Genequant 1300

Total mRNA of white adipose tissues was isolated from samples using TRIzol Reagent (Invitrogen, Life Technologies Corp., Carlsbad, CA, USA) and cDNA was synthesized using a cDNA Reverse Transcription Kit (Applied Biosystems Inc., Foster City, CA, USA) according to the manufacturer’s instructions. The purity and quantity of the isolated RNA were spectrophotometrically determined using the GeneQuant 1300 spectrophotometer (GE Healthcare Bio-sciences AB, Uppsala, Sweden). The mRNA levels were determined using the Applied Biosystems 7500 Fast Real-Time PCR System with SYBR Green PCR Master mix (Applied Biosystems Inc., Foster City, CA, USA). The quantitative real-time PCR amplification procedure was briefly described as follows: samples were predenatured at 95 °C for 10 min followed by 40 cycles of amplification consisting of 15 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C; β-actin was used as the internal control. To verify the accuracy and specificity, all reactions were performed at least in triplicate, followed by melting curve analysis. The relative quantification of mRNA was evaluated using the 2^−ΔΔCt method [42 (link)]. Dilution curves were adopted to assess the PCR efficiency as proposed by Bustin et al. [43 (link)]. The sequences of primers (Sangon Biotech Co., Ltd., Shanghai, China) were given in Table S3.

For the EB permeability assay, 500 µL of freshly prepared EB solution (10 mg/mL in NaCl 0.9% saline solution; Sigma-Aldrich, NJ, USA) were placed on top of each mucosa fragment, previously placed upward in a Petri dish. After 10 min, samples were washed twice in saline solution (NaCl 0.9%): The first wash consisted of two rapid dips in 100 mL of saline solution, while the second one, in 50 mL of saline, lasted 5 min. At the end, all samples were trimmed and divided into two aliquots to get technical duplicates. Aliquots were then dried at 37°C in a thermostatic hood for 30 min and the mucosal layer was isolated, immediately weighed, placed in 3 mL of formamide (Sigma-Aldrich, NJ, USA) and incubated for 48 h at 50°C to extract EB dye. Colorimetric measurements were performed using a spectrophotometer (Gene Quant 1300; GE Healthcare, UK) at the maximum absorption for EB (620 nm). Micrograms of EB per mg of tissue were quantified using a standard curve (0.025-25 μg/mL EB in formamide) and samples’ weights. Data of the two technical replicates were averaged.

The total amount of phenolic compounds from the extracts was determined according to the Folin-Ciocalteu procedure with in house modifications (Singleton et al., 1965 (link)). Briefly, samples (200 μL) were introduced into test tubes with 1.0 mL of Folin–Ciocalteu reagent (1:1 v/v) and 2.5 mL of sodium carbonate (20%). The mixture was incubated for 30 min at room temperature and allowed to stand still for additional 30 min. The absorbance from the blue colored mixture was measured at 765 nm (Gene Quant 1300, GE Healthcare). The amount of total phenol was calculated as milligrams (mg) of Gallic Acid Equivalents (GAE)/g of dry mass from calibration curve of Gallic acid standard solution. For the Gallic acid, the curve absorbance versus concentration is described by the equation y = 1.5221x + 0.0081 (r² = 0.9712).

Fluorescence (475 nm–575 nm excited at 480 nm) of 10 µM sfGFP, m1–m5, and m7–m11 in 20 mM Pipes (pH 7.2) containing 100 mM NaCl, 0.1% CHAPS, and 10% sucrose was measured by using GeneQuant 1300 (GE Healthcare Life Sciences) and F-2700 Fluorescence spectrophotometer (Hitachi High-Technologies, Tokyo, Japan), respectively. Fluorescence intensity of 25 µM sfGFP, mH1–mH5, and mH1C–mH5C in PBS was measured using Infinite F500 (Tecan, Männedorf, Switzerland) in 96 well plates with the specific filters (Ex/Em  = 485 nm/535 nm).