Fig. 6

ERAT4.01 reveals ATP depletion in the ER of HeLa cells after SLC35B1 knockdown. a–d HeLa cells were transfected with control siRNA or with SLC35B1- or SLC35B1-UTR-targeting siRNA for 72 h, then transfected with ERAT4.01. After 24 h, they were imaged by fluorescence microscopy. Where indicated, Thapsigargin (Tg, 1 µM) or 2-deoxy-glucose (2-DG, 10 mM) were added. a Images were recorded using transmission light or fluorescence. Representative images are shown (scale bar 10 µm). b Time-resolved live cell recordings of ER luminal ATP levels are shown as FRET-ratio F₅₃₅/F₄₈₀. Data are presented as means for ctrl, n = 33 cells, UTR siRNA, n = 20, SLC35B1 siRNA, n = 21, from at least three independent experiments. c Statistical analysis of the resting ATP levels in the experiments shown in b. Three time points before Tg addition were averaged (indicated as 1) and subtracted from the MAX-values (indicated as 2) following Tg addition for each single cell. Data are presented as mean with SEM. The indicated pairs were assessed by unpaired, two-sided standard Student´s t-test (*P < 0.05, **P < 0.01, **P < 0.001). d Statistical analysis of the Tg-induced ATP increase in the experiments shown in b. e, f HeLa cells were transfected with control siRNA or with SLC35B1- or SLC35B1-UTR-targeting siRNA, transfected with ATeam, and imaged. The mean values of time-resolved live cell recordings of cytosolic ATP levels (e) and the corresponding statistical analysis (f) are shown for ctrl, n = 116 cells, UTR siRNA, n = 95, SLC35B1 siRNA, n = 57 from at least three independent experiments. g HeLa cells were transfected with control siRNA or with SLC35B1-targeting or SLC35B1-UTR-targeting siRNA for 96 h, and total cellular ATP was determined using ApoSensor according to the manufacturer´s protocol. Data from three independent experiments are reported together with the individual data points as % of control with SEM