Fig 2: MAT decreases the migratory capacity of MDA-MB-231 cells potentially by targeting ITGB1. (A) Migration was analyzed in MDA-MB-231 and MCF-7 cells with or without MAT treatment (1 and 2 mg/ml) for 48 h. (B) Reverse Transcription-quantitative PCR and (C) western blot analysis of ITGB1 mRNA and protein levels in MDA-MB-231 and MCF-7 cells following MAT treatment. *P<0.05 and **P<0.01 MAT vs. CTL by one-way ANOVA. The expression of ITGB1 was reduced in (D) MDA-MB-231 and (E) MCF-7 cells transfected with ITGB1 siRNA. β-actin was used as a loading control. (F) In the presence of siRNA targeting ITGB1, Transwell® assay was conducted to evaluate MDA-MB-231 and MCF-7 cell migration following transfection. Silencing ITGB1 results in decreased MDA-MB-231 and MCF-7 cell migration. *P<0.05, **P<0.01 and ***P<0.001 MAT vs. NC by one-way ANOVA followed by Student-Newman-Keuls post hoc test. MAT, matrine; ITGB1, integrin β1; ANOVA, analysis of variance; CTL, control; NC, negative control; si, small interfering.

Fig 3: Schematic representation for MARVELD1-mediated neuronal migration.MARVELD1 deletion in glial cells induced the abnormality of glial fibres. Meanwhile, the expression of ITGB1 was increased in the pre-mRNA process in neurons, which further activated FAK through increasing its Tyr397 phosphorylation level. This regulation activated FAK-mediated downstream signalling that resulted in neuronal migration. The regulation process of MARVELD1 during brain development was in a glia cell-dependent manner. Furthermore, this process affected the neurodegeneration and behaviour in adult mice

Fig 4: rAj-Tspin inhibited the growth and promoted the apoptosis of Huh7 cells. A Measurement of cell viability upon rAj-Tspin treatment with a CCK-8 assay. B Migration, invasion and adhesion capacity of Huh7 cells treated with increasing concentrations (0, 0.2, 0.4, 0.8 μM) of rAj-Tspin for 24 h. C Apoptotic cells were examined via a TUNEL assay; scale bar = 50 μm. D Immunoblotting of ITGB1 and ZYX in LO2 and Huh7 cells. E and F Immunoblotting of ITGB1, ZYX, p-FAK, FAK, p-AKT, AKT, Bcl-2, Bax, cleaved caspase-3, vimentin, N-cadherin, and E-cadherin in Huh7 cells treated with increasing concentrations (0, 0.2, 0.4, 0.8 μM) of rAj-Tspin for 24 h (n = 3). *P < 0.05, **P < 0.01

Fig 5: Cellular uptake and tissue penetration. (a) Time‐dependent cellular uptake of CDVs and EVs was analyzed in BT549 cells by flow cytometry. BT549 cells were seeded on a 24‐well plate at 4 × 104 cells/well. The next day, DiR‐labelled CDVs (DiR‐labeled UCMSC‐CDV) and EVs (DiR‐labelled UCMSC‐EV) were added to the cells at 1 × 105 particles per cell concentration and incubated at 37°C for 1, 3, 6, 18, 24 and 48 h, along with control groups: dye only (DiR only) or unlabelled CDVs (UCMSC‐CDV only) or EVs (UCMSC‐EV only). After harvesting cells at each time point, the fluorescence intensity was measured using a flow cytometer to determine the uptake efficiency. Data represent the mean ± SEM from two independent experiments using different batches of CDVs or EVs. The p‐value was determined by a two‐way analysis of variance (ANOVA). **p < 0.01. (b) Enhanced CDV uptake was observed from diverse recipient cells. BT549, HUVEC, NHDF and HEK293 cells were used, and the uptake efficiency was analysed 24 h after incubating with UCMSC‐CDVs or UCMSC‐EVs as described above. All uptake assays were repeated using two different batches of CDVs and EVs. Data represent the mean ± SD. An unpaired t‐test was employed. **p < 0.01, ***p < 0.001. ns. Not significant. (c) For confocal microscopy, BT549 cells treated with DiR‐UCMSC‐CDVs or DiR‐UCMSC‐EVs, as described above, were collected at 24 h after incubation. Cells were fixed with paraformaldehyde and subjected to immunostaining and confocal imaging. Representative images are shown here. CDVs or EVs are shown in red (DiR); cytoplasm in green (anti‐β‐tubulin antibody); nucleus in blue (Hoechst counterstain). Scale bar: 25 μm. (d) Digestion (proteinase K) or (e) blockade by antibodies of membrane proteins resulted in a target‐specific reduction in cellular uptake. (d) To digest surface proteins, UCMSC‐CDVs and UCMSC‐EVs were treated with proteinase K at 0.2 μg proteinase K/μg protein before labelling with DiR. DiR‐labelled CDVs and EVs were then added to BT549 cells at 1 × 105 particles per cell concentration and incubated at 37°C for 24 h. (e) For the surface protein blocking, 2 × 109 particles of DiR‐labelled CDVs and EVs were treated with antibodies against CD63, CD81, CD9, CD29 and LAMP‐1 at 1:100 (high), 1:1000 (medium) or 1:10,000 (low) dilution for 30 min at 4°C in the dark. The fluorescence intensity was measured using a flow cytometer to determine the uptake efficiency. The fluorescence intensity of proteinase K‐untreated (d) or antibody‐untreated (e) DiR‐UCMSC‐CDVs was used to normalise each data for comparative analysis. Data represent the mean ± SD (N = 2 for proteinase K; N = 3 for antibody blockade). An unpaired t‐test was employed. *p < 0.05, **p < 0.01, ***p < 0.001. (f) Retinal penetration of CDVs and EVs. A total of 1.5 μl of DiD‐labelled UCMSC‐CDVs or UCMSC‐EVs (approximately 1 × 1011 particles/ml) were injected into the right eye (intravitreal injection) along with PBS control. The mice were sacrificed at 6 and 24 h post injection. The eyeball was removed, fixed in paraformaldehyde, sectioned at 7 μm thickness, and subjected to immunostaining and imaging under the epifluorescence microscope. Representative fluorescence images after 6 and 24 h of intravitreal injection are shown. CDVs or EVs colocalised in the RPE and choroid tissue are shown in bright yellow (Merged, combining red and green channels), while the relative quantity of CDVs or EVs in the retinal tissues can be visualised better without a green channel (Red). Nuclei were stained with DAPI (blue), RPE with anti‐RPE65 (green) and CDVs or EVs with DiD (red). ONL (outer nuclear layer); INL (inner nuclear layer); RPE (retinal pigment epithelium). Scale bar: 100 μm. (g) Quantitation results show the relative intensity of the fluorescence signal in the retina normalised by the total input signal. Data represent the mean ± S.D. An unpaired t‐test was employed. **p < 0.01