(A) Calu-3 lung epithelial cells were incubated with the noted concentrations of recombinant CHI3L1 (rCHI3L1; ng/mL) or vehicle control (rCHI3L1 = 0) for 24 hours and were then subjected to RT-qPCR to quantitate the levels of mRNA encoding ACE2 and the SPP. (B) Western blot evaluations of the dose response and kinetics of CHI3L1 stimulation of ACE2 and SPP protein accumulation in Calu-3 cells. (C and D) Calu-3 cells were incubated with vehicle (PBS; CHI3L1^–) or rCHI3L1 (500 ng/mL) for 24 hours, recombinant S protein of SC2 was added, and the incubation continued for an additional 2 hours. The cell lysates were prepared, Co-IP and immunoblot assays were undertaken (C), and the uncleaved S and cleaved S1 and S2 proteins were evaluated using Western immunoblotting (D). (E) Calu-3 cells were incubated with vehicle (rCHI3L1^–) or the noted concentrations of rCHI3L1 for 24 hours. They were then transfected with a pseudovirus containing the S protein (PS; D614 variant) from SC2 and a GFP expression construct and incubated for additional 24 hours and then evaluated using fluorescence microscopy. Quantification of mean fluorescence intensity (MFI) can be seen in the dot plot on the right. (F) Calu-3 cells were incubated with rCHI3L1 or vehicle control for 48 hours in the presence or absence of antibody against CHI3L1 (FRG) or control antibody (IgG). Pseudovirus S (PS; D614 and G614 variants) that expressed GFP was added as noted, and GFP-positive cells (%) were evaluated by flow cytometry. Values in A and E are mean ± SEM. B–F are the representative data obtained from at least 3 independent experiments. β-Actin or GAPDH was used as an internal control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA with post hoc Dunnett’s multiple comparison test). Scale bar: 50 μm.