Proteinase k solution

Isogenic E. coli K-12 strains (see Table S1 in the supplemental material) were obtained from the Keio collection (52 (link)) or constructed using CRISPR genome editing technology (53 (link)). Plasmids and primers used for this work are listed in Table S4. E. coli strains were cultured by shaking at 200 rpm aerobically at 37°C in Luria-Bertani (LB) broth or by plating on LB agar incubated at 37°C. Yeast extract, tryptone, agar, and 5(6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA) were obtained from Thermo Fisher Scientific Corp. (Waltham, MA, USA). Ciprofloxacin, ampicillin, and kanamycin were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Vancomycin, erythromycin, lysine monohydrochloride, arginine hydrochloride, histidine hydrochloride, malonate, propidium iodide, proteinase K solution, 5× protein loading dye, dimethyl sulfoxide (DMSO), and 2.2′-bipyridyl were purchased from Sangon Biotech Inc. (Shanghai, China). Lipopolysaccharide (LPS) was bought from Beyotime Biotech Inc. (Jiangsu, China). Ceftriaxone (Roche, Shanghai, China), and meropenem (Shenghuaxi Pharmaceutical Co., Chongqing, China) were gifts from the Zhongshan Hospital (Xiamen, China) pharmacy.

Prepared paraffin-embedded sections of myocardial tissue were routinely dewaxed in xylene, dehydrated in gradient ethanol and incubated with proteinase K solution (20 μg/ml) (Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 20 min. According to the instructions of the In Situ Cell Death Detection Kit (Roche, Basel, Switzerland), working solution was added to the cells to detect apoptosis. In addition, fluorescence microscopy was employed to observe apoptotic cells; the TUNEL-positive cells fluoresced green and nuclei fluoresced blue. The percentage of apoptotic cells was calculated. Five visual fields were selected from each section, and the average percentage of apoptotic cells was used to calculate the apoptosis index.

Casein, Casein-Na, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Shanghai, China). All oligonucleotides—including the forward primer 5′-biotin-GCTCAACCAGGAACGATC-3′ and reverse primer 5′-FITC-GCAGCATTTGACTACGTACCA-3′ (with expected amplified length of 112 bp); 4S Red Plus nucleic acid stain (1000×); Taq DNA polymerase (5 U/μL); 10 × PBS; streptavidin; agarose; dNTP mix (25 mM); and proteinase K solution (20 mg/mL)—were all obtained from Sangon Biotech (Shanghai, China). A S1000 Thermal Cycler PCR (Bio-Rad, Hercules, CA, USA) and the portable thermal controller (Hangzhou Ao-Min Biological Co., Ltd., Hangzhou, China) were used for the amplification. All components of the LFS, including a plastic adhesive backing, sample pad, conjugate pad, nitrocellulose membrane CN 95 and absorbent pad were ordered from the Shanghai Jie-ning Biotechnology Co., Ltd. (Shanghai, China). All solvents and other chemicals of analytical reagent grade were used without further purification.