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1 Supplementary Materials for Plasma membrane Ca 2+ -ATPases can shape the pattern of Ca 2+ transients induced by store-operated Ca 2+ entry Katalin Pászty, Ariel J. Caride, Željko Bajzer, Chetan P. Offord, Rita Padányi, Luca Hegedűs, Karolina Varga, Emanuel E. Strehler, Agnes Enyedi* This PDF file includes: *Corresponding author. Published 17 February 2015, Sci. Signal. 8, ra19 (2015) DOI: /scisignal Text S1. The system of rate equations describing the kinetic model. Fig. S1. A23187-induced Ca 2+ signaling of control HeLa cells or HeLa cells expressing mcherry-pmca4b-la, mcherry-pmca4a, or mcherry-pmca2b. Fig. S2. HEK293 cells generate the typical shape of the Ca 2+ signals of the slow and fast PMCAs. Legends for movies S1 and S2 Other Supplementary Material for this manuscript includes the following: (available at Movie S1 (.mov format). SOCE signal in sh-pmca4 HeLa cells. Movie S2 (.mov format). SOCE-mediated transients in HeLa cells cotransfected with mch-pmca4b-la and GCaMP2.
2 Text S1. The system of rate equations describing the kinetic model. When the law of mass action is applied to the reaction mechanism described in Table 2 the following system of differential equations is obtained. d[a] = 6k 1 '[AB]+k 3 '[CaiB] 6k 1 [A] 6 k 3 [CaiCaM][A] (1) d[ab] 1 [A] 6 k 1 '[AB] (2) d[cae] 5 [CaiP]+k 10 [PCaiCaMCai] k 2 [Cae][AB] (3) d[cai] 2 [Cae][AB]+k 4 '[CaiP]+2k 6 '[CaiCaMh]+2k 7 '[CaiCaM]+ +k 9 '[PCaiCaMCai]+4k 11 [PCaiCaM] k 4 [Cai][P] d[caicam] 2k 6 [CaM][Cai] 2 k 9 [PCaiCaM][Cai] 4k 11 '[PCaM][Cai] 4 (4) 3 '[CaiB]+k 7 [CaiCaM][Cai] 2 +k 8 '[PCaiCaM]+ +k 13 '[TCaiCaM] k 3 [CaiCaM][A] k 7 '[CaiCaM] k 8 [CaiCaM][P] k 13 [CaiCaM][T] (5) d[caib] 3 [CaiCaM][A] k 3 '[CaiB] (6) d[p] = (k 4 '+k 5 )[CaiP]+k 8 '[PCaiCaM]+k 12 [PCaM] k 4 [Cai][P] k 8 [CaiCaM][P] (7) d[caip] 4 [Cai][P] (k 4 '+k 5 )[CaiP] (8) d[cam] 6 '[CaiCaMh]+k 12 [PcaM] k 6 [CaM][Cai] 2 (9) d[caicamh] 6 [CaM][Cai] 2 +k 7 '[CaiCaM] k 6 '[CaiCaMh] k 7 [CaiCaM][Cai] 2 (10) d[pcaicam] 8 [CaiCaM][P]+(k 9 '+k 10 )[PCaiCaMCai]]+ +k 11 '[PcaM][Cai] 4 (k 8 '+k 11 )[PCaiCaM] k 9 [PCaiCaM][Cai] (11) d[pcaicamcai] 9 [PCaiCaM][Cai] (k 9 '+k 10 )[PCaiCaMCai] (12) d[pcam] 11 [PCaiCaM] k 11 '[PcaM][Cai] 4 k 12 [PCaM] (13) d[t] 13 '([T] 0 [T]) k 13 '[CaiCaM][T] (14) In the last equation, we used the mass conservation equation: [T](0)=[T] 0 =[T]+[TCaiCaM] (15) where [T](0) is the initial concentration of the generic calmodulin target other than channel and pump. Channel A is assumed to be initially in equilibrium with channel in clustered state AB. Consequently, the equilibrium concentration [A] e is given as the solution of equation:
3 (6/K 1 )[A] 6 e+[a] e [A] T =0, K 1 =k 1 '/k 1 (16) where [A] T is the total concentration so that 6[AB]+[A]=[A] T. K 1 is the dissociation constant. The initial value for [A] is the equilibrium concentration [A] e, and the initial value for [AB] is [AB](0)=[AB] e =([A] T [A] e )/6. Total concentration [A] T is estimated by fitting to data. For the initial concentrations of other species, it was assumed: [CaiCaM](0)=[CaiB](0)=[CaiP](0)=[PCaiCaM](0)=[PCaiCaMCai](0)=0 (17) [CaiCaMh](0)=[PCaM](0)=[TCaiCaM](0)=0, [Cae](0)=2000µM (18) Initial concentrations [T](0), [Cai](0), [P](0), [CaM](0) were estimated by the fitting process. The signal S(t) obtained in measurements is related to [Cai](t) by the Hill equation: q([cai](t)) h S(t)= 1+([Cai](t)/K) h (19) where q is a fitting parameter and h=3.16, K=0.171µM (69).
4 Figure S1. A23187-induced Ca2+ signaling of control HeLa cells or HeLa cells expressing mcherry-pmca4b-la, mcherry-pmca4a, or mcherry-pmca2b. Ca2+ signals were detected with GCaMP2 and A23187 (2 μm) was added where indicated. (A) A23187-induced Ca2+ signals in HeLa cells (control). Traces from >15 cells are shown. (B) A23187-induced Ca2+ signals in mcherry-pmca4b-la cells. Traces from >15 cells are shown. (C) Average A induced Ca2+ signals from cells expressing the indicated mcherry-pmca isoforms. Data are shown as the mean ± 95% CI from cells.
5 Figure S2. HEK293 cells generate the typical shape of the Ca2+ signals of the slow and fast PMCAs. (A) Western blot analysis of whole cell lysates of HeLa (40 μg) and HEK293 (40 μg) cells. All PMCAs blot: The first lane shows a mixture of 2 μg PMCA1b (endogenous) and 0.05 μg PMCA4b overexpressing microsomal membrane preparations from COS-7 cells to serve as standards for the PMCA1b and PMCA4b. Proteins were detected with the antibody 5F10. PMCA1 blot: The first lane shows 2 μg of a microsomal membrane preparation of COS-7 cells to provide a standard for PMCA1. Proteins were detected with an antibody specific for PMCA1. Coomassie staining: the same blots were stained with Coomassie Brilliant Blue R-250 to show equal amounts of loaded proteins. Data are representative of 4 experiments. (B) Ca2+ transients in untransfected control HEK293 cells using the Ca2+ re-addition procedure and GCaMP2. (C, D, E) mcherry fluorescence (upper panels) and Ca2+ transients (graphs) in HEK293 cells expressing the indicated mcherrypmca proteins. Data in B-E represent the average ± 95% CI of >15 cells.
6 Movie S1. SOCE signal in sh-pmca4 HeLa cells. A representative experiment of the SOCE-mediated Ca 2+ transients in sh-pmca4 HeLa cells expressing GCaMP2. The recording shows SOCE that was induced by the re-addition protocol adding 2 mm Ca 2+ to cells after store depletion. Movie S2. SOCE-mediated transients in HeLa cells cotransfected with mch-pmca4b- LA and GCaMP2. A representative experiment of the SOCE-mediated Ca 2+ transients in mcherry-pmca4b-la and GCaMP2 expressing HeLa cells. The recording shows SOCE that was induced by the re-addition protocol adding 2 mm Ca 2+ to cells after store depletion.
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Supplementary Figure 1 Supplementary Figure 1 SNARE Probes for FRET/2pFLIM Analysis Used in the Present Study. mturquoise (mtq) and Venus (Ven) are in blue and yellow, respectively. The soluble N-ethylmaleimide-sensitive
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DOI:.38/ncb2822 a MTC02 FAO cells EEA1 b +/+ MEFs /DAPI -/- MEFs /DAPI -/- MEFs //DAPI c HEK 293 cells WCE N M C P AKT TBC1D7 Lamin A/C EEA1 VDAC d HeLa cells WCE N M C P AKT Lamin A/C EEA1 VDAC Figure
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d e f Spatiotemporal quantification of subcellular ATP levels in a single HeLa cell during changes in morphology Supplementary Information
Ca 2+ level (a. u.) Area (a. u.) Normalized distance Normalized distance Center Edge Center Edge Relative ATP level Relative ATP level Supplementary Information Spatiotemporal quantification of subcellular
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Supplementary Figure 1 a γ-h2ax MDC1 RNF8 FK2 BRCA1 U2OS Cells sgrna-1 ** 60 sgrna 40 20 0 % positive Cells (>5 foci per cell) b ** 80 sgrna sgrna γ-h2ax MDC1 γ-h2ax RNF8 FK2 MDC1 BRCA1 RNF8 FK2 BRCA1
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p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
A.. Relative # of ECs associated with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 ol b p < 0.001 Relative # of blood vessels that formed with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 l b p = 0.002 Control IHC:
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Shao-Ming Shen et al Role of I in MT of cancers MO reports xpanded View igures igure V1. nalysis of the expression of I isoforms in cancer cells and their interaction with PTN. RT PR detection of Ish and
+ + + IP: Anti-Flag. Catalytic domain. Regulatory domain. Myr SH3 SH2 Kinase domain PP2. Flag-HK1. c-src ΔSH3. HK1 N-half: C-half:
a d h ΔSH2 Δ(SH3SH2) Myr SH3 SH2 Kinase domain 85 5 249 55 5 csrc ΔSH3 FlagHK HAcSrc HAcSrcΔSH2 HAcSrcΔSH3 HAcSrcΔ(SH3SH2) WB: FlagHK HAcSrc HAcSrcKD HAcSrcY529F WB: HisHK2HK2 Input GSTcSrc GST : : GST
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Supplementary Figure 1. DTPA does not interfere with the activity of catalase. Dependency of CAT activity on DTPA concentration at 25 C.
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