Fig 1: Stable, functional AMPfret expression in HaCaT C1 keratinocytes. (a) Epifluorescence microscopy images of HaCaT C1 cells showing fluorescence at specific excitation (ex) and emission (em) wavelengths for CFP (ex: 440 nm, em: 460 nm, blue), Venus (ex: 510 nm, em: 535 nm, yellow) and the FRET channel (ex: 440 nm, em: 535 nm, yellow). (b) Representative immunoblots showing the expression of endogenous AMPK and AMPfret AMPK subunits in HaCaT C1 cells (two independent experiments; for uncropped immunoblots, refer to Suppl. Figure 1a-d). (c) Time course of normalized FRET ratio (AMPfret signal) after addition of 20 mM 2-DG (blue) or vehicle (water; black). Data represent mean ± SEM (n = 3, about 140 cells per single experiment); * p < 0.05, ** p < 0.001 (two-way ANOVA test). (d) Microscopy images showing the non-normalized FRET ratio of AMPfret in a HaCaT C1 cell population at t = 0 min (T0) and t = 90 min (T90) from one representative experiment in (c). The AMPfret signal is given in a false color scale. (e) HPLC determination of ATP/ADP ratios in HaCaT C1 cells grown under conditions as used in FRET experiments, after a treatment with 20 mM 2-deoxyglucose (2-DG; blue) or vehicle (water; black) for 60 min. Data represent mean ± SEM (n = 3); ** p < 0.001 (unpaired Student’s t-test). All scale bars represent 20 μm.