Dataset from "A triangulenum-based biosensor platform for mapping kinase activity in T cells"

Table S1. Peptide sequences and HPLC-MS results of triangulene biosensors. Figure 2. Fluorescence emission spectra of the biosensors and their fully phosphorylated controls. (A) Src-OH and Src-P; (B) Akt1-OH and Akt1-P. All spectra (lex = 530 nm) were collected from solutions of the peptides at 5 µM, diluted in PBS pH 7.4 NaCl 100 mM. Figure S1. Fluorescent emission at 560 nm of the Src biosensor hydroxylated (Src-OH, black) and phosphorylated (Src-P, orange) biosensor at different ATP concentrations (0-1 mM). (A) MgCl2 1 mM; (B) MgCl2 5 mM. Figure S3. Michaelis-Menten kinetics with recombinant enzyme. Kinetic profiles used to determine initial phosphorylation velocity for the (A) Src-OH biosensor, with 100 ng recombinant kinase; and (B) Akt1-OH biosensor, with 50 ng recombinant kinase. Figure 4. Michaelis–Menten kinetic analysis of the Src-OH (A) and Akt1-OH (B) biosensors. The initial velocity (min–1) values were obtained through the analysis of the kinetics traces in Figure S3. Lines represent nonlinear fittings to the Michaelis–Menten equation. Error bars represent s.d. from 3 independent repetitions. Figure S4. Endpoint in extracto assays. Biosensor concentration was fixed at 5 μM (BS-OH in gray, BS-P in dark orange/red and BS-OH with cells extracts in light orange/red), and increasing amounts of cell extract were added: (A) 0.5–40 μg of HeLa-membrane extract for Src; (B) and 0.5–20 μg of HeLa-cytoplasmic extract for Akt1. Figure 5 and S5. Endpoint extract assays for endogenous kinase activity of Src (A) and Akt1 (B) were performed using the peptide biosensors. The biosensor concentration was fixed at 5 μM in all assays. Emission intensity values were normalized to the phosphorylated biosensors in the presence of standard cell extracts (dark-colored bars). The signals of the Src OH (dark orange) and Akt1 OH (dark blue) biosensors decreased with increasing concentrations of their respective inhibitors (light-colored bars), reaching the same levels as the unphosphorylated controls, Src OH or Akt1 OH (grey bars), at 50 nM Das or 10 μM LY. (A) 10 μg of HeLa-membrane extract (Ext) with 0–200 nM of Dasatinib (Das). (B) 10 μg of HeLa-cytoplasmic extract (Ext) with 0–20 µM of LY294002 (LY). Fluorescence emission at 560 nm (λex = 530 nm[AO16.1]). Error bars represent s.d. from 3 independent experiments. Figure S6. Viability test by flow cytometry. Cytotoxicity profile of the Akt1-Pep-OH biosensor in mouse primary T cells after 24 h incubation at increasing concentrations (0–100 μM), measured by flow cytometry. Data are presented as mean ± s.d. from three independent experiments. Figure S7. Time-dependent cellular uptake kinetics. Cells were incubated with the Akt1-Pep1-OH biosensor (25 µM), and uptake was measured as normalized biosensor emission relative to untreated cells over time. Data are presented as mean ± SD (n = 3). The solid red line represents a nonlinear fit to a one-phase exponential association model, indicating a saturable uptake process. Figure 6 and S8. A) TG intensity (ITG, top) and τFL (bottom) imaging of the Src activity biosensor in HeLa cells: i) untreated HeLa cells; ii) non-phosphorytalable Src-Pep1-Ala control; iii) Src-Pep1-OH biosensor; iv) Src-Pep1-OH biosensor in the presence of 40 nM Das. Scale bars represent 5 µm. B) Box plots of ITG (left) and τFL values (right) from different images for each condition. ITG was defined in in a detection time widow of 5 ns after the excitation pulse. Boxes represent s.d. while whiskers represent 90% of the population. Figure 7 and S9. A) TG intensity (ITG, top) and τFL (bottom) imaging of the Akt1 activity biosensor in HeLa cells: i) untreated HeLa cells; ii) non-phosphorytalable Akt1-Pep1-Ala control; iii) Akt1-Pep1-OH biosensor; iv) Akt1-Pep1-OH biosensor in the presence of 10 µM LY; v) Akt1-Pep1-OH biosensor in the presence of 50 nM insulin (Insu). Scale bars represent 5 µm. B) Box plots of ITG (left) and τFL values (right) from different images for each condition. ITG was defined in in a detection time widow of 5 ns after the excitation pulse. Boxes represent s.d. while whiskers represent 90% of the population. Figure 8. Live-cell confocal microscopy images of isolated mouse T cells. The images illustrate the intracellular localization of the Akt1-Pep1-OH sensor shortly after exposure (step 1–2) and the subsequent Akt1 activation induced by αCD3/αCD28 stimulation (step 2–3). The background-subtracted mean fluorescence intensity (MFI) is plotted, with error bars representing the s.d. of 3 independent measurements. ** indicates significant differences (p < 0.01) using an ANOVA test. Figure 9. Application of the Akt1-Pep1-OH sensor for monitoring Akt1 activity in primary T cells. (A) Representative scatter plot showing gating of CD4⁺ and CD8⁺ T cell populations, and overlaid histograms of Akt1 sensor fluorescence (MFI in the FITC channel) after 30 min of incubation with sensor or control (background without sensor). Pair 1: For CD4⁺ T cells: control, gray; sensor, blue. For CD8⁺ T cells: control, gray; sensor, green; Pair 2: MFI distributions for control (gray, prior activation) and stimulated cells (αCD3/αCD28, green; PMA, red). (B) Flow cytometry analysis of sensor uptake in splenocytes. Bar graph summarizing the mean fold-change in fluorescence intensity in both subsets, compared to no sensor. (C) Flow cytometry analysis of Akt1 activation in CD4⁺ (blue) and CD8⁺ (green) T cells, showing MFI distributions for control (prior activation) and stimulated cells (αCD3/αCD28, PMA), with the corresponding bar graph summarizing fold-changes relative to the values of non-stimulated conditions. Statistical significance was determined by ANOVA; ** p < 0.01.