Natural and synthetic lipids were dissolved in chloroform. Lipids with indicated compositions were mixed in a glass vial. The lipid films were obtained by evaporating under a stream of nitrogen and the dry lipid film was then hydrated at room temperature with constant mixing in 500 μl buffer A (20 mM HEPES (pH 7.5) and 150 mM NaCl). Liposomes were generated by extrusion of the hydrated lipids through 100 nm polycarbonate filter (Whatman) 20 times using a Mini-Extruder device (Avanti Polar Lipids). To prepare Tb3+-encapsulated liposomes, the lipid film was hydrated with 500 μl buffer B (20 mM HEPES (pH 7.5), 100 mM NaCl, 50 mM sodium citrate and 15 mM TbCl3). After the extrusion process, Tb3+ ions outside the liposome were removed by washing with buffer A on a centrifugal filter device (Amico Ultra-4, 100 K MWCO, Millipore). The liposomes were subjected to buffer A for use. To prepare FITC–dextran-encapsulated liposomes, the lipid film was hydrated in buffer A supplemented with 2 mg ml−1 FITC-dextran. After the extrusion process, the liposomes were repeatedly washed with buffer A to remove external dextran by centrifugal filter device (Amico Ultra-4, 100K MWCO, Millipore). All the liposomes were stored at 4 °C and used within 48 h.
Amico ultra 4
Manufactured by Merck Group
Amico Ultra-4 is a compact, high-performance laboratory centrifuge designed for efficient sample processing. It features a maximum speed of 4,000 RPM and a maximum RCF of 2,510 x g, making it suitable for a variety of common laboratory applications.
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4 protocols using amico ultra 4
1
Liposome Preparation and Encapsulation
2
Western Blot Protein Analysis
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Standard Western-blot assays were used to analyze the levels of protein in cell lysates and CM. CM which was cultured with 1/3 number of the cells used for Western-blot assays was concentrated with an Amico Ultra-4 centrifugal filter device (Millipore) after a brief centrifugation to remove any cell debris. Antibodies against p53 (FL393, Santa Cruz; 1:1,000 dilution), anti-p-p53 (Ser15) (9284, Cell Signaling; 1:1,000 dilution), anti-LIF (AF-250-NA, R&D; 1:1,000 dilution), anti-PARP1/2 (H250, Santa Cruz; 1:1,000 dilution), anti-cleaved-caspase 3 (D175, Cell Signaling; 1:1,000 dilution), anti-MDM2 (2A10; 1:1,000 dilution), anti-p-Stat3 (Tyr705) (9131, Cell Signaling; 1:1,000 dilution), anti-Stat3 (C-20, Santa Cruz; 1:2,000 dilution), anti-ID1 (C-20, Santa Cruz; 1:2,000 dilution), anti-p21 (Ab-6, Calbiochem; 1:1,000 dilution, and anti–β-actin (A5441, Sigma; 1:125,000 dilution) antibodies were used in this study. The full blots are shown in Supplementary Fig. 10 – Supplementary Fig. 14 .
3
Western Blot Protein Analysis
4
Recombinant Protein Expression and Purification
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Expression and purification were performed by Nexomics Inc. Prior to gene synthesis, the sequence was optimized by codon optimization software. The designed gene was synthesized by Synbio-Tech (www.Synbio-tech.com) and subcloned into pET21-NESG vector.
Protein expression was performed as previously reported (85) . Briefly, the recombinant pET21-NxSC1 plasmid was transformed into E. coli BL21 (DE3) cells and the cells were cultured in 13 C 15 N-MJ9 medium containing 100 μg/mL of ampicillin. The culture was further incubated at 37 o C and protein expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the final concentration of 1 mM at logarithmic phase. Cells were harvested after overnight culture at 18 o C and protein expression was evaluated by SDS-PAGE.
The protein was purified using a standard Ni affinity followed by size exclusion two-step chromatography method first as previously reported (85) . Since the purified NxSC1 sample presented as 2 bands, an additional ion exchange chromatography was performed. The NxSC1 sample from the two-step purification was pooled and dialyzed against buffer A (Buffer A: 20 mM Tris-HCl, pH 7.5), and loaded onto a HiTrap Q HP 5 ml column. A gradient of NaCl from 0 to 1 M was applied (Buffer B: 20 mM Tris-HCl, pH 7.5, 1 M NaCl). The NxSC1 was pooled and concentrated to 1 mM using Amico Ultra-4 (Millipore).
Protein expression was performed as previously reported (85) . Briefly, the recombinant pET21-NxSC1 plasmid was transformed into E. coli BL21 (DE3) cells and the cells were cultured in 13 C 15 N-MJ9 medium containing 100 μg/mL of ampicillin. The culture was further incubated at 37 o C and protein expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the final concentration of 1 mM at logarithmic phase. Cells were harvested after overnight culture at 18 o C and protein expression was evaluated by SDS-PAGE.
The protein was purified using a standard Ni affinity followed by size exclusion two-step chromatography method first as previously reported (85) . Since the purified NxSC1 sample presented as 2 bands, an additional ion exchange chromatography was performed. The NxSC1 sample from the two-step purification was pooled and dialyzed against buffer A (Buffer A: 20 mM Tris-HCl, pH 7.5), and loaded onto a HiTrap Q HP 5 ml column. A gradient of NaCl from 0 to 1 M was applied (Buffer B: 20 mM Tris-HCl, pH 7.5, 1 M NaCl). The NxSC1 was pooled and concentrated to 1 mM using Amico Ultra-4 (Millipore).
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