Fusion fx image system

The changes of nuclear morphology for assessing apoptosis were assessed using DAPI staining. Briefly, cells were cultured with or without different concentrations of isorhamnetin for 48 h, and were then fixed with 4% paraformaldehyde (Sigma-Aldrich Chemical Co.) for 10 min at RT. The cells were rinsed with PBS, and incubated with 1 μg/mL DAPI solution (Sigma-Aldrich Chemical Co.) at 37 °C for 10 min. Stained cells were visualized and photographed using fluorescence microscopy (Carl Zeiss, Oberkochen, Germany). For DNA fragmentation assay, the collected cells were lysed in a buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, and 0.5% Triton X-100 for 30 min. The fragmented DNA in the supernatant was extracted using an equal volume of neutral phenol:chloroform:isoamyl alcohol (25:24:1, Sigma-Aldrich Chemical Co.), analyzed electrophoretically on 1% agarose gel containing EtBr (Sigma-Aldrich Chemical Co.), and photographed under a Fusion FX Image system (Vilber Lourmat, Torcy, France).

As described previously, total protein was extracted from the cells using the Bradford Protein assay kit [27 (link)]. The nuclear protein extracts obtained from the cells were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc., Waltham, MA, USA). An equal amount of protein from the samples was separated by 10–13% sodium-dodecyl sulfate gel electropThermoTherhoresis, and transferred onto polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH, USA). The membranes were blocked with 5% non-fat skim milk in Trisbuffered saline containing 0.1% Triton X-100 (TBST) for 1 h, and probed with specific primary antibodies, which were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Merck Millipore (Temecula, CA, USA), BD Biosciences (Franklin Lakes, NJ, USA), and Abcam Inc. (Cambridge, UK), at 4 °C overnight. After washing 3 times with TBST, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (Santa Cruz Biothechnology, Inc.,) for 2 h at RT. The expression of protein was detected by enhanced chemiluminescence kit (GE Healthcare Life Sciences, Little Chalfont, UK), and visualized by Fusion FX Image system (Vilber Lourmat).

RAW 264.7 cells were seeded on 100 mm culture dish at a density of 2 × 10⁶ cells/well, and incubated for 24 h. The cells were treated with CSP32 and l ng/mL LPS for 24 h, and then, the total protein was extracted from the cells using a Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). A total of 40 μg of protein was separated by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore, Bedford, MA, USA). The membranes were blocked using 5% skim milk in Tris-buffered saline containing 0.1% Triton X-100 (TBST) at room temperature (RT) for 1 h and probed with specific primary antibodies at 4 °C overnight. Subsequently, the membranes were incubated with the corresponding secondary antibodies for 1 h at RT, developed, and analyzed using a Fusion FX Image system (Vilber Lourmat, Torcy, France). Densitometric analysis of the data was performed using the ImageJ^® software (v1.48, NIH, Bethesda, MD, USA). The information about the antibodies is provided in Table S1.