6 diazo 5 oxo l norleucine don

The following drugs were used: 6‐Diazo‐5‐oxo‐l‐norleucine (Don, D2141, Sigma‐Aldrich); Sodium dichloroacetate (DCA, 347795, Sigma‐Aldrich); NCT‐502 and PHGDH inactive (19716 and 19717, Cayman); Doxycycline (Dox, D9891, Sigma‐Aldrich), Oligomycin (sc‐203342, Santa Cruz Biotechnology); FCCP (sc‐203578, Santa Cruz Biotechnology); Antimycin (sc‐202467, Santa Cruz Biotechnology); Rotenone (sc‐203242, Santa Cruz Biotechnology); Cyt.C (C2037, Sigma‐Aldrich) CB‐839 (10‐4556, Focus Biomolecules); Adenosine diphosphate (ADP, A2754, Sigma‐Aldrich).

CL1-5 cells, which were kindly provided by Dr. Pan-Chyr Yang, and A549 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium. Culture media were supplemented with 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂. The cells were treated with 5 μM OGA inhibitor Thiamet G (TMG; cat. no. 13237, Cayman Chemical, Ann Abor, MI, USA) or 50 μM PUGNAc (A7229, Sigma-Aldrich, St. Louis, MO, USA), in the complete medium for 24 h to increase O-GlcNAc levels. To manipulate the hexosamine biosynthetic pathway (HBP), 10 mM Glucosamine (G1514, Sigma-Aldrich, St. Louis, MO, USA), or 40 μM 6-Diazo-5-oxo-L-norleucine (DON) (D2141, Sigma-Aldrich, St. Louis, MO, USA) were used in the complete medium for 24 h.

Orlistat (Psicofarma), lonidamine (Sigma), 6-Diazo-5-oxo-L-norleucine (DON) (Sigma), growth hormone (GH) (Merck), insulin (Lilly), and indomethacin (Sigma) were employed. Orlistat and indomethacin were dissolved in absolute ethanol (Sigma), lonidamine in DMSO (Sigma), and DON, GH and insulin in complete medium. The compounds were administered alone or in the anti-anabolic (Orlistat + lonidamine + DON, named ‘OLD’), anti-catabolic (GH + insulin + indomethacin, named ‘GII’), or 6 drugs (OLD + GII, named ‘6 drugs’) schemes.

We prepared single-cell suspensions of mouse splenocytes as previously described^{23 (link)}. We purified murine B cells from splenocytes using a B-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s guidelines.
We cultured B cells in RPMI complete medium (Thermo Fisher Scientific, Waltham, MA, USA). For murine B-cell activation, purified B cells were seeded into 96-well flat-bottom tissue culture plates at a density of 5 × 10⁵ cells/well with or without TLR2 ligand (P3C, 2 μg/mL; InvivoGen, San Diego, CA, USA)/HBV particles [Multiplicity of infection (MOI): 1,000] stimulation for 24 h. In some experiments, we treated the cells with the indicated inhibitors, such as 2-deoxy-d-glucose (2-DG), oligomycin, 6-diazo-5-oxo-l-norleucine (DON), Rapamycin, and Akti-1/2 (Sigma, St. Louis, MO, USA).

COS-7 cells from green monkey kidney and HeLa cells from human cervical cancer were obtained from the American Tissue Culture Collection (Manassas, VA) and cultured in 8-well Culture Slides (BD Falcon™; CA, USA) for transfection, drug treatment, and immunofluorescence (IIF) analyses. Alternatively, these cells were cultured on round 22 mm-diameter coverslips for microinjection procedures. Both cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Calf Serum (FCS) at 37°C and 5% CO₂. Adherent cell lines were maintained at 50% confluence.
COS-7 and HeLa cells were treated with compounds reported to induce RR structures [2 (link), 12 (link)]. Ribavirin (Sigma-Aldrich; R9644,) and mycophenolic acid (MPA, Sigma-Aldrich; M3536) were solubilized in tissue culture grade water to a 50 mM stock and 6-diazo-5-oxo-L-norleucine (DON, Sigma-Aldrich; D2141) was solubilized in water to a 100 mM stock and stored at -80°C until use. Drugs at various concentrations were added to cell culture 24 h prior to fixation.

Calu-3 cells were seeded in 24-well plate, and after 72 h of seeding, the cells were infected with SARS-CoV-2 at multiplicity of infection (MOI) of 0.001 for 1 h. Following infection, the cells were treated with Dulbecco's modified Eagle's medium (Gibco), which contained pyruvate (1 mM) and glutamine (4 mM) as the basal carbon source and were supplemented with 5% fetal bovine serum and different concentrations of glucose (11.1, 22.2, and 44.4 mM) (Gibco) and keeping basal glucose concentration at 11.1 mM, different concentrations of mannose (11.1, 22.2, and 44.4 mM) (Sigma–Aldrich). Inhibitors of glycolysis, 2-DG (Sigma–Aldrich), and glutaminolysis, 6-diazo-5-oxo-l-norleucine (DON; Sigma–Aldrich), were reconstituted in water, and cytotoxicity at different concentrations and 24 h time point was determined in Calu-3 cells using alamarBlue Cell Viability Reagent (Invitrogen) according to the manufacturer's instructions. To determine the effect of these drugs on viral replication, following 1 hpi (MOI = 0.001), the Calu-3 cells were treated with 2-DG (10 mM) and DON (200 μM), respectively. The supernatants were collected after 24 hpi, and the cells were lysed in TRI reagent (Zymo Research) and stored in −70 °C for RNA extraction.