clr selection invivogen hb csm normocin tm invivogen ant nr 1 puromycin invivogen ant pr  (InvivoGen)

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: STAT5A activation dynamics of HEK-Blue IL-2 cells expressing STATeLight5A.

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques:

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: a , Schematic representation of FRET-based biosensor to detect STAT5A dimerization in the IL-2–JAK–STAT5 pathway. STAT5A monomers are fused to mNG and mSC-I. Upon IL-2 stimulation, a switch in STAT5A mode of dimerization changes the distance between mNG and mSC-I, resulting in FRET occurrence. This image was created using BioRender.com . b , AlphaFold model of human STAT5A. NTD, dark blue; CCD, yellow; DBD, light blue; LD, orange; SH2 domain, purple; TAD, gray. c , AlphaFold models showing parallel and antiparallel dimeric conformations of full-length STAT5A (interface predicted template modeling (ipTM) = 0.642 and 0.611, respectively). In red are residues from which distances were measured. d , Quantification of mNG mean fluorescence lifetime of STAT5A biosensor variants transfected in HEK-Blue IL-2 cells. Schematic representation of STAT5A variants. N, NTD; C, C-terminal domain. Data were acquired on the same cells before and 30 min after stimulation with 3.5 μg of IL-2. 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 = 14, 44, 28, 50, 24, 8, 15 and 8 cells, respectively. A two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted P values: P = 0.48 (not significant (NS); variant 1), P = 0.27 (NS; variant 2), **** P = 3.75 × 10 −8 (variant 3), **** P = 1.1 × 10 −16 (variant 4), **** P = 3.01 × 10 −6 (variant 5), P = 0.051 (NS; variant 6), P = 0.051 (NS; variant 7) and * P = 0.014 (variant 8). e , Representative images of best-performing biosensor (STATeLight5A). Scale bars, 10 μm.

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Fluorescence, Transfection, Variant Assay

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: HEK-Blue TM IL-2 cells were transfected with different biosensor variants and mNG fluorescence lifetime was evaluated before and 30 min after stimulation with 3.5 µg of IL-2. ( a ) Representative fast FLIM images of HEK-Blue TM IL-2 cells transfected with different biosensor variants. mNG fluorescence lifetime is represented by a color-coded scale. Scale bars, 10 µm. ( b ) Mean difference in mNG fluorescence lifetime in the same cells before and after IL-2 stimulation. Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. From left to right, n = 14, 44, 28, 50, 24, 8, 15, 8 cells. ( c ) FRET efficiency of biosensor variants before and after IL-2 stimulation. Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. From left to right, n = 14, 44, 28, 50, 24, 8, 15, 8 cells. Two-sided Wilcoxon’s rank-sum test was used to calculate false discovery rate (FDR)-adjusted p values: 0.482, not significant (ns, variant 1), 0.313, ns (variant 2), **** p = 3.75 ×10 −8 (variant 3), **** p = 1.10 × 10 −16 (variant 4), **** p = 3.01 ×10 −6 (variant 5), 0.051, ns (variant 6), 0.051, ns (variant 7), * p = 0.014 (variant 8). ( d ) Quantification of mNG fluorescence lifetime in the cytoplasm (n = 35 cells) and nucleus (n = 12 cells) of unstimulated and IL-2-stimulated cells expressing STATeLight5A. Boxplots show the median (center line), 25 th and 75 th 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 = 1.82 × 10 −12 (cytoplasm), **** p = 2.96 × 10 −6 (nucleus).

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Transfection, Fluorescence, Variant Assay, Expressing

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: 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).

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Expressing, Fluorescence, Western Blot, Concentration Assay, Reporter Assay, Transfection

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: ( a ) Representative fast FLIM images of HEK-Blue TM IL-2 cells transfected with STATeLight5A 30 min after stimulation with titrated concentrations of IL-2. Fluorescence lifetime is represented by a color-coded scale. Scale bars, 10 µm. ( b ) Percentage of pSTAT5 + HEK-Blue TM IL-2 cells treated with varying IL-2 concentrations, as assessed flow cytometry. ( c ) Correlation between mNG fluorescence lifetime and pSTAT5 + cells. Statistical significance was tested with Pearson correlation coefficient, R = 0.91, *** p = 0.0003. ( d ) NativePAGE-western blot showing STAT5 dimerization at different IL-2 concentrations. ( e ) HEK-Blue TM reporter assay showing STAT5 transcriptional activation at different IL-2 concentrations. Data are shown as mean ± SEM, n = 3 biological replicates. ( f ) Correlation between the dimer/monomer ratio, assessed by western blot and STAT5 transcriptional activation, assessed by HEK-Blue TM reporter assay. Statistical significance was tested with Pearson correlation coefficient, R = 0.98, **** p = 9.17 × 10 −6 . ( g ) Correlation between fluorescence lifetime and STAT5 transcriptional activation, assessed by HEK-Blue TM reporter assay. Statistical significance was tested with Pearson correlation coefficient, R = -0.98, *** p = 0.0002.

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Transfection, Fluorescence, Flow Cytometry, Western Blot, Reporter Assay, Activation Assay

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: ( a ) Representative fast FLIM images of HEK-Blue TM IL-2 cells transfected with STATeLight5A and stimulated with 3.5 µg of IL-2 over a time course of 90 minutes. Scale bars, 10 µm. ( b ) Immunoprecipitation coupled with western blot detecting endogenous STAT5B which interacts with STAT5-mSC-I-FLAG in HEK-Blue TM IL-2. ( c ) Immunoprecipitation coupled with western blot detecting endogenous STAT5B which interacts with STAT5-mNG-I-Myc in HEK-Blue TM IL-2. ( d ) Representative flow cytometry plots showing endogenous pSTAT5 time dynamics in IL-2–stimulated HEK-Blue TM IL-2 cells expressing either STATeLight5A (gated on mNG⁺mSC-I⁺ cells) or wild-type (WT) STAT5A-Myc (gated on Myc⁺ cells). ( e ) Quantification of pSTAT5 levels shown in ( d ). ( f ) HEK-Blue TM IL-2 cells were transduced with either scrambled short-hairpin RNA (scr shRNA) or STAT5B shRNA. STAT5B abundance after knock-down was assessed by western blot using anti-STAT5B antibodies. ( g ) Quantification of mNG fluorescence lifetime in untreated cells (n = 27), scr shRNA-expressing cells (n = 26), or STAT5B knockdown cells (n = 15), either in unstimulated or IL-2-stimulated conditions. 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 = 1.21 × 10 −14 (Untransduced), **** p = 1.21 × 10 −14 (scr shRNA), **** p = 1.29 × 10 −8 (STAT5B shRNA).

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Transfection, Immunoprecipitation, Western Blot, Flow Cytometry, Expressing, Transduction, shRNA, Knockdown, Fluorescence

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: HEK-Blue TM IL-2 or HeLa cells were transfected with indicated homodimeric and heterodimeric STAT variants, including STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. mNG fluorescence lifetime was evaluated before and 30 min after stimulation with 3.5 µg of IL-2 (for STAT3, STAT5A and STAT5B), IFNα (for STAT1, STAT2 and STAT4) and IL-4 (for STAT6). a , Representative fast FLIM images of transfected cells before and after cytokine stimulation. Scale bars, 10 µm. b , Quantification of mNG mean fluorescence lifetime before and after cytokine stimulation of indicated homodimeric STATeLights. 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 = 64, 25, 14, 13, 20, 17 and 14 cells. A two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted P values: **** P = 1.16 × 10 −20 (STAT5A), **** P = 1.66 × 10 −12 (STAT5B), *** P = 1.77 × 10 −4 (STAT1), **** P = 3.57 × 10 −12 (STAT2), **** P = 4.10 × 10 −10 (STAT3), **** P = 4.80 × 10 −9 (STAT4) and * P = 0.016 (STAT6). c , Quantification of mNG mean fluorescence lifetime before and after cytokine stimulation of indicated heterodimeric STATeLights. Box plots show median (center line), 25th and 75th percentiles (box limits) and whiskers extending 1.5× the interquartile range. From left to right, n = 23, 12, 12 and 21 cells. A two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted P values: **** P = 8.45 × 10 −11 (STAT1–STAT2), ** P = 0.007 (STAT3–STAT5A), *** P = 2.75 × 10 −4 (STAT3–STAT5B) and **** P = 3.72 × 10 −11 (STAT5A–STAT5B).

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Transfection, Fluorescence

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: 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.

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Expressing, Fluorescence, Western Blot

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: ( 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-Blue TM IL-2 cells. ( c ) Quantification of pSTAT5 in cells expressing cancer-associated STAT5B gain-of-function mutations. ( d ) Western blot analysis of HEK-Blue TM 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), 25 th and 75 th 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-Blue TM 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), 25 th and 75 th 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-Blue TM IL-2 cells co-expressing full-length cancer-associated STAT5B mutants and PTPN1.

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Binding Assay, Flow Cytometry, Expressing, Western Blot, Fluorescence, Reporter Assay

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: ( 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), 25 th and 75 th 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-Blue TM 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), 25 th and 75 th 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).

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Fluorescence, Expressing, Mutagenesis, Western Blot

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: HEK-Blue TM IL-2 cells were transfected with constructs containing a single-point mutation (Thr-to-Ala, Ser-to-Ala or Tyr-to-Phe). mNG fluorescence lifetime was evaluated before and 30 min after stimulation with 3.5 µg of IL-2. ( a ) Quantification of mNG fluorescence lifetime in cells expressing STATeLight5A with point mutations at known phosphorylation sites (Thr or Ser). Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. From left to right, n = 47, 36, 26, 29, 33, 12, and 19 cells. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: **** p = 3.88 × 10 −25 (WT), **** p = 4.76 × 10 −19 (T92A), **** p = 4.03 × 10 −15 (S127A), **** p = 6.65 × 10 −17 (S128A), **** p = 5.40 × 10 −17 (S193A), **** p = 7.40 × 10 −7 (T647A), **** p = 6.60 × 10 −11 (T648A). ( b ) Quantification of mNG mean fluorescence lifetime in cells expressing STATeLight5A with Tyr-to-Phe mutations. Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. From left to right, n = 50, 26, 26, 21, 26, 27, 29, 31, 34, 37, 42, 27, 32, 24, and 25 cells. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: **** p = 4.11 × 10 −17 (WT), **** p = 4.33 × 10 −11 (Y22F), **** p = 6.05 × 10 −15 (Y33F), **** p = 4.65 × 10 −12 (Y90F), **** p = 6.05 × 10 −15 (Y98F), **** p = 2.21 × 10 −15 (Y114F), **** p = 1.66 × 10 −16 (Y171F), **** p = 2.15 × 10 −17 (Y176F), **** p = 2.55 × 10 −17 (Y225F), **** p = 8.32 × 10 −19 (Y405F), **** p = 8.32 × 10 −19 (Y548F), **** p = 6.51 × 10 −10 (Y568F), **** p = 4.27 × 10 −15 (Y668F), **** p = 8.45 × 10 −14 (Y682F), **** p = 7.64 × 10 −11 (Y683F).

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Transfection, Construct, Mutagenesis, Fluorescence, Expressing, Phospho-proteomics

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: HEK-Blue TM IL-2 cells were transfected with STATeLight5A and incubated with indicated STAT5 inhibitors for 2 hours. Subsequently, cells were stimulated with 3.5 µg of IL-2 for 30 min. mNG fluorescence lifetime was measured. ( a ) Representative fast FLIM images. Scale bars, 10 µm. ( b ) Quantification of mNG fluorescence lifetime in cells treated with different STAT5A inhibitors. Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. From left to right, n = 21, 22, 20, 17 and 21 cells. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: **** p = 9.30 × 10 −12 (DMSO), **** p = 4.76 × 10 −12 (AC-4-130), 0.277, ns (SH-4-54), **** p = 1.42 × 10 −9 (Sigma 573108), ** p = 1.85 × 10 −3 (Pimozide). ( c ) Mean difference in mNG mean fluorescence lifetime. Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. From left to right, n = 21, 22, 20, 17 and 21 cells. Differences between DMSO-treated and conditions treated with STAT5 inhibitors were analyzed using one-way ANOVA, followed by Dunnett’s post-hoc test. p values: 0.993, ns (AC-4-130), **** p < 1 × 10 −15 (SH-4-54), 0.721, ns (Sigma 573108), **** p < 1 × 10 −15 (Pimozide). ( d ) Western blot analysis of STATeLight5A-expressing cells treated with STAT5 inhibitors. ( e ) Quantification of STAT5A dimer/monomer ratio in STAT5 inhibitor-treated cells by western blot using anti-STAT5A antibodies. Shown are mean ± SEM from 4 biological replicates. Differences between DMSO-treated and conditions treated with STAT5 inhibitors were analyzed using repeated-measures ANOVA, followed by Dunnett’s post-hoc test. p values: 0.614, ns (AC-4-130), **** p = 1.25 × 10 −8 (SH-4-54), 0.633, ns (Sigma 573108), ** p = 0.002 (Pimozide).

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Transfection, Incubation, Fluorescence, Western Blot, Expressing

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: HEK-Blue IL-2 cells expressing STATeLight5A were treated with JAK inhibitors overnight, followed by stimulation with 3.5 µg of IL-2. a , Dose–response curve of STATeLight5A treated with titrated amounts of upadacitinib (purple) and filgotinib (orange). From lowest to highest JAK inhibitor concentration, n = 28, 24, 18, 19, 26, 24, 17 and 15 cells (filgotinib) and n = 28, 15, 30, 19, 22, 27, 29 and 20 cells (upadacitinib). Data are shown as the mean ± s.e.m. b , Heat map representation of normalized lifetime ratio of JAK inhibitors compared to DMSO-treated condition. The mNG fluorescence lifetime ratio was calculated as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\rm{mNG}}\; {\rm{lifetime}}\; {\rm{after}}\; {\rm{IL}}{{\mbox{-}}2}\;{\rm{stimulation}}}{{\rm{mNG}}\; {\rm{lifetime}}\; {\rm{before}}\; {\rm{IL}}{{\mbox{-}}2} \;{\rm{stimulation}}}$$\end{document} mNG lifetime after IL - 2 stimulation mNG lifetime before IL - 2 stimulation . The lifetime ratio of DMSO-treated cells was normalized to 0. c , Western blot analysis of cells treated with JAK inhibitors. d , Quantification of STAT5A dimer-to-monomer ratio in JAK inhibitor-treated cells by western blot using anti-STAT5A antibodies. Data are shown as the mean ± s.e.m. ( n = 3 biological replicates). Differences between DMSO-treated condition and conditions treated with JAK inhibitors were analyzed using a one-way ANOVA, followed by Dunnett’s post hoc test: P = 0.957 (NS; filgotinib), ** P = 0.002 (upadacitinib), P = 0.063 (NS; abrocitinib), * P = 0.017 (itacitinib), * P = 0.020 (fedratinib), P = 0.680 (NS; pacritinib), * P = 0.044 (gandotinib), * P = 0.011 (decernotinib), ** P = 0.005 (peficitinib), P = 0.903 (NS; momelotinib), ** P = 0.003 (baricitinib), ** P = 0.002 (ruxolitinib), ** P = 0.003 (tofacitinib), ** P = 0.007 (gusacitinib), * P = 0.029 (delgocitinib) and * P = 0.049 (cerdulatinib). e , Correlation between fluorescence lifetime ratio and dimer-to-monomer ratio, assessed by western blot. Statistical significance was tested using the Pearson correlation coefficient ( R = 0.81); **** P = 8.083 × 10 −5 .

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Expressing, Concentration Assay, Fluorescence, Western Blot

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: Human PBMCs or HEK-Blue TM IL-2 cells were pre-treated with titrated amounts of Filgotinib or Upadacitinib, followed by stimulation with IL-2. ( a ) Gating strategy to identify CD4 + T cells in human PBMCs. ( b ) Representative pSTAT5 histograms of CD4 + T cells treated with Filgotinib or Upadacitinib. ( c ) Percentage of pSTAT5 + cells in CD4 + T cells treated with Filgotinib (yellow) or Upadacitinib (purple). Data are represented as mean ± SEM of n = 3 replicates. ( d ) Dose-response of HEK-Blue TM IL-2 cells treated with Filgotinib (yellow) or Upadacitinib (purple) using HEK-Blue TM IL-2 reporter assay. Data are represented as mean ± SEM (n = 3 replicates). ( e) Time-course of mNG fluorescence lifetime in HEK-Blue™ IL-2 cells expressing STATeLight5A, untreated (n = 75 cells) or treated with Upadacitinib (n = 39 cells) or Filgotinib (n = 33 cells) and stimulated with IL-2. Data are shown as mean ± SEM.

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Reporter Assay, Fluorescence, Expressing

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: HEK-Blue TM IL-2 cells were transfected with STATeLight5A and incubated with indicated JAK inhibitors overnight. Subsequently, cells were stimulated with 3.5 µg of IL-2 for 30 min and mNG fluorescence lifetime was measured. ( a ) Quantification of mNG fluorescence lifetime before and after IL-2 stimulation. Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. From left to right, n = 20, 22, 12, 22, 26, 26, 28, 31, 22, 14, 21, 22, 20, 21, 33, 24, 54. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: **** p = 2.24 × 10 −6 (Filgotinib), * p = 0.014 (Upadacitinib), *** p = 1.69 × 10 −4 (Abrocitinib), * p = 0.029 (Itacitinib), * p = 0.035 (Fedratinib), **** p = 1.50 × 10 −5 (Pacritinib), 0.173, ns (Gandotinib), ** p = 1.50 × 10 −3 (Decernotinib), *** p = 1.99 × 10 −4 (Peficitinib), ** p = 1.50 × 10 −3 (Momelotinib), * p = 0.026 (Baricitinib), ** p = 3.72 × 10 −3 (Ruxolitinib), * p = 0.035 (Tofacitinib), ** p = 8.10 × 10 −3 (Gusacitinib), *** p = 3.57 × 10 −4 (Delgocitinib), **** p = 3.72 × 10 −5 (Cerdulatinib), **** p = 1.06 × 10 −17 (DMSO). ( b ) Mean difference in mNG mean fluorescence lifetime. Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Differences between untreated and conditions treated with JAK inhibitors were analyzed using one-way ANOVA, followed by Dunnett’s post-hoc test. p values: **** p = 7.20 × 10 −12 (Filgotinib), **** p < 1 × 10 −15 (Upadacitinib), * p = 0.011 (Abrocitinib), **** p < 1 × 10 −15 (Itacitinib), **** p < 1 × 10 −15 (Fedratinib), **** p < 1 × 10 −15 (Pacritinib), **** p < 1 × 10 −15 (Gandotinib), **** p < 1 × 10 −15 (Decernotinib), **** p < 1 × 10 −15 (Peficitinib), **** p = 4.15 × 10 −11 (Momelotinib), **** p < 1 × 10 −15 (Baricitinib), **** p < 1 × 10 −15 (Ruxolitinib), **** p < 1 × 10 −15 (Tofacitinib), **** p < 1 × 10 −15 (Gusacitinib), **** p < 1 × 10 −15 (Delgocitinib), **** p < 1 × 10 −15 (Cerdulatinib).

Article Snippet: HEK-Blue IL-2 cells obtained from InvivoGen were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco), penicillin–streptomycin (100 U per ml; Gibco), l -glutamine (2 mM, Gibco), Normocin (100 μg ml −1 ; InvivoGen), HEK-Blue CLR selection (1×; InvivoGen) and puromycin (1 μg ml −1 ; InvivoGen).

Techniques: Transfection, Incubation, Fluorescence