Tween 80

Fresh soybean residue was provided by Kangle Tofu Workshop (Changchun, China). Soybean residue should be dried for 24 h at 55 °C before reuse. Saccharomyces cerevisiae strain BNCC337309 was obtained from the BeNa Culture Collection (Beijing, China). The KC205 LAB strain was isolated and screened from homemade pickled cabbage juice by the food microbiology team of the Agro-product Process Institute of the Jilin Academy of Agricultural Sciences, and has the characteristics of strong acid production and acid resistance.
High temperature-resistant α -amylase (2 × 10⁴ U/mL), papain (800 U/mg), and amyloglucosidase (1 × 10⁵ U/mL), were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Peptone, yeast extract fermentation, glucose, NaCl, beef extract, anhydrous sodium acetate, sodium citrate, K₂HPO₄, MgSO₄, MnSO₄ and Tween 80 were all purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All chemicals were of analytical grade or better.

As per a slightly modified version of a protocol published by Yang et al. (2014) [27 (link)], overnight cultures (4 μL of 0.4 OD_600nm) were inoculated into the center of NA plates supplemented with 1% Tween80 (Sangon Biotech, Shanghai, China) and varying concentrations (1/16 × MIC–2 × MIC) of oridonin. After incubation for 48 h at 28 °C, the appearance of opalescent regions surrounding these colonies was assessed, and the zone diameters were measured.

For all in vitro experiments, ML604440 (Probechem, PC-60968), ONX -0914 (MCE, HY-13207) were dissolved at a concentration of 10 mM in DMSO and stored at −80°C. A final DMSO concentration of 0.3% was used. For proteasome inhibition in mice, ML604440 was diluted in PBS with 5% polyethylene glycol (PEG-400; Sangon Biotech) and 1% Tween-80 (Sangon Biotech) immediately before use. ONX 0914 was formulated in an aqueous solution of 10% (w/v) sulfobutylether-beta-cyclodextrin and 10 mM sodium citrate (pH 6) and administered to mice as a subcutaneous bolus dose of 10 mg/kg.

This study used strains of P. oxalicum, S. cerevisiae and E. coli (Supplementary Table S2). P. oxalicum strains were cultured on PDA (Becton, Dickinson and Company, Sparks, MD) plates for 5 days at 28 °C to produce asexual spores. Tween 80 (Sangon Biotech Co. Ltd., Shanghai, China) was used to harvest asexual spores for reproduction.
Asexual spores (1 × 10⁸) were cultured on modified minimal medium containing SCS (Sigma-Aldrich, St. Louis, MO, USA)^{5 (link)}, then used for measurement of RSDE and SSDE activities and for RT-qPCR assay. Solid modified minimal medium containing glucose, SCS or PDA were used for investigation of colony and mycelial development.
S. cerevisiae and E. coli strains were grown on yeast extract peptone dextrose (YPD) medium and Luria-Bertani (LB) medium for preservation and/or reproduction, respectively.

M. bovis BCG (strain 1173P2), M. marinum (ATCC BAA-535), M. smegmatis mc2155, M. abscessus (ATCC 19977), M. aurum, M. avium (ATCC 25291), and M. tuberculosis H37Rv (ATCC 27294) were obtained from the collection of the Institut Pasteur. M. tuberculosis clinical isolates, including three susceptible, three Rif-resistant and one MDR strain were collected from hospitals in China. Mycobacteria were cultured in Middlebrook 7H9 broth containing 10% (v/v) ADC enrichment (albumin, dextrose, catalase; Becton Dickinson, Franklin Lakes, USA), 0.05% glycerol (Sangon Biotech, Shanghai, China), and 0.05% Tween 80 (Sangon Biotech, Shanghai, China), or Middlebrook 7H11 agar medium supplemented with 10% (v/v) OADC enrichment (oleic acid, albumin, dextrose, catalase; Becton Dickinson, Franklin Lakes, USA). All mycobacteria were cultured under aerobic conditions at 37 °C, except for M. marinum, which was cultured at 30 °C. E. coli ATCC 25922 and S. aureus ATCC 25923 were cultivated at 37 °C in LB medium.

Levofloxacin (LVX, 98%) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Peroxide (H₂O₂, 3%) was purchased from Guangdong Hengjian Pharmaceutical Co., Ltd. Polyhexamethylene biguanide (PHMB, 99%), sodium chloride (AR), soybean lecithin (BR), glacial acetic acid, paraffin, formaldehyde, xylene, absolute ethanol, and ammonia water were obtained from Sinopharm Chemical Reagent Co., Ltd. Chitosan (CS, DD: 90%, Mw:700,000–800,000) was obtained from Shanghai Lanji Biotechnology Co., Ltd. Sodium thiosulfate pentahydrate, Tween 80, magnesium sulfate (AR), and bovine serum albumin (BR) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Tryptone physiological solution (TPS) (BR) was bought from Guangdong Huan Kai Microbial Technology Co., Ltd. Hematoxylin, eosin, BASO, and neutral resin were purchased from Beijing Solarbio Technology Co., Ltd. Masson trichromatic staining solution was bought from Beijing Huayueyang Biotechnology Co., Ltd. Then, 3% sodium chloride alkaline peptone broth, 3% sodium chloride soybean–casein digest agar, thiosulfate–citrate–bile salts–sucrose agar, nutrient broth, nutrient agar, Sabouraud dextrose broth, potato dextrose agar (PDA), cation-adjusted Mueller–Hinton broth (CAMHB), and Mueller–Hinton Agar were acquired from Qingdao Hope Biotechnology Co., Ltd.

CYH33 was provided by Shanghai HaiHe Biopharma Co., Ltd. (Shanghai, China). Alpelisib (Novartis, Cambridge, MA, USA) and EPZ6438 (Epizyme, Cambridge, MA, USA) were supplied by Selleck (Houston, TX, USA). GDC‐0077 (Genentech, San Francisco, CA, USA) was obtained from MedChemExpress (Monmouth Junciton, NJ, USA). Dimethyl sulfoxide (DMSO) and polybrene were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Tween 80 and CMC‐Na were purchased from Sangon Biotech (Shanghai, China) and Sinopharm (Beijing, China), respectively. Compounds were dissolved in DMSO at 10 mM (CYH33, Alpelisib and GDC‐0077) or 50 mM (EPZ6438) and then stored at −20℃. Stock solutions were diluted to the desired concentrations freshly in fetal bovine serum‐free medium before experiments. The final concentration of DMSO did not exceed .1% (v/v). For animal studies, compounds were dissolved in normal saline containing .5% Tween 80 (v/v) and .5% CMC‐Na (m/v).