Ultrasonic homogenizer

Fermenter−cultured MSR−∆F−BF was collected by centrifugation at 10,000 rpm at 4 °C for 15 min. The collected cells were resuspended in 20 mM phosphate−buffered saline (PBS) with a mass volume ratio of 1:10 (pH 7.4) and disrupted using an ultrasonic homogenizer (Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China) for 20 min at 3 s/time with a time interval of 5 s at 400 W to lyse the cells. BMPs were collected from the disrupted cell suspension using a columnar neodymium−boron magnet. The supernatant was discarded, and the separated magnetosomes were resuspended in 10 mM PBS (pH 7.4), sonicated for 15 min at 3 s/time with a time interval of 5 s at 150 W, and washed twice. Then, we reduced the power to 100 W and washed the sample several times until the supernatant protein concentration was lower than 0.1 mg/mL. The purified BMP−∆F−BF was resuspended in 25% glycerol and stored at 4 °C.
Purified BMP−∆F−BF and wild-type BMP were observed under a TEM. For this purpose, a small number of BMPs was suspended in 1 mL of deionized water and thoroughly dispersed by ultrasonication for 10 min. Then, 10 μL of the suspension was dropped onto a copper mesh for 10 min, air-dried, and observed under a TEM. Hydrated radii and zeta potential of BMP−∆F−BF were detected at Nanjing Dongna Biological Technology Co., Ltd., Nanjing, China.

ChIP assay using mouse retinas was performed according to procedures described previously [44 (link)]. Briefly, three retinas were pooled together and homogenized in 250 μl of ice-cold PBS containing proteinase cocktails, then another 750 μl of PBS was added and cross-linking was performed with 1% formaldehyde (final concentration). Sonication was performed by using SCIENTZ ultrasonic homogenizer (Amplitude: 60%, 2 s on and 2 s off for 5 min in total) and 6 μg of chromatin were used for each immunoprecipitation. The antibodies and the dilutions are listed in Table 1, and the primers used are listed in Table 2.

The PCR-amplified cpxR gene from A. pleuropneumoniae strain S4074 was cloned into the pET30a vector by digesting with Nde I and Xho I. Then, the CpxR expression plasmid was transformed into E. coli BL21(DE3), and their expression was induced by the addition of 0.5 mM IPTG and incubated at 16°C overnight. The cells were disrupted using a Ultrasonic Homogenizer (SCIENTZ, Ningbo, China), and centrifuged at 12,000 g for 20 min to remove cellular debris. The supernatant was extracted with Ni-nitrilotriacetic acid (Ni-NTA) resin affinity chromatography following the manufacturer’s instructions. The purified protein concentration was determined by a BCA protein assay kit (Beyotime, Shanghai, China), and the purity was checked by 12% SDS-PAGE.

The chromatin immunoprecipitation (ChIP) assay was accomplished as previously described (Bowler et al., 2004 (link); Zheng et al., 2021 (link)). Briefly, fresh in vitro-grown materials were collected and subjected to vacuum infiltration in 1% (v/v) formaldehyde for 15 min to crosslink the chromatin proteins to the DNA. Fixation was stopped by adding glycine to a final concentration of 0.125 M. The fixed samples were washed three times with ddH₂O and ground to a powder in liquid nitrogen. Chromatin was extracted and sonicated using an Ultrasonic Homogenizer (SCIENTZ, JY 92-IIDN, Ningbo, China). The anti-GFP antibody was purchased from Abmart Shanghai Co., Ltd. (M20004M, Shanghai, China). The immunoprecipitated DNA was then analyzed with quantitative PCR (qPCR), and the amplified DNA from the chromatin fractions prior to antibody incubation was used as the control. PCR reactions were performed in triplicate for each sample, and the enrichments were normalized to the input sample. The β-actin gene was used as an endogenous control. The gene-specific primers used for ChIP–qPCR are listed in Supplementary Table S1.

After the last administration, tissue proteins were gently resuspended in cytoplasmic buffer (10 mM HEPES, 0.5 mM DTT, 1.5 mM MgCl₂, 10 mM Na₂MoO₄, 10 mM KCl, 25 mM NaF, 20 mg/ml aprotinin, 0.1% NP-40, 2 mM PMSF, and 0.2 mM Na₃VO₄) and were centrifuged at 900 × g for 10 min. The nuclear pellets were prepared as described below. The cytoplasmic supernatant was recentrifuged at 900 g, and the acquired supernatant was further centrifuged at 1000 × g for 30 min. Then, the cytosolic supernatant was subjected to immunoblotting. In addition, different subcellular fractions were separated using endoplasmic reticulum extraction kit (Bjbalb, China) and Golgi body extraction kit (Bjbalb, China). These fractions were broken with a cell disruptor (Scientz Ultrasonic Homogenizer, China).

Different concentration of Doxorubicin (Yuanye Biology, China) and Rose Bengal (Sigma-Aldrich, USA) were added to the exosomes (1 mg mL⁻¹) and the mixture was sonicated (10% amplitude, 6 cycles, 8 s on/off, 2 min duration) by Ultrasonic Homogenizer (Scientz, China). The mixture was then incubated at 4 °C for 2 h to recover the exosome membrane to form drug loaded exosomes (Natural Exo@RB/Dox). Then Natural Exo@RB/Dox was obtained by centrifugation and free Dox and RB were discarded with the supernatant.