Supplemental DATA for:
UNCOUPLING PROTEIN 3 (UCP3) MODULATES THE ACTIVITY OF SARCO/ENDOPLASMIC RETICULUM Ca2+ATPase (SERCA) BY DECREASING MITOCHONDRIAL ATP PRODUCTION
Umberto De Marchi, Cyril Castelbou, and Nicolas Demaurex1
From Department of Cell Physiology and Metabolism, University of Geneva, rue Michel-Servet, 1,
CH-1211 Genève, Switzerland
Supplementary figure legends
Fig.S1. Detection of UCP3 knockdown by Western blot. 48 h post-transfection, mitochondria were isolated and 50 g of proteins from mitochondrial samples and cell extracts were analyzed. The bars of UCP3 expression (upper) indicate optical density of the respective bands in the bottom, which are normalized according the corresponding mitochondrial Tom20 band. They are the mean ± S.E.M of 3 independent experiments. Representative blot from three independent experiments is shown.
Fig. S2.Effect of UCP3 over-expression on cellular Ca2+ handling and ATP production.HeLa cells were transiently co-transfected with 4mtD3cpv (A), YC3.6cyto(B), or ATeammito(C), and with either Ctrl mtDsRed or UCP3/mtDsRed constructs for 48h. A) Left: Averaged [Ca2+]mit elevation evoked by 100 µM histamine. Right: averaged [Ca2+]mitamplitude (peak) and integrated Ca2+ responses (area). Bars are mean ± S.E.M of 61 (n= 6) and 74 (n= 6) cells for Ctrl/mtDsRed and UCP3/mtDsRed, respectively. B) Statistical evaluation of the effects of UCP3/mtDsRed over-expression on the integrated [Ca2+]cyto responses during histamine-induced Ca2+release (release) and subsequent Ca2+ readmission (influx), recorded with the protocol shown in Fig. 2A. Bars are mean ± S.E.M of 36 (n=3) and 48 cells (n= 3) for Ctrl/mtDsRed and UCP3/mtDsRed, respectively. C) Statistical evaluation of the histamine-induced, oligomycin A-sensitive [ATP]mit changes, , recorded with the protocol shown in Fig. 6A. Bars are mean ± S.E.M of n= 5 (39 cells) and n= 4 (41 cells) for Ctrl/mtDsRed and UCP3/mtDsRed, respectively.
Fig. S3.Effect of UCP2 over-expression on cellular Ca2+ handling in UCP3-depleted cells.HeLa cells were transiently co-transfected with 4mtD3cpv (A) or YC3.6cyto(B) together with either Ctrl siRNA, UCP3 siRNA, or UCP3 siRNAand UCP2 cDNA for 48 h. A) Averaged [Ca2+]mit elevation (left) evoked by 100 µM histamine and averaged integrated [Ca2+]mitresponses (right). Bars are mean ± S.E.M of 47 (n= 5), 47 (n= 5) and 34 (n= 4) cells, for Ctrl, UCP3 siRNA and UCP3 siRNA + UCP2, respectively. B) Left: averaged [Ca2+]cyto response during the readmission of Ca2+to cells depleted with histamine, as shown in Fig. 2A. Right: statistical evaluation of the integrated [Ca2+]cyto responses. Bars are mean ± S.E.M of 38 (n= 3), 32 (n= 3) and 40 (n= 3) cells, for Ctrl, UCP3 siRNA and UCP3 siRNA + UCP2, respectively.
Fig. S4.Effect of UCP3 knockdown on mitochondrial and cytosolic Ca2+ elevations, measured simultaneously.HeLa cells were transiently transfected with the mitochondrial calcium probe 4mtD3cpv together with the indicated siRNA for 48 h, and loaded with Fura2 to enable concurrent [Ca2+]mit and [Ca2+]cyt recordings. A) Left: 4mtD3cpv signal recorded at 535 nm. Middle: Averaged [Ca2+]mit elevation evoked by 100 µM histamine. Right: averaged amplitude (i) and integrated Ca2+ response (ii) of cells expressing scrambled or UCP3 siRNA. B) [Ca2+]cyt elevations recorded with fura-2 in the cells shown in A.Bars are mean ± S.E.M of 25 (n= 4) and 29 cells (n= 5) for Ctrl and UCP3 siRNA, respectively.
Fig. S5.[Ca2+]ER calibration and effect of SERCA inhibition on ER Ca2+ release. HeLa cells were transiently transfected with the ER Ca2+ probe D1ER and, where indicated (B,C), with Ctrl siRNA or UCP3 siRNA for 48 h. A)Change in D1ER fluorescence ratio during calibration of the probe. Where indicated, the saline solution was replaced with solutionscontaining digitonin and ionomycin and equilibrated at pCa of >8 and ~2 to determine the maximal(Rmax) and minimal (Rmin)fluorescence ratio, as described in (36). B) Statistical evaluation of the basal [Ca2+]ER. Bars are mean ± S.E.M of 27 (n= 4) and 28 (n= 4) cells, for Ctrl siRNA and UCP3 siRNA, respectively.C) Left: average of recording of [Ca2+]ER release in HeLa cells stimulated with 100 µM histamine and 1 µM TG at the same time. Right: statistical evaluation (inset) of the UCP3 siRNA on the kinetics of Ca2+ release. For the latter statistics, the halflife of [Ca2+]ER responses was extracted as described in Fig. 3. Bars are mean ± S.E.M of 35 (n=5) and 42 cells (n= 4) for Ctrl and UCP3 siRNA, respectively. D) Average of recordings of [Ca2+]ER in HeLa cells stimulated with 1 µM TG (73 cells). To evaluate the time necessary to inhibit SERCA pumps, basal R/R0 fluorescence was compared each 2 seconds versus basal R/R0 fluorescence (=1). Student t-test was applied. E) Average of recordings of [Ca2+]ER in HeLa cells stimulated with 1 µM TG (73 cells) or 15 M BHQ (40 cells).
Fig. S6.Effect of SERCA inhibition on cytosolic Ca2+ elevation.HeLa cells were transiently co-transfected with the Ca2+ probe YC3.6cyto and with the indicated siRNAs for 48 h and washed. ER Ca2+ stores were depleted with 1 M TG in Ca2+-free medium and Ca2+ was then readmitted to monitor Ca2+ influx. A) Average of [Ca2+]cytorecordings from Ctrl (scramble) siRNA and UCP3 siRNA cells. B) Statistical evaluation of the UCP3 knock down on rate of both Ca2+ entry after TG stimulation and influx component after Ca2+ readmission. The slopes were extrapolated by fitting the linear phase of increasing [Ca2+] signals after TG stimulation or Ca2+-readmission. Bars are mean ± S.E.M of 96 (n=9) and 104 cells (n= 11) for Ctrl and UCP3 siRNA, respectively.
Fig. S7.Effect of UCP3 knockdown on the BHQ-dependent store-operated Ca2+ entry. HeLa cells were transiently co-transfected with the YC3.6cyto and with the indicated siRNAs for 48 h and washed. Cytosolic Ca2+ responses were measured. SERCA pumps were reversibly inhibited with15 M BHQ in Ca2+-free medium to deplete Ca2+ stores and, after wash, Ca2+ was then readmitted to monitor Ca2+ influx. A) Average of cytosolic Ca2+ responses elicited BHQ and by Ca2+ readmission. B) Statistical evaluation of the UCP3 siRNA on the integrated [Ca2+]cyto response during stimulation and Ca2+ readmission. Bars are mean ± S.E.M of 38 (n=4) and 31 cells (n= 4) for Ctrl and UCP3 siRNA, respectively.
Fig. S8.Effect of inhibition of ATP production on Ca2+ elevation.A)HeLa cells were transiently co-transfected with the YC3.6cyto probe and with the Ctrl siRNA or the UCP3 siRNA for 48 h and were used to determine Ca2+ influx component, by applying the same protocol as in Fig. 2A. Inhibitors of mitochondrial ATP production were pre-incubated for 3 minutes before Ca2+ readmission. Statistical evaluation of the effect of 5 g/ml oligomycin (A) or 10 M antimycin-A (B) on integrated [Ca2+]cyto responses, during Ca2+ influx. Bars are mean ± S.E.M of 3 independent experiments for each condition; the number of cells is indicated into each histograms.
1