Additional file 1 of Neutrophil membrane engineered HucMSC sEVs alleviate cisplatin-induced AKI by enhancing cellular uptake and targeting
Creators: name: Wu, Peipei
name: Tang, Yuting
name: Jin, Can
name: Wang, Min
name: Li, Linli
name: Liu, Zhong
name: Shi, Hui
name: Sun, Zixuan
name: Hou, Xiaomei
name: Chen, Wenya
name: Xu, Wenrong
name: Qian, Hui
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Space Science
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Marine Biology
subject: Cancer
subject: Virology

description: Additional file 1: Table S1. PCR primer sequences of genes. Fig. S1. Morphological characteristics, multipotential differentiation and surface marker identification of HucMSCs. a Inverted microscope was used to observe the morphological characteristics of primary and third generation hucMSCs (40×). b Oil red O staining and alizarin red S staining were performed to evaluate adipogenic differentiation (400×) and osteogenic differentiation of P3 hucMSCs (200×). c Flow cytometry were applied to detect the expression of hucMSCs surface specific antigens. Fig. S2. HucMSC-sEVs are highly enriched in various of beneficial molecules for AKI repair. a LC/MS–MS was performed to detect the proteomics of hucMSC-Ex. b Several molecules have been shown to be used for the repair of different acute kidney injury diseases. Fig. S3. Preparation and characterization of Neu-NVs. a Schematic diagram of preparation of human peripheral blood neutrophil membrane derived Neu-NVs. b Coomassie blue staining was performed to identify neutrophils and their cell membrane protein components. c The particle size and morphology of Neu-NVs were detected by TEM. d, e The appearance and height of Neu-NVs were observed by AFM. f Western blot was used to detect protein molecules on the surface of Neu-NVs. g, h NTA was applied to verify the particle size, concentration and Zeta potential of Neu-NVs. Fig. S4. Preparation and characterization of neutrophils membrane vesicles. a Neutrophils cell membrane precipitates were collected by differential supercentrifugation. b Immunofluorescence staining was perform to detect DIO and DIL-labeled Neu-NVs (600×). Fig. S5. DIO and DIL fluorescence emission spectra. a,b Automatic microplate reader cytation 5 was performed to detect DIO and DIL fluorescence emission spectra, respectively. Fig. S6. Fusion efficiency detection of Neu-NVs and hucMSC-sEVs. a The particle size and concentration of DIO-DIL-labeled hucMSC-sEVs and Neu-NVs were detected by NTA. b Fluorescence expression of DIO and DIL-labeled hucMSC-sEVs after fusion was detected by laser scanning confocal microscope (600×). Fig. S7. The particle size and concentration detection of DIO labeled Neu-NVs and DIL labeled hucMSC-sEVs. a The particle size and concentration of DIO labeled Neu-NVs were detected by NTA. b The particle size and concentration of DIL labeled hucMSC-sEVs were detected by NTA. Fig. S8. Construction of AKI model induced by cisplatin in vivo. a Biochemical assay was used to measure serum Cr and BUN of mice in normal group and AKI model group. b HE staining was performed to evaluate the pathological changes of renal tissues in normal group and AKI model group (100 μm). c Immunofluorescence staining as used to detect renal cell apoptosis in normal group and AKI model group (200×). Fig. S9. Construction of NRK52E cell injury model induced by cisplatin in vitro. a Cell apoptosis was detected by flow cytometry after NRK52E cells were treated with different concentrations of cisplatin. b CCK8 assay was applied to measure the proliferation activity of NRK52E cells treated with different concentrations of cisplatin for 12 h and 24 h. c qRT-PCR was used to detect the expression of proinflammatory cytokines IL-1β and IL-6 in NRK52E cells treated with 10 ng/mL cisplatin for 12 h. Fig. S10. DIO-labeled hucMSC-sEVs and DIO-labeled Neu-NVs were internalized by NRK52E cells. a-c Confocal microscopy was used to detect the cell internalization of DIO-labeled hucMSC-sEVs and DIO-labeled Neu-NVs after co-incubation with NRK52E cells for 6 h, 12 h and 24 h. Fig. S11. Detection of CD47 protein expression in sEVs derived from HucMSCs. Western blotting was used to detect characteristic protein markers and CD47 protein expression levels in HucMSC-sEVs.