Coomassie plus protein assay reagent

Cells were rinsed with PBS and lysed as previously described (Feng et al., 2015 (link)). Protein concentration was determined using Coomassie Plus Protein Assay Reagent (Thermo Scientific). Equal amounts of cell lysate proteins (50 μg) were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes (PerkinElmer Life Sciences). Membranes were blocked in 3% BSA/TBST for 1 h and incubated with primary antibodies (overnight at 4°C), with shaking. Following three TBST washes, membranes were incubated for 1 h at room temperature with secondary antibodies (1:10,000). Detection and quantifications were made using an Odyssey Infrared Imaging System (LICOR Biosciences). Antibodies to OC43 N-protein and the spike protein were obtained from EMD Millipore (cat# MAB9012) and CUSABIO (cat# CSB-PA336163EA01HIY), respectively. Antibodies against eIF4G1, eIF4E and 4EBP1 were purchased from Cell Signaling Technology (respective cat# 8701, cat# 2067, and cat# 9644), and HSP90 antibody was obtained from Santa Cruz Biotechnology (cat# sc-13119). Secondary antibodies were goat anti-rabbit Alexa Fluor 680 (cat# A21076) and goat anti-mouse Alexa Fluor 680 (cat# A21057) (Invitrogen). All antibodies were used according to suppliers’ recommendations.

An l-Lactic acid (LA) colorimetric assay kit (ACE biolabs, Taoyuan, Taiwan) was used to measure LA levels. Briefly, 4T1 or MDA-MB-231 cells (4 × 10⁵) were cultured in medium containing 10% FBS, treated with the indicated agents for 24 h and incubated in medium containing 3% FBS for another 24 h. The cells were collected and resuspended in 100 μL of PBS, sonicated on ice and collected the supernatants after centrifugation at 1500×g for 10 min at 4 °C. Protein concentrations were determined by Coomassie Plus Protein Assay Reagent (Thermo Fisher Scientific). The samples were mixed with the enzyme working solution and chromogenic agent and incubated at 37 °C for 10 min. The stop solution was added, and the absorbance was measured at 530 nm by a microplate reader (TECAN). LA levels were calculated by the following formula: LA (mmol/gm protein) = (ΔOD1 sample/ΔOD2 standard) × 3 (the concentration of standard, mmol/l) × dilution factor of sample/concentration of protein in sample (gm of protein/l).

MM cells after wash in PBS were lysed in lysis buffer (300 mM NaCl, 50 mM Tris HCl pH 7.6, 0.1% Triton X-100, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 4 mM EDTA, phosphatase inhibitors). After 1.5 h of incubation on ice, total cell lysates were clarified using high-speed centrifugation for 15 min and an aliquot of the supernatant was assayed to determine protein concentration by Coomassie plus protein assay reagent (Thermo Fisher Scientific, Rockford, IL, USA). Proteins were separated by SDS-polyacrylamide gel electrophoresis (Bolt Bis-Tris gel 4–12%, Thermo Fisher Scientific), and transferred to nitrocellulose membrane (GE Healthcare, Milan, Italy). The membrane was incubated in blocking solution (5% non-fat dry milk, 20 mM Tris, 140 mM NaCl, 0.1% Tween-20), and probed overnight at 4 °C with specific antibodies against HIF1α (Millipore) and IPO7 (Pierce, Thermo Fisher Scientific); Histone H3 and VHL (Santa Cruz Biotechnology, SantaCruz, CA, USA); β-actin (Cell Signalling Technology, Beverly, MA, USA). After three washes with 20 mM Tris, 140 mM NaCl, 0.1% Tween-20, the membrane was incubated 1 h with secondary antibody daylight 488 (Thermo Fisher Scientific) diluted in blocking solution. After signal detection by Chemidoc (Biorad, Milan, Italy), densitometric analysis was done with Image J software (National Institute of Health, Bethesda, MA, USA).

For analysis of detergent-soluble and -insoluble fractions, HEK 293T cells and CaCo-2 cell monolayers were washed with 1× PBS and lysed in ice-cold Nonidet P40 lysis buffer (40 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% (v/v) Nonidet-P40, 10 mM β-glycerophosphate, 10 mM NaF, 1 mM EDTA, 1 mM PMSF, 1 μg/mL pepstatin, 1 μg/mL aprotinin, and 1 μg/mL leupeptin) for 10 min on ice. Cell extracts were centrifuged at 10,000× g and 4 °C for 20 min. The supernatants, representing the detergent-soluble fraction, were collected, and the detergent-resistant pellets were washed once with the lysis buffer, solubilized in 2× SDS-PAGE sample buffer (125 mM Tris-HCl pH 6.8, 20% (v/v) glycerol, 2% (v/v) SDS, 100 mM DTT, 0.005% bromophenol blue), sonicated, and boiled at 95 °C for 5 min. To normalize the samples, protein concentration was determined in supernatants using Coomassie Plus Protein Assay Reagent (Thermo Scientific, Waltham, MA, USA). Protein extracts in sample buffer were separated by SDS–PAGE, transferred to a nitrocellulose membrane (Amersham Protran Premium 0.2 µm NC), blocked with 5% milk protein in TBST (20 mM Tris-HCl pH 7.6, 137 mM NaCl, 0.05% (v/v) Tween), and immunoprobed with the antibodies indicated according to manufacturer’s recommendations. Enhanced chemiluminescence detection (Biozym Scientific, Oldendorf, Germany) was used in immunoblot assays.