Fig 2: Enzymatic response of synthetic compounds. (a) Chemical structures of 1 (HADP) and its analogues 2–4. (b) Fluorescence responses of probe HADP and control compounds 2–4 (each 5 µM) towards human heparanase (HPA) (1 µg) in 40 mM NaOAc buffer (pH 5.0) at 37 °C, fluorescence intensity was recorded on a Spectramax M5 Multimode plate reader (Molecular Devices, USA) at each timepoint. (c) UV-vis absorption and (d) fluorescence (?ex = 365 nm) spectra of probe HADP (5 µM) before (blue line) and after (red line) incubation with human heparanase (HPA) (2 µg) in 40 mM NaOAc buffer (pH 5.0) for 2 h at 37 °C. Fluorescence spectra were recorded on a Fluorolog TAU-3 spectrofluorometer with a xenon lamp (Jobin Yvon-Spex, Instruments S. A., Inc.) (e) Fluorescence quantification of (d). Turn-on ratio is defined as the ratio of fluorescence intensity after activation to that before activation. (f) Time dependence of fluorescence spectra of HADP (5 µM) with human heparanase (HPA) (1 µg) from 0 min to 3 h in 40 mM NaOAc buffer (pH 5.0). Inset of (f) quantification of fluorescence intensity at 455 nm over time. ?ex = 365 nm. Vertical bars indicate mean ± SD. ****p < 0.0001.

Fig 3: ROS is required for GA-induced autophagy.(A) Intracellular ROS in HCT116 cells treated with 1‰ DMSO (Control), 1.0 µM GA (GA), or 1.0 µM GA in the presence NAC (10 mM) (GA+NAC) for 24 h were detected by staining cells with 2',7'-dichlorofluorescein diacetate under blue (B), or blue and white excitation (B+A). The DCFH-DA signal was measured using a Molecular Devices SPECTRAMAX M5 fluorimeter. (B) HCT116 cells transfected with a pEGFP-LC3 plasmid with or without GA (1.0 µM) or/and NAC (10 mM) for 24 h. Cells were defined as positive if they had 5 or more GFP-LC3 dots in the cytoplasm. The percentage of the cells with GFP-LC3 dots and the average number of GFP-LC3 dots per cell were analyzed from at least 100 random fields. ** p<0.01. (C) Immunoblot analysis of the conversion of LC3-I to LC3-II and expression of cleaved-caspase 3. (D) HCT116 cells treated with or without GA (1.0 µM) or/and NAC (10 mM) for 24 h, cell apoptosis was detected by TUNEL assay. The TUNEL-positive cells were counted from at least 100 random fields. (E) HCT116 cells were treated with or without GA or/and the antioxidants Apocynin, rotenone and nordihydroguaiaretic acid (NDGA) for 24 h, and then the intracellular ROS were measured using a Molecular Devices SPECTRAMAX M5 fluorimeter. (F) HCT116 cells were treated with or without GA or/and the antioxidants Apocynin, rotenone and nordihydroguaiaretic acid (NDGA) for 24 h, and then the conversion of LC3-I to LC3-II were detected by immunoblot analysis.

Fig 4: Effect of Pluronic on pDNA uptake in various cell types in vitro. RAW 264.7 MØ, C2C12 MBs and C2C12 derived D7 MTs (50,000 cells/well in 96 well plates, 24 h after plating) cells were exposed to YOYO-1 labeled gWIZ™ Luc pDNA (0.25 µg and 1 µg) in the absence (white bars) or presence of 1% w/v P85 (black bars) in SFM for 2 h. After that cells were rinsed thrice with PBS and lysed using 50 µl M-PER® cell lysis reagent (ThermoFisher Scientific, Vernon Hills, IL) for 5–10 min at 4 °C. Total fluorescence was quantified in cell lysates using Spectramax M5 plate reader (Molecular devices, Sunnyvale, CA) at ? Ex/Em 490/591. Data are mean±SEM (n=6). Statistical comparisons were made by Pair-wise t-test with Welch?s correction: *p<0.05, n.s. – non significant.

Fig 5: Hemolytic activities of the peptides against fresh human erythrocytes. The release of hemoglobin was monitored with a Microplate Autoreader (SpectraMax M5, Molecular Devices, San Francisco, CA, USA) by measuring the absorbance at 570 nm. Data are the averages of three independent experiments.