Fig 2: VEGF-induced intracellular Ca2+ oscillations are initiated by the PLCγ/InsP3 pathway in breast cancer-derived endothelial colony forming cells(A), U73122 (10 μM, 10 min), a selective PLC blocker, prevented the Ca2+ response to VEGF, while U73343 (10 μM, 10 min), an inactive analogue of U7322, was without effect. (B), 2-APB (50 μM, 20 min), an InsP3R blocker, suppressed VEGF-induced Ca2+ oscillations under 0Ca2+ conditions to prevent any contaminating effect on plasmalemmal channels. (C), the depletion of the InsP3-sensitive ER Ca2+ store with CPA (10 μM) in the absence of external Ca2+ (0Ca2+) prevented the following Ca2+ response to VEGF (10 ng/mL). Note the transient increase in [Ca2+]i caused by CPA due to the depletion of the ER Ca2+ pool. (D), mean±SE of the percentage of BC-ECFCs responding to VEGF (10 ng/mL) under the designated treatments. The asterisk indicates p<0.05. NoR: No response.

Fig 3: InsP3-dependent signalling in mRCC cells and endothelial progenitor cells. (a) Thimerosal triggered a brief train of intracellular Ca2+ spikes in mRCC cells when applied at 50 μM (black tracing), while it was almost ineffective at 25 μM (grey tracing). (b) Prolonged oscillations in intracellular Ca2+ levels stimulated by thimerosal (same solution as that used in (a), recording performed on the same day) in EPCs isolated from mRCC patients. (c) Percentage of mRCC cells and EPCs responding to thimerosal. *P < 0.05 (Student's t-test). (d) 100 μM ATP stimulated InsP3-dependent Ca2+ mobilization from EPCs (black tracing), but not from mRCC cells (grey tracing). The same solution was used in both experiments and the recordings were conducted on the same day. (e) Fraction of mRCC cells and EPCs responding to ATP. *P < 0.05 (Student's t-test). (f) InsP3R transcripts in primary mRCC cells. *P < 0.05 versus InsP3R1, °P < 0.05 versus InsP3R2. The inset illustrates InsP3R expression detected by an InsP3R-I/II/III antibody. A major band that corresponds to the sum of the three single 313/260/250 kDa bands for InsP3R1/2/3 was found.

Fig 4: Working scheme of the modulatory roles of TMEM16A in myoblastIntracellular TMEM16A in myoblast modulates the Ca2+ release from ER through IP3R calcium channels to coordinate the processes of proliferation and myogenic differentiation. Cl- effects as counter ion by binding Ca2+ and modulates the calcium homeostasis in ER and global calcium transients. TMEM16A deletion causes the disturbance of ER calcium homeostasis and global calcium.

Fig 5: APP and a-synuclein converge on the IP3R1-Grp75-VDAC2 axis.A The sensitivity of hippocampal mitochondria to the calcium channel antagonists 2-APB and erastin was assessed. Ten milligrams of isolated hippocampal mitochondria from 3-month-old male rats were loaded with Fluo 4-AM and then incubated with 200 µM 2-APB or 20 µM erastin. With 16 µM calcium stimulation, mitochondria in which calcium channels were inhibited showed a smaller increase in fluorescence count than untreated controls. The percentages of net fluorescence increase in the antagonist-treated mitochondria relative to its untreated controls were determined. Three independent experiments including at least five rats per genotype were performed. Data from individual rats are indicated by dots, and the mean ± SEM for each genotype is presented. T-tests were used. *p < 0.05 and ***p < 0.001 indicate significant differences, n.s. indicates no significance. B Coimmunoprecipitation of Grp78 and FL-APP in hippocampal lysates from 3-month-old WT male rats. Anti-Grp78 antibody was used for immunoprecipitation, and Grp78 protein pulled down from the lysate was used as the loading control. Anti-APP antibody was used for western blotting. The stray bands are indicated by asterisks. C Comparison of the IP3R1-Grp78 interaction in the hippocampi of 3-month-old WT and APP KO male rats. An anti-Grp78 antibody was used for immunoprecipitation, and Grp78 protein pulled down from the lysate was used as the loading control. IP3R1 levels were measured by western blotting, and the stray bands are indicated by asterisks. D Three independent experiments in (C) including three rats per genotype were used for quantification. The mean ± SEM for each genotype is presented. T-test was used for statistical analysis, *p < 0.05 indicates a significant difference. E Interactions of VDAC2 with FL-APP and a-synuclein in the hippocampi of 3-month-old WT and SNCA KO male rats. A VDAC2 antibody was used for immunoprecipitation, and VDAC2 protein pulled down from the lysate was used as the loading control. FL-APP and a-synuclein were detected through western blotting. Immunoprecipitation was performed in SNCA KO hippocampal lysates as a negative control. The stray bands are indicated by asterisks. The arrowheads indicate the IgG heavy/light chain. Three independent experiments including 3 rats per genotype were performed. F Schematic diagram of the mechanism by which APP and a-synuclein regulate calcium flow via the IP3R1-Grp75-VDAC2 axis in MAMs. FL-APP interacts with Grp78 to disrupt the binding of Grp78 to IP3R1, prohibiting calcium flow released from the ER lumen into the MAM. Both FL-APP and a-synuclein interact with VDAC2 to promote calcium flow into the matrix. At OMMcyto, only a-synuclein binds to VDAC channels to regulate calcium flux. G Representative fluorescence traces showing mitochondrial calcium uptake after a series of extra calcium stimulations ranging from 0.75 mM to 12 mM. Ten milligrams of crude hippocampal mitochondria isolated from 3-month-old male rats and loaded with Fluo4-AM were used for the assay. The fluorescence count was measured every 30 s and six times before and after each stimulation. The average fluorescence count of unstimulated mitochondria of each genotype was used as an internal reference (F0), and all fluorescence measurements were divided by F0 for normalization (F1/F0). The F1/F0 value is plotted on the y-axis, and the detection time is plotted on the x-axis. The red arrow indicates the addition of CaCl2 buffer for stimulation. H Quantification of mitochondrial calcium release after 12 mM extra calcium stimulation. Three independent experiments including at least four male rats per genotype at 3 months old were performed as described in (G). The percentage of net reduction in the average F1/F0 value after 12 mM calcium stimulation relative to the plateau level of 3–6 mM calcium stimulation was determined. The data of individual rats are indicated by dots, and the mean ± SEM for each genotype is presented. T-tests were used for statistical analysis, and differences are indicated by asterisks. ***p < 0.001 indicates significant differences.