Fig 1: VSIR-ERK1/2 signaling induced by the SARS-2-S protein activates ISX-kynurenine-AA metabolic pathways. (a) The protein levels of VSIR, ISX, ERK1/2, p-ERK1/2, p38, PI3K, IκBα, p50, p52, and p65 in BEAS-2B cells were analysed using Western blot. The analysis included BEAS-2B and A549 cells with and without VSIR expression. (b) The protein levels of Spike, VSIR, ISX, ERK1/2, p-ERK1/2, IDO, IDO2, TDO2, PI3K, AKT, COX1, COX2, and ALOX12 in BEAS-2B cells were analyzed using Western blot in SARS-2-S-expressing BEAS-2B cells transfected with either VSIR shRNA or control. (c-e) Kynurenine, PGE2, and TXB2 were detected in the culture medium of BEAS-2B and A549 cells treated with SARS-2-S and either ISX or VSIR shRNA. (f-g) The protein levels of Spike, VSIR, ISX, ERK1/2, p-ERK1/2, IDO, IDO2, TDO2, PI3K, AKT, COX1, COX2, and ALOX12 in BEAS-2B (f) and A549 (g) cells were analyzed using Western blot in ISX-expressing BEAS-2B and A549 cells transfected with either VSIR shRNA or a control. (h) Endogenous ISX levels in cytoplasmic and nuclear fractions were detected in cells transfected with GFP-tagged VSIR, either alone or in combination with U0126. (i) Confocal immunofluorescent imaging of ISX and VSIR was conducted in BEAS-2B (left) and A549 (right) cells. ISX was visualized in magenta, VSIR in green, and DAPI was used to indicate nuclei. Yellow and blank arrows indicate nuclear and cytosolic localization of ISX, respectively. (j) Kynurenine was detected in the culture medium of ISX-expressing BEAS-2B and A549 cells transfected with either VSIR shRNA or a control. The results are shown as the mean ± s.d., using unpaired t-tests based on three to five biological replicates (c-e and j)