Fig 1: Cancer-associated STAT5B mutants show differential dynamics of activation and deactivation.(a) Schematic representation of cancer-associated STAT5B mutations. Blue, SH2 domain; violet, linker domain (LD); orange, DNA-binding domain (DBD); and green, coiled coil domain (CCD). (b) Flow cytometry plots showing pSTAT5 in cancer-associated STAT5B mutants, expressed in HEK-BlueTM IL-2 cells. (c) Quantification of pSTAT5 in cells expressing cancer-associated STAT5B gain-of-function mutations. (d) Western blot analysis of HEK-BlueTM IL-2 cells expressing full-length cancer-associated STAT5B mutants. (e) Quantification of mNG fluorescence lifetime in cells expressing STATeLight5B biosensors harboring cancer-associated mutations. From left to right, n = 19, 18, 31, 27, 33, 27, 23, 30, and 28 cells. Boxplots show median (center line), 25th and 75th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: ****p = 1.56 × 10−5 (Y712E), ****p = 5.50 × 10−6 (Y665F), ****p = 2.11 × 10−6 (N642H), ****p = 4.22 × 10−7 (A510V), ****p = 1.81 × 10−11 (E438Q), ****p = 4.93 × 10−10 (S434L), ****p = 3.20 × 10−9 (N418K), ****p = 1.85 × 10−13 (P267A), ****p = 4.13 × 10−12 (WT). (f) HEK-BlueTM IL-2 reporter assay in cells expressing cancer-associated STAT5B mutants and using titrated concentrations of IL-2. Shown are mean ± SEM of 3 biological replicates. (g) Quantification of mNG fluorescence lifetime in cells co-expressing STATeLight5B biosensors with cancer-associated mutations and protein tyrosine phosphatase non-receptor type 1 (PTPN1). From left to right, n = 17, 16, 47, 27, 19, 20, 19, 25, and 52 cells. Boxplots show median (center line), 25th and 75th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: ****p = 4.01 ×10−6 (Y712E), ****p = 1.20 × 10−7 (Y665F), ****p = 1.25 × 10−14 (N642H), ****p = 7.70 × 10−5 (A510V), ***p = 7.92 × 10−4 (E438Q), **p = 4.80 × 10−3 (S434L), ****p = 1.64 × 10−5 (N418K), 0.803, ns (P267A), ****p = 1.70 × 10−8 (WT). (h) Western blot analysis of HEK-BlueTM IL-2 cells co-expressing full-length cancer-associated STAT5B mutants and PTPN1. Source data
Fig 2: Characterization of STATeLight5A biosensor.a, Representative fast FLIM images of HEK-Blue IL-2 cells expressing STATeLight5A before and after stimulation with 3.5 μg of IL-2, IL-15 and IL-4, as indicated. Scale bars, 10 μm. b, Quantification of mNG mean fluorescence lifetime in response to IL-2 (n = 50 cells), IL-15 (n = 36 cells) and IL-4 (n = 34 cells). Box plots show the median (center line), 25th and 75th percentiles (box limits) and whiskers extending 1.5× the interquartile range. A two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted P values: ****P = 1.37 × 10−17 (IL-2), ****P = 6.28 × 10−19 (IL-15) and P = 0.253 (NS; IL-4). c, Western blot analysis of STATeLight5A-expressing cells stimulated with IL-2, IL-15 and IL-4. d, Correlation between mNG fluorescence lifetime and dimer-to-monomer ratio, assessed by western blot. e, Dose response of STATeLight5A-expressing cells stimulated with IL-2. Shown is the mNG mean fluorescence lifetime ± s.e.m. from two experiments. From lowest to highest IL-2 concentration, n = 56, 60, 57, 63, 52, 55, 52 and 59 cells. f, mNG fluorescence lifetime of STATeLight5A was assessed over 90 min after IL-2 stimulation. Shown are the mean ± s.e.m. of n = 12 cells. g, Endogenous STAT5 was measured in STATeLight5A-expressing HEK-Blue IL-2 reporter assay. Shown are the mean ± s.e.m. from three biological replicates. h, STATeLight5A-expressing cells were stimulated with IL-2, followed by transfection with PTPN1. mNG mean fluorescence lifetime was quantified. Box plots show the median (center line), 25th and 75th percentiles (box limits) and whiskers extending 1.5× the interquartile range. From left to right, n = 57, 62 and 72 cells. A one-way ANOVA followed by Tukey’s post hoc test was used to calculate P values: ****P = 6.2 × 10−14 (IL-2 versus unstimulated) and ****P = 6.2 × 10−14 (IL-2 versus IL-2 and PTPN1).Source data
Fig 3: Characterization of C-terminally labelled full-length STAT5A biosensor variant.(a) Quantification of mNG fluorescence lifetime in cells expressing C-terminally labelled full-length STAT5A biosensor variants, including WT (black, n = 27 cells), loss-of-function (LOF) mutant (Y694F, magenta, n = 24 cells), and gain-of-function (GOF) mutant (S710F, green, n = 35 cells). Boxplots show the median (center line), 25th and 75th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: ****p = 6.17 × 10−15 (WT), 0.282, ns (Y694F), ****p = 7.89 × 10−16 (S710F). (b) Western blot analysis using anti-STAT5A and anti-pSTAT5 antibodies in HEK-BlueTM IL-2 cells expressing the full-length STAT5A biosensor variants shown in (a). (c) Western blot analysis using anti-STAT5A and anti-pSTAT5 antibodies in HEK-Blue™ IL-2 cells co-expressing PTPN1 and full-length STAT5A biosensor variants, including WT (black) and GOF S710F mutant (green). (d) mNG fluorescence lifetime measured in cells co-expressing PTPN1 and full-length STAT5A biosensor variants, including WT (black, n = 23 cells) and the GOF S710F mutant (green, n = 25 cells). Boxplots show the median (center line), 25th and 75th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: 0.965, ns (WT), 0.242, ns (S710F). Source data
Fig 4: Mutational analyses of STAT5A using the biosensor platform.a, Schematics of SH2 domains of STAT5A in dimeric complexes. LOF and GOF substitutions are indicated in magenta and green, respectively. Structures were predicted using AlphaFold simulation. b, Representative fast FLIM images of HEK-Blue IL-2 cells expressing LOF and GOF STATeLight5A biosensors before and after IL-2 stimulation. c, Quantification of mNG fluorescence lifetime in cells expressing LOF and GOF STATeLight5A. Box plots show the median (center line), 25th and 75th percentiles (box limits) and whiskers extending 1.5× the interquartile range. From left to right, n = 50, 33, 32, 30, 20 and 14 cells. A two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted P values: ****P = 8.22 × 10−17 (WT), P = 0.368 (NS; Y694F), P = 0.694 (NS; W641A), P = 0.368 (NS; F706A), ****P = 4.35 × 10−11 (S710F) and ****P = 1.99 × 10−7 (Y665F). d, Western blot analysis of HEK-Blue IL-2 cells expressing LOF and GOF STATeLight5A biosensors using anti-STAT5A and anti-pSTAT5 (pY694) antibodies. Dimeric pSTAT5 observed for LOF mutants stemmed from endogenous STAT5B of HEK-Blue IL-2 cells. e, Quantification of mNG fluorescence lifetime in cells coexpressing GOF mutants and PTPN1. Box plots show the median (center line), 25th and 75th percentiles (box limits) and whiskers extending 1.5× the interquartile range. From left to right, n = 35, 32 and 20 cells. A two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted P values: *P = 0.028, ****P = 1.64 × 10−12 (S710F) and ****P = 3.48 × 10−10 (Y665F). f, Western blot analysis using anti-pSTAT5 antibodies in indicated GOF mutants.Source data
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