Extended Data Fig. 2: STAT3 palmitoylation promotes STAT3 nuclear translocation and synergically enhances STAT3 signalling activity with phosphorylation.

a, HEK293A cells were pretreated with ruxolitinib (1 μM) for 0.5 h and then labelled with 50 μM chemical probe (alk-C₁₆) for 2 h, with the incubation with IFNγ (1 ng ml⁻¹). Endogenous STAT3 palmitoylation was analysed by click reaction and streptavidin bead pulldown, and subjected to western blotting. b, HEK293A cells were transfected with Flag-tagged wild-type STAT3 or Flag–STAT3(C687S) and then labelled with 50 μM chemical probe (alk-C₁₆) for 2 h, with the incubation with IL-6 (20 ng ml⁻¹). STAT3 palmitoylation was analysed by western blot, after click reaction and streptavidin bead pulldown. c, HEK293A cells were transfected with MYC-tagged wild-type STAT3, MYC–STAT3(C687S) or MYC–STAT3(Y705F), and treated with IL-6 (20 ng ml⁻¹, 1 h). Whole-cell lysates were immunoprecipitated with anti-MYC antibody, followed by immunoblotting using indicated antibodies. d, Confocal microscopy showing changes in subcellular localization of wild-type STAT3 and STAT3(C687S/C712S) mutant in HEK293A stable cell lines. Cells were stained with anti-Flag antibody (green), anti-lamin B1 antibody (red) and DAPI (blue). Lamin B1 displays the nuclear membrane. The red line indicates the position of the Z-stack. Western blot showing that the wild-type STAT3 and STAT3(C687S/C712S) mutant were expressed at comparable levels in HEK293A stable cell lines. The levels of nuclear-localized STAT3 were quantified by measuring the Flag (STAT3) fluorescence intensity in the nucleus, using the confocal software to define the selected region of interest on the basis of nuclear DAPI signal. The data are presented as mean ± s.e.m. n = 225 cells with wild-type STAT3 and n = 300 cells with STAT3(C687S/C712S) mutant. P value was determined by two-tailed Student’s t-test. Scale bars, 20 μm. e, HEK293A cells were transfected with Flag-tagged wild-type STAT3, or Flag–STAT3(C687S/C712S) or Flag–STAT3(K685S), with or without CBP. Whole-cell lysates were analysed by western blot using indicated antibodies. f, HEK293A cells were co-transfected with Flag-tagged wild-type STAT3 or Flag–STAT3(C687S/C712S) along with GFP–STAT1. Whole-cell lysates were analysed by anti-Flag immunoprecipitation, followed by immunoblotting using the indicated antibodies. g, Analysis similar to that in f was performed in HEK293A cells transfected with Flag–STAT3 and HA–JAK1, followed by anti-HA immunoprecipitation. h, HEK293A cells were co-transfected with Flag–STAT3 and MYC–STAT3, pretreated with 5,15-DPP (10 μM) and then treated with IL-6 (20 ng ml⁻¹, 1 h). Whole-cell lysates were analysed by anti-Flag immunoprecipitation, followed by immunoblotting using the indicated antibodies. i, HEK293A cells were transfected with Flag–STAT3 treated with 5,15-DPP alone or in combination with IL-6 (20 ng ml⁻¹, 1 h), and labelled with 50 μM chemical probe (alk-C₁₆). STAT3 palmitoylation was analysed by click reaction and streptavidin bead pulldown, and subjected to western blotting. j, The acyl chain-binding pocket according to the STAT3 crystal structure (RCSB Protein Data Bank code 4E68). Hydrophobic amino acids are shown in yellow. k, Mutational analysis of selected residues involved in STAT3 palmitoylaiton. l, HEK293A cells were transfected with MYC- and Flag-tagged STAT3 as indicated. Whole-cell lysates were immunoprecipitated with anti-MYC antibody, followed with immunoblotting using indicated antibodies. m, STAT3-null (Stat3^−/−) mouse embryonic fibroblast (MEF) cells were transfected with vector control, Flag-tagged wild-type STAT3 or STAT3(C687S/C712S) mutant. Cells were labelled with 50 μM alk-C₁₆ probe for 2 h, with the incubation with IL-6 (20 ng ml⁻¹). Western blotting using anti-Flag antibody in the streptavidin bead pulldown samples indicates the palmitoylation levels of STAT3. n, Stat3^−/− mouse embryonic fibroblast cells were co-transfected with STAT3 reporter construct (m67-luciferase reporter) and Renilla luciferase control construct, and a vector control, a Flag-tagged wild-type STAT3 or STAT3(C687S/C712S) mutant. Cells were then treated with IL-6 (20 ng ml⁻¹) for 8 h. Luciferase activity was obtained from triplicate experiments and normalized to the Renilla luciferase. Relative fold induction was plotted as shown. o, Experiment similar to that in n was performed in HEK293A cells. p, Stat3^−/− mouse embryonic fibroblast cells were transfected with vector control, Flag-tagged wild-type STAT3 or STAT3(C687S/C712S) mutant. Cells were treated with IL-6 (20 ng ml⁻¹) for 8 h. The mRNA levels of STAT3 target genes (BCL2, BCL2L1 and MMP9) were analysed by quantitative PCR with reverse transcription (qRT–PCR). q, Experiment similar to that in p performed in HEK293A cells. In n–q, the data are presented as mean ± s.e.m. n = 3 biologically independent samples. P value were determined by two-tailed Student’s t-test. In a–c, e–i, k–m, the experiments were independently repeated at least three times with similar results. For gel source data, see Supplementary Fig. 1.

Source data