Additional file 1 of Novel plant-derived exosome-like nanovesicles from Catharanthus roseus: preparation, characterization, and immunostimulatory effect via TNF-α/NF-κB/PU.1 axis

Additional file 1: Figure S1. Isolation of exosome-like extracellular vesicles from Catharanthus roseus.C. roseus picked from Guangzhou, China.TEM analysis of C. roseus-derived exosome-like nanovesicles. Scale bar = 100 nm.Particle size distribution of C. roseus-derived exosome-like nanovesicles. Peak 1: 200.80 ± 87.22 nm, with an intensity of 65.0%; Peak 2: 14.65 ± 3.40 nm, with an intensity of 34.0%; Peak 3: 4881.00 ± 682.00 nm, with an intensity of 1.0%.TEM analysis of exosome-like nanovesicles derived from C. roseus leaves, stems, and flowers. Scale bar = 100 nm.Protoplasts that settle at the bottom of the beaker after enzymatic digestion.Pictures of nanovesicles obtained by different ultracentrifugation methods. a. Juicing then differential ultracentrifugation; b. enzyme digestion then differential ultracentrifugation; c. juicing then sucrose cushion ultracentrifugation; d. enzyme digestion then sucrose cushion ultracentrifugation. Figure S2. CLDENs with membrane fusion in an acidic environment.The larger vesicle was fusing with the smaller vesicles.Two vesicles with similar particle sizes were merging. Figure S3. Biodistribution of CLDENs.Biodistribution of CLDENs in the organs after oral administration. a. Fluorescent signals in the brain, heart, liver, spleen, thymus, lung and kidney. b. Fluorescent signals in the gastrointestinal tract. A strong fluorescence signal exceeding the detection threshold of the instrument was observed in the stomach until the 12th hour.Biodistribution of CLDENs in the organs after tail vein injection. a. Fluorescent signals in the brain, heart, liver, spleen, thymus, lung and kidney. b. Fluorescent signals in the gastrointestinal tract. Figure S4. CLDENs were found in the lymph nodes in the neck of animals after intraperitoneal injection. Figure S5. Molecular weights distribution of the identified proteins in the PLANT group. Figure S6. Effects of different treatments on CLDENs’ immunostimulatory activity. CLDENs were treated with proteinase K at 37 °C, RNase A at 37 °C, DNase I at 37 °C, strong acid and strong base at 25 °C, 0.1% Triton X-100 at 25 °C, and 0.1% SDS at 25 °Cfor 30 min or heated at 100 °C for 10 min. Next, CLDENs’ ability to encourage nitric oxide secretion from RAW264.7 cells was investigated. CLDENs’ activity was not significantly altered by proteinase K at 37 °C, RNase at 37 °C, strong acid/strong base at 25 °C, and DNase I at 37 °C treatment, but their immunostimulatory activity was lost by 0.1% Triton X-100 at 25 °C, 0.1% SDS at 25 °C, and 100 °C heating treatment. Data were mean ± SD, n = 3; **P < 0.01 and ***P < 0.001 vs. Blank. ##P < 0.01 and ###P < 0.001 vs. CLDENs group. ns, not significant. Figure S7. Induction of C. roseus cells.Germinating dedifferentiated cells in the leaves of C. roseus.Germinating cambial meristematic cells in the stems of C. roseus.C. roseus dedifferentiated cells viewed through a 20 × optical microscope. Scale bar = 100 μm.C. roseus cambial meristematic cells viewed through a 20 × optical microscope. Scale bar = 100 μm.. Comparison of the yield of three nanovesicles. Data were mean ± SD, n = 3, **P < 0.01 vs. CLDENs group. Table S1. List of identified substances in lipidomic analysis. Table S2. List of identified substances in metabolomic analysis. Table S3. List of disease phenotypes that correlated with CLDENs. Table S4. Proteins significantly up-regulated in differential proteomics. Table S5. Real-time quantitative PCR primer sequence.