfected with siItgb3/5 for 48 h prior to 18 h of LPS (1µg/mL) treatment. (E and F) Western blot analysis of MMP-9 protein levels from PMVECs transfected with control siRNA (siCon) or siItgb3/5 for 48 h prior to 18 h of PBS or LPS (1µg/mL) treatment. (G and H) Western blot analysis of MMP-9 protein levels from PMVECs transfected with Itgb3/5 plasmid in a dose course and then exposed to LPS (1µg/mL). ELISA analysis of MMP-9 secretion from PMVECs transfected with Itgb3/5 plasmid (I) or siItgb3/5 (J) and then exposed to PBS or LPS (1µg/mL). *P<0.05, **P<0.01. All the results are from at least three independent experiments; Data represent means ±SEM.
Fig 5: HOXA10 overexpression rescues METTL3-impaired embryo attachment in vitro. a The mRNA levels of METTL3, HOXA10, ITGB3 and EMX2 in the METTL3-overexpressing Ishikawa cells with or without HOXA10 overexpression were analyzed by qRT-PCR. b The protein levels of METTL3, HOXA10, ITGB3 and EMX2 in the METTL3-overexpressing Ishikawa cells with or without HOXA10 overexpression were analyzed by Western blotting. c The levels of m6A RNA methylation in the METTL3-overexpressing Ishikawa cells with or without HOXA10 overexpression were evaluated by the m6A RNA Methylation Assay Kit. d The levels of m6A in the METTL3-overexpressing Ishikawa cells with or without HOXA10 overexpression were evaluated by dot blotting assay. e A vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids was used to evaluate the embryo attachment. Bar 100 µm. The data are the average of three independent experiments (n=3). *P<0.05, **P<0.01, **P<0.001 versus the indicated group
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