Figure 3. Reduced phospho-ERK and ACE2 expression by the combination of MEK inhibitor and remdesivir.

(A) Effects of 5 μM remdesivir (RDV), RAF/MEK inhibitor VS-6766, or the combination on ACE2, ERK1/2, and pERK protein expression. HCT116 human colorectal cancer cells, H1975 human NSCLC cells and BEAS-2B normal human bronchial epithelial cells were treated with 5 μM VS-6766 or/and 5 μM Remdesivir for 48 hr. Remdesivir alone increased pERK protein expression in all three cell lines, which was completely depleted by VS-6766 treatment. Effects of 5 μM RDV, VS-6766, or the combination on ACE2, ERK1/2, and pERK protein expression in (B) normal human lung cells and (C–E) human lung cancer cells are shown. Serum-deprived cells were plated and cultured in medium containing 1% FBS for the indicated amount of time. For serum-stimulated cells, FBS was added to a final concentration of 10% for 24 hours (B–E, left 2 panels). Cells were plated in 10% serum for 16 hours, then media was removed and replaced with 1% FBS for serum-deprived cells. For serum-stimulated cells, FBS was added to a final concentration of 10% for 4 hours (E, right panel). ACE2 (PAB), ACE2 (CS), ERK1/2, and pERK were probed with Abnova PAB13444, Cell Signaling 4355, Cell Signaling 9102, and Cell Signaling 4370 antibodies. Ran was probed with BD Biosciences 610341 antibody as a loading control. (F) Modulation of pRb and ACE2 expression in H1299 cells with serum starvation, stimulation, and MEKi treatment. Control cells were grown with the normal 10% serum throughout the treatment. Serum starved cells were plated in 10% serum and incubated for 16 hours, then grown in media containing 0% serum for 72 hours. Serum stimulated cells were similarly starved for 72 hours, then stimulated with media containing 20% serum for 48 hours. All drug treated cells received 5 mM RDV, VS-6766, or the combination 48 hours before harvesting. (G) Modulation of ACE2 expression with serum starvation and stimulation. Four different cell lines (2 lung, 1 colorectal, and 1 breast cancer) were grown in 10% serum for 16 hours then were starved in 0% FBS for 0 (no starvation), 24, 48, or 72 hours. Cells were stimulated with 20% FBS and harvested either 0 (no stimulation), 12, or 24 hours later. An increase in pRb relative to total Rb was seen upon stimulation and this correlated with ACE2 levels. pERK correlation with ACE2 was heterogeneous, with a correlation seen in MCF7 cells but not H1299 or HCT-116 cells (three left-most panels). pRb relative to total Rb decreased upon starvation and increased upon stimulation, which correlated with ACE2 expression (H460 and HCT-116 in right-most panels).