Ab52298

RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with the protease inhibitors (Roche, Basel, Switzerland) to lysed cells. The equivalent amounts of protein (20 μg) was denatured at 100 °C in loading buffer for 15 min. Afterwards, load samples containing equal amount of proteins and prepared in sample buffer into 8–12% SDS/PAGE wells and transferred to PVDF membranes by voltage gradient transfer. The blots were blocked overnight in 5% nonfat milk. The membranes were incubated with the primary antibodies against Bcl-2 (ab32124), cleaved-Casapse-3 (ab49822), cleaved-Caspase-7 (ab32522), cleaved-Caspase-9 (ab52298), MMP-2 (ab37150), MMP-9 (ab73734), Vimentin (ab8978), MEK (ab32576), p-MEK (ab96379), ERK (ab32537), p-ERK (ab131438), p65 (ab16502), p-p65 (ab86299), IκBα (ab32518), p-IκBα (ab32518), and β-actin (ab8227) purchased from Abcam (Cambridge, UK) at the dilution of 1:1000. Incubate the membrane in primary antibody solutions overnight at 4 °C with gentle rocking. Wash the membrane with 1× TBST three times for 10 min and then incubate the membrane in the appropriate diluted secondary antibody (Abcam). Then the signal was captured and the intensity of the bands was analyzed. Finally result was quantified using Image Lab™ Software (Bio-Rad, Shanghai, China).

Briefly, HeLa cells (2×10⁴ cells/well) were grown on plastic coverslips inside 24-well plates and incubated at 37°C with 5% CO₂, before being treated with 384.10, 768.2, and 1536.4 µg/ml of Nano-PMBE for 24 h. Then, they were fixed in 500 µl of methanol (absolute, 32.04 g/mol Sigma-Aldrich, USA) for 10 min, washed with 1× PBS (pH 7.4, Sigma-Aldrich, USA), and treated with 1 ml of 1% hydrogen peroxide solution (1-5%, 34.01 g/mol, Sigma-Aldrich, USA) in absolute methanol (1:10) for 10 min. Subsequently, the cells were blocked with background sniper followed by staining with 1:200 p53 mouse monoclonal antibody (53 kDa, p53 Ab-1, Thermo Scientific™ Lab Vision™) and 1:50 anti-caspase-9 antibody (ab52298, Abcam) for 1 h in the dark. The stained cells then were washed with 1× PBS (pH 7.4, Sigma-Aldrich, USA) twice. The protein expression was detected by the Starr Trek Universal HRP Detection System. Counterstaining of the cells was done using Mayer’s hematoxylin solution (Sigma-Aldrich, USA), and the cells dipped in ethanol (≥99.8%, 46.07 g/mol, Sigma-Aldrich, USA) and dried. Then, the coverslips were mounted onto glass slides and visualized using Nikon Eclipse Ci microscope (Nikon Corp., Tokyo, Japan). The expression of p53 and caspase-9 was evaluated semi-quantitatively based on H-score method [24 ].

Tissue samples of the small intestine were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4 μm sections, and dewaxed with xylene. The sections were evenly covered with goat serum and sealed for 30 min at room temperature. After discarding the sealing fluid, the slices were incubated overnight at 4°C with a primary antibody at an appropriate dilution, including anti-γ-H2AX antibody (1 : 100, bs-3185R; Bioss), anti-p53 antibody (1 : 100, ab26; Abcam), anti-caspase-8 antibody (1 : 50, ab25901; Abcam), or anti-caspase-9 antibody (1 : 100, ab52298; Abcam), followed by incubation with secondary antibodies for 50 min at room temperature. Subsequently, the nuclei were restained using DAPI and analyzed under a microscope (OLYMPUS BX51). Hematoxylin stained the nuclei blue, and DAB produced a brown stain for positive expression of the targeted proteins. DAPI-stained nuclei were blue under UV excitation, and the positive expression of targeted proteins was detected as a red or green light signal, depending on the fluorescein label.