Figure 3. Metabolic reprogramming by influenza confirmed and distinct from TLR stimulation.

(A–C) NHBE cells were grown and differentiated in 24-well Seahorse plates and left untreated (control), infected for 17 hours at MOI 1 with either viable virus or a β-propriolactone–inactivated virus (CA04 or BPL, respectively) or stimulated with TLR agonists lipopolysaccharide (LPS), polyinosinic polycytidylic acid (PolyIC), or Resiquimod (R848). After treatment, the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), and proton production rate (PPR) of NHBE cells were monitored for 2 hours. Error bars represent standard error of the mean of quadruplicate (control & CA04) or triplicate (BPL, LPS, PolyIC, & R848) samples, representative date from 3 independent experiments. Basal rate was established by measuring technical replicates measure 13 times/per well, to establish basal rate giving n=52 (control & CA04) or n=39 (BPL, LPS, PolyIC, & R848) for SEM.(D) NHBE cells were treated as in A–C and glucose uptake was monitored using a standard blood glucometer in 2 independent experiments, in quadruplicate with SEM. (E) NHBE cells were infected with CA04 for up to 17 hours at MOI 1, harvested at indicated time and lysates subjected to immunoblotting with a c-Myc protein standard from cMyc positive tumor lysate (“ctrl”) and probed for c-Myc and β-actin. (F–G) NHBE cells were grown and differentiated in 96-well Seahorse plates and left untreated (control), infected for 17 hours with CA04 viable virus at MOI 1, treated with cMyc inhibitor at 0.5 uM, 1.0 uM, and 2.0uM respectively or infected and treated. After treatment, the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of NHBE cells were monitored for 2 hours. Error bars represent standard error of the mean of quintuplicate (control & CA04) and (cMyc 2.0uM & CA04+2.0uM) samples, representative plate from 5 independent experiments. Basal rate was established by measuring technical replicates measure 12 times per well, to establish basal rate giving n=60 (control, CA04, cMyc 2.0uM & CA04+2.0uM) for SEM. (H) BGEM was replaced with complete DMEM (red dotted line) or metabolite deplete DMEM for 2–4 hours followed by infection for 17 hours when viability was assessed using trypan blue exclusion. Two independent experiments were done with each control and virus in triplicate. (B–H) Error bars reflect the S.E.M. and significances are indicated as * p≤0.05, ** p≤0.001, *** p≤0.0001, and **** p<0.0001 from a Dunnett’s multiple comparison adjusted T test that followed ANOVA (<0.0001).