Fig 1: Knockout JAK2 and SRC reduced GC cells' sensitivity to vortioxetine hydrobromide.A CRISPR/Cas9 system was used to knockout both JAK2 and SRC in HGC27 and AGS cells. Knockout efficiency in GC cells were assessed by Western blotting. B Cell viability after both JAK2 and SRC knockout was assessed by MTT assay. C Colony numbers of JAK2 and SRC knockout cells were measured by colony formation assay. D The protein levels of p-STAT3 Y705 and STAT3 in JAK2 and SRC double knockout cells by Western blotting. E The protein levels of p-STAT3 Y705 and STAT3 in sgControl, sgJAK2, sgSRC and sgJAK2 + sgSRC cells by Western blotting. F The inhibitory effect of vortioxetine hydrobromide on JAK2 and SRC knockout cells was detected by proliferation assay after 96 h. Cell viability was evaluated by MTT assay and normalized to that of the sgcontrol. G JAK2 and SRC knockout cells were plated into 6-well plates and treated with 4 μM vortioxetine hydrobromide for 10 days, followed by crystal violet staining to monitor colony formation. Mean ± S.D. (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001).
Fig 2: Vortioxetine hydrobromide suppressed STAT3 signaling pathway by targeting JAK2 and SRC kinases.A In vitro kinase assay of active JAK2 and inactive STAT3. The active JAK2, inactive STAT3, and ATP mixture were treated with vortioxetine hydrobromide or DMSO at 30 °C for 30 min. p-STAT3 Y705 and STAT3 were visualized by Western blotting. B Vortioxetine hydrobromide suppressed SRC kinase activity in a dose-dependent manner. C The protein levels of JAK2/SRC-STAT3 signaling pathway in GC cells after vortioxetine hydrobromide (0, 0.5, 1, 2, 4 μM) treatment. The quantitative analyses of fluorescence intensity of p-STAT3 (D) and STAT3 (E) in GC cells. F The changes of STAT3 dimer formation after vortioxetine hydrobromide treatment in GC cells. G The nucleus localization variation of STAT3 after treatment of various concentrations of vortioxetine hydrobromide in HGC27 cells. H The protein levels of Bcl2, Mcl1 and c-Myc by Western blotting after treatment of various concentrations of vortioxetine hydrobromide in HGC27 cells. Mean ± S.D. (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001).
Fig 3: Vortioxetine hydrobromide bound with JAK2 and SRC.A The interaction between vortioxetine hydrobromide (pink) and JAK2 (green) was predicted using a computational docking model. Hydrogen bonds were represented as a yellow dash line. B The interaction between vortioxetine hydrobromide (pink) and SRC (blue) was predicted using a computational docking model. Hydrogen bonds were represented as a yellow dash line. C–E Vortioxetine hydrobromide directly bound to JAK2 and SRC. The recombinant proteins (C) or GC cell lysates (D, E) were incubated with vortioxetine hydrobromide-conjugated Sepharose 4B beads or with Sepharose 4B beads alone. The results were analyzed by Western blotting. Data from three independent experiments were shown. The binding capacity of vortioxetine hydrobromide to JAK2 and SRC in AGS (F) and HGC27 (G) intact cells. The cells were treated with vortioxetine hydrobromide or DMSO for 24 h and incubated in different temperatures. The protein bindings were visualized by Western blotting. Mean ± S.D. (n = 3).
Fig 4: JAK2 knockout reduced GC cells sensitivity to vortioxetine hydrobromide.A CRISPR/Cas9 system was used to knockout JAK2 in HGC27 and AGS cells. Knockout efficiency in GC cells was assessed by Western blotting. B Cell viability after JAK2 knockout was assessed by MTT (96 h after seeding cells) assay. C Colony numbers of JAK2 knockout cells were measured. D The protein levels of p-STAT3 Y705 and STAT3 in JAK2 knockout cells by Western blotting. E The inhibitory effect of vortioxetine hydrobromide on JAK2 knockout cells was detected by proliferation assay after 96 h. Cell viability was evaluated by MTT assay and normalized to that of the sgcontrol. F JAK2 knockout cells were plated into 6-well plates and treated with various concentration of vortioxetine hydrobromide (0, 0.5, 1, 2, 4 μM) for 10 days, followed by crystal violet staining to monitor colony formation. Mean ± S.D. (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001).
Supplier Page from Abcam for Recombinant human JAK2 protein (His tag N-Terminus)