Bca kit

A fragment encoding MtrA (Rv3246c) was amplified from the M. tuberculosis H37Rv genomic DNA using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). The primers (5′-cagcaaatgggtcgcggatccATGGACACCATGAGGCAAAGG-3′), (5′-gtggtggtggtggtgctcgagTCACGGAGGTCCGGCCTT-3′) were designed using Primer3 (https://www.primer3plus.com, accessed on 23 November 2020) and synthesized by Tsingke Biotechnology (Beijing, China). The purified fragment was cloned into pET28a and linearized by BamH I and Xho I (NEB, England) double-digestion using Exnase (Vazyme, Nanjing, China).
E. coli BL21 (DE3) transformed with pET28a-MtrA was induced with 0.5 mM IPTG at 16 °C for 20 h when OD600 reached 0.6. For protein purification following routine procedures, a 250 mM final concentration of Imidazole was used for elution after being loaded on a Ni-NTA 6FF prepacking chromatographic column (Sangon Biotech, Shanghai, China), and eluted MtrA was desalted and concentrated using a 10 Kda protein concentrator (Millipore, MA, USA) in storage buffer (20 mM HEPES, 100 mM NaCl and 5% glycerol pH 7.5). The purified protein was stored at −80 °C for further experiments after the purity and concentrations were assayed by 12% SDS-PAGE (Yeasen Biotechnology, Shanghai, China) and a BCA kit (Sangon Biotech, Shanghai, China).

The level of PARP, c-PARP, and Bcl-2 24 h after treatment with the hit compound was assessed using western blot analysis (Supplementary Material). The standard protocol involved treating cells with varying concentrations of the hit compound for 24 h, extracting whole cell lysates with the RIPA lysis buffer, and determining sample concentrations using the BCA kit from Sangon Biotech (Shanghai, China). Subsequently, 20.0 g of protein was loaded into a 10% gel and transmembrane PVDF (Millipore, Burlington, MA, USA, IPVH00010). After sealing with 5% skim milk for 1 h at room temperature, membranes were incubated with primary antibodies against PARP (CST, Danvers, MA, USA, 9542S), c-PARP, and Bcl-2 (Proteintech, Rosemont, IL, USA, 12789-1-AP) overnight at 4 °C. The membranes were then washed three times with TBST before incubating with anti-rabbit or anti-mouse antibodies conjugated with HRP tags for 1.5 h at room temperature. Finally, the blots were overlaid using the ECL detection kit and visualized under the ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA). Anti-rabbit and anti-mouse IgG were purchased from Sigma (Burlington, MA, USA).

The level of PARP, c-PARP, and Bcl-2 24 h after treatment with the hit compound was assessed using western blot analysis (Supplementary Material). The standard protocol involved treating cells with varying concentrations of the hit compound for 24 h, extracting whole cell lysates with the RIPA lysis buffer, and determining sample concentrations using the BCA kit from Sangon Biotech (Shanghai, China). Subsequently, 20.0 g of protein was loaded into a 10% gel and transmembrane PVDF (Millipore, Burlington, MA, USA, IPVH00010). After sealing with 5% skim milk for 1 h at room temperature, membranes were incubated with primary antibodies against PARP (CST, Danvers, MA, USA, 9542S), c-PARP, and Bcl-2 (Proteintech, Rosemont, IL, USA, 12789-1-AP) overnight at 4 °C. The membranes were then washed three times with TBST before incubating with anti-rabbit or anti-mouse antibodies conjugated with HRP tags for 1.5 h at room temperature. Finally, the blots were overlaid using the ECL detection kit and visualized under the ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA). Anti-rabbit and anti-mouse IgG were purchased from Sigma (Burlington, MA, USA).

RIPA buffer (Solarbio) was utilized for extracting total protein, and then the protein was quantified by BCA Kit (Sangon, Shanghai, China). Next, the proteins were separated by SDS-PAGE gel and electro-blotted to PVDF membrane (Beyotime, Shanghai, China). The membrane was incubated with the primary antibodies against SIK1 (51045–1-AP, 1:1000), E-cadherin (20874–1-AP, 1:5000), N-cadherin (22018–1-AP, 1:2000), matrix metalloproteinase 9 (MMP9, 10375–2-AP, 1:1000), Bcl2-associated x (Bax, 50599–2-Ig, 1:5000) or GAPDH (10494–1-AP, 1:20,000). After further incubating with Goat Anti-Rabbit IgG (SA00001–2, 1:10,000), the protein bands in the membranes were visualized using Ultrasensitive ECL Dection Kit (Proteintech, Rosemont, IL, USA). All antibodies were obtained from Proteintech.

The correct integrant colony was grown in 20 ml of BMGY medium at 30°C, 220 rpm for 24 h. The yeast cells were collected by centrifugation at 1,500×g for 5 min at 4°C, and subsequently resuspended in 750-ml BMMY medium (flask, 220 rpm) to a final OD₆₀₀ of 1.0. The expression of rFIP-mco was induced by adding 100% methanol to a final concentration of 0.5% every 12 h for 72 h. The secreted rFIP-mco was collected and purified via His-affinity Ni-NTA chromatography (Qiagen, California, USA) according to the manufacturer’s protocols, and was dialyzed third times against PBS in the total period of 18 h. The total rFIP-mco protein mass was detected using BCA kit (Sangon, Shanghai, China), whereas the protein mass of the purified rFIP-mco was adjusted with extinction coefficient predicted according to the amino acid sequence (54 (link)).