Additional file 1 of Inhibition of DAMP actions in the tumoral microenvironment using lactoferrin-glycyrrhizin conjugate for glioblastoma therapy
Creators: name: Kim, Hyung Shik
name: Park, Seok Chan
name: Kim, Hae Jin
name: Lee, Dong Yun
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: 19999 Mathematical Sciences not elsewhere classified
subject: FOS: Mathematics
subject: Developmental Biology
subject: Cancer
subject: Plant Biology
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. FT-IR spectra of GL, Lf and Lf-GL respectively. The conjugation between GL and Lf was confirmed by Fourier transform infraredanalysis. The amide I and amide II vibrations observed at 1,523 cm-1 and 1,630 cm-1 in both Lf and Lf-GL. The 1,035 cm-1 indicates primary alcohol stretch of GL. Figure S2. SDS-PAGE result of Lf and Lf-GL. The successful synthesis of Lf-GL could be assumed from the sodium dodecyl sulfate polyacrylamide gel electrophoresisresults as a molecular weightrise in the band above 80 kDa, the MW of native Lf. Figure S3. HPLC result of GL and Lf-GL. High performance liquid chromatographyusing gel permeation chromatographycolumn was performed to verify GL content in Lf-GL conjugate. Mobile phase is composed of methanol, acetonitrile, water, and acetic acid in a ratio of 55:23.69:19.63:0.68. GL was dissolved in mobile phase in different concentrationand Lf-GLwas also dissolved in mobile phase. Ultrahydrogel 120 Columnwas used as column, flow rate was 1 mL min-1. Then, absorbance was measured in 254 nm and results were calibrated with Empower software. Figure S4. MALDI-TOF result of Lf and Lf-GL. MALDI-TOF was conducted at Seoul National Universityusing MALDI-TOF Voyager DE-STRand sinapinic acidaqueous solution containing about 30% acetonitrile in 0.15% trifluoroacetic acidwas used as a matrix. Average molecular weight of Lf-GL is 86,727.3 ± 98.1 Da. Considering the molecular weight of Lf is 78,497.9 Da and GL is 822.9 Da, binding ratio of Lf:GL is 1:9.6. Figure S5. Binding affinity against HMGB1. Surface Plasmon Resonancewas conducted at WoojungBSC. The Reichert SR7500DC systemand Scrubber2 softwarewere used and the CMDH chipwas used for immobilization of the human recombinant HMGB1 protein. HMGB1 protein was used as ligand and analytes were HMGB1 antibody, GL, Lf and Lf-GL conjugate. Immobilization buffer was 10 mM S.A.and running buffer was 1× PBS. The flow rate of the analyte was 30 μL min-1. The association and dissociation time were 3 min each and conducted at room temperature. Results are expressed in response unitsover time. Figure S6. Live and dead assay of U87MG in the Control, GL, Lf, and Lf-GL group. The cells were treated with GL equivalent concentration of 200 μM for 24 h. Thereafter, cells were treated with 1 μM of Calcein AM and EthD-1. Magnification: X100. Figure S7. Cell viability assay of intestinal epithelial celltreated with GL, Lf, and Lf-GL, respectively.Optical image of Caco-2 cell that treated with GL equivalent concentration of 50 μM for 24 h. Scale bar: 500 μmCell viability of Caco-2 cell that treated with GL equivalent concentration of 25 μM to 100 μM for 24 h. Data were expressed as mean ± S.E.M. Figure S8. GBM spheroid growth inhibition of Lf-GL. To form U87MG spheroid, cells were seeded at density of 1X105 cells in concave mold. After spheroids were formed, they were transferred to 24-well plate coated with 2% agarose gel. The constructed spheroids were treated with conditioned medium at 2 days interval after a washing step with PBS.Morphology of U87MG spheroid treated with Lf-GL of GL equivalent concentration of 50 μM on day 0, 2, 4 and 6, respectively. Scale bar: 200 μmGBM spheroids time-related volume after treated with Lf-GL of GL equivalent concentration of 50 μM. Data were expressed as mean ± S.E.M. **P &lt;0.01 versus Control. Figure S9. Non-apoptotic GBM spheroid growth inhibition of Lf-GL. Live and dead staining of GBM spheroids on day 6 that treated with GL equivalent concentration of 50 μM. Scale bar: 200 μm. Figure S10. Non-apoptotic U87MG growth inhibition of Lf-GL. Annexin V-DY-634 / PI apoptosis staining to detect the apoptosis and necrosis in the Control, GL, Lf, and Lf-GL treated group at all GL equivalent concentration of 50 μM for 24 h. However, the cell population of annexin V/PIin the Lf-GL treatment group was 0.28%, which was even lower level to 1.40% in the Control group. Figure S11. Significant cellular uptake of Lf-GL in U87MG.Flow cytometry to evaluate cellular uptake of FITC tagged-Lf-GLin U87MG for 2,6, and 18 h. The 2 h pre-treatment of Lf and Pitstop® were treated in the concentration of 500 nM and 75 nM, respectively.Quantification of cellular uptake for 18 h. Data were expressed as mean ± S.E.M. Figure S12. Live and dead assay of HUVEC in the Control, GL, Lf, and Lf-GL group. The cells were treated with GL equivalent concentration of 100 μM. for 24 h. Thereafter, cells were treated with 1 μM of Calcein AM and EthD-1. Magnification: X100. Figure S13. HUVEC growth inhibition of Lf-GL. Annexin V-DY-634/PI apoptosis staining to detect the apoptosis and necrosis in the Control, GL, Lf, and Lf-GL treated group at all GL equivalent concentration of 100 μM for 24 h. Figure S14. Significant inhibitory effect of Lf-GL in aortic ring angiogenesis. Optical images of vessel regression in aortic ring with Lf-GL treatment. Aortic ring assay was carried out in 48-well plate coated with Matrigel. VEGF-non treated control represents that aortic ring incubated with normal medium. VEGF-treated control represents that aortic ring incubated with 25 ng mL-1 VEGF-conditioned medium. GL, Lf and Lf-GL groups represents that aortic ring incubated with VEGF-conditioned medium containing GL, Lf, and Lf-GL with GL equivalent concentration of 200 μM, respectively. Magnification: X100. Scale bar: 100 μm. Figure S15. Fluorescence tracer images of intravenously injected GL and Lf-GL. Balb/c mice were administered either FITC-tagged GL or FITC-tagged Lf-GL at a GL equivalent concentration of 50 mg kg-1 body weight via tail vein injection. The fluorescence signals of FITC-tagged GL or FITC-tagged Lf-GL in organs were imaged using an in vivo imaging system. The exposure time was fixed to 200 sec for analyzing fluorescent signals from tissues. The quantified fluorescence signal of brain at each time point was measured with Intensity Unit. Figure S16. Histopathological analysis of heart, liver, lung, spleen, and kidney after 28 doses. The tissues were histologically analyzed after intravenous administration of daily dosesto GBM-modeled mice for 28 days. Scale bar: 50 μm.