Asymmetric post-translational modifications regulate the nuclear translocation of STAT3 homodimers in response to leukemia inhibitory factor

Figures

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  Fig. 1. Asymmetric PTMs can modulate the subcellular localization of STAT3 homodimers upon cytokine stimulation. a Representative western blot images of total protein extracts from reference HeLa and STAT3-/- strains transfected with wild-type VN-STAT3 construct, expressing STAT3 levels similar to endogenous STAT3 in reference HeLa cells. b Our Venus-STAT3 BiFC system consists of two constructs where STAT3α was fused to either the N-terminus (VN, amino acids 1-158) or C-terminus (VC, amino acids 159–222) of the Venus fluorescent reporter. STAT3 dimerization brings the complementary Venus fragments together, restoring fluorescence. Fluorescence intensity is proportional to STAT3 homodimerization. c Representative frames from time-lapsed videos of STAT3-/- HeLa cells transfected with the indicated BiFC pairs of Venus-STAT3 constructs (green) at 0, 10 and 20 min of incubation with LIF (200 ng/ml). Red line marks the nuclear perimeter. Scale bar, 40 μm. d Average kinetic curves of LIF-induced nuclear translocation of STAT3 dimers in > 50 cells (Nr. of independent experiments: WT pair, N = 11; Y705F pair N = 5; any other pair N = 4). For each cell, the fluorescence intensity was measured in…

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  Fig. 2. Expression of different STAT3 variants has impact on cell’s proteome and on the functional profile of regulated targets. a Venn diagrams describing the overlaps between the sets of identified proteins in cells expressing the three STAT3 variants (WT-WT, WT-Y705F and Y705F-Y705F) with and without LIF stimulation. Proteins identified uniquely in one condition are potentially specific targets of the corresponding STAT3 variant. b Table showing the number of proteins uniquely identified in each of the 6 conditions studied (3 STAT3 variants with and without LIF stimulation), and within those sets, how many are codified by genes known to be regulated by STAT3 according to the DoRothEA database or to unphosphorylated STAT3 (USTAT3) ChIP-seq data. c Heatmap representing GO terms significantly associated with targets of specific STAT3 variants with or without LIF stimulation. Color represents the logarithm (base 2) of the Fold Enrichment (logFE) of a given GO term (rows) in one experimental condition (columns, WWL – WT-WT + LIF, WW – WT-Wt, WYL – WT-Y705F + LIF, WY – WT-Y705F, YYL – Y705F-Y705F + LIF, YY – Y705F-Y705F). The Fold Enrichment is the ratio of the observed frequency of a GO…