(A) Left panel: representative Fura-2AM based cytosolic Ca2+ measurements in primary hepatocytes isolated from wild type (WT) and leptin-deficient (Lepob/ob) obese mice. ER Ca2+ stores were depleted with 1 µM thapsgargin (Tg), a SERCA inhibitor. SOCE was evaluated by substituting the extracellular media (0 mM Ca2+) with media containing 2 mM Ca2+. Dashed lines show Ca2+ measurements in the presence of 50 µM 2-Aminoethoxydiphenyl borate (2-APB). Right panel: quantification of Tg-induced Ca2+ release (reflecting ER Ca2+ content) and SOCE based on the measurements shown in (A), n = 160 WT and n = 250 Lepob/ob cells for Tg response and n = 234 WT and n = 303 Lepob/ob cells for SOCE response. Data were pooled across six independent experiments. *p<0.0001 (B) Left panel: Immunoblot analysis of protein expression levels in total liver lysate from WT and Lepob/ob mice, Right panel: quantification of the western blots n = 4, representative of 4–5 experiments. (C and D) Confocal images of immunofluorescence staining for endogenous STIM1 in primary hepatocytes from WT and Lepob/ob animals, treated with DMSO (vehicle, NT) or 1 µM Tg for 10 and 30 min. NT refers to ‘not Tg treated’ (E) Quantification of STIM1 puncta/cluster number in the bottom and middle cross-section of non-treated (DMSO) hepatocytes from WT and Lepob/ob animals.n = 5–6 fields (WT) and 4–5 fields (Lepob/ob), representative of 4 independent experiments, *p=0.02 #p=0.003 (F) Quantification of pixel intensity in a set area (125 × 125 pixels) of the middle section of the cells from WT and Lepob/ob animals, treated with 1 µM Tg or vehicle (quantification methods depicted in Figure 1—figure supplement 1 F), n = 4–9 cells, representative of 4 independent experiments, *p=0.02 #p=0.008 (G) Representative profile plots of STIM1 levels (pixel intensities) in a defined area (box) across cells treated with DMSO or 1 µM Tg for 10 or 30 min. Left: cells from WT animals, right: cells from Lepob/ob animals. (H) Quantification of STIM1 translocation by calculating the ratio between the mean STIM1 pixel intensity at a selected area of the edge of the cell relative to the same measurement performed in the cytosolic area near the edge of the cell, n = 3–4 ratios per cell, quantified in 2–8 cells for each condition *p<0.0001 **p=0.009 (I and J) Representative TIRF images of STIM1 and Na+K+-ATPase (PM marker) in cells from WT (I) and Lepob/ob mice (J) treated with 1 µM Tg or vehicle for 10 min. NT refers to “not Tg treated. For all graphs, error bars denote s.e.m. Scale: 10 µm.