Ip3r1

VEGF-A165 was obtained from Sigma-Aldrich (St. Louis, MO; V5765). SiRNA for FUNDC1, IP3R1, VEGFR2, serum response factor (SRF), and controls were obtained from Santa Cruz Biotechnology (sc-36563, sc-91118, sc-42475, and sc-29318; Dallas, Texas). Antibodies against the following proteins were used as the primary antibodies: CD31 (BD Pharmingen, San Jose, CA; 550274; Cell Signaling Technology, Danvers, MA;77699); β-actin and GAPDH (Santa Cruz Biotechnology, Dallas, Texas; sc-47778 and sc-137179); PCNA, Calreticulin, Cyt C, VDAC1, mitofusin-2 (MFN2), SRF, phosphorylated (p) SRF, VEGFR1, VEGFR2, pVEGFR2, and VEGFR3 (Cell Signaling Technology, Danvers, MA; 13110, 12238, 4280,4661, 9482, 5147,4261, 2893, 2478, 2479, 3408); IP3R1 (Abcam, Cambridge, MA; ab5804; ThermoFisher, Waltham, MA, PA1-901); calnexin (Abcam, Cambridge, MA; ab22595); FUNDC1 (Aviva Systems Biology, San Diego, CA; ARP53281), FUNDC1 (Novus Biologicals, LLC, Littleton CO, NBP1-81063; EMD Millipore Corporation, Temecula, CA, ABC506).

Glucose, streptozocin (STZ), and Evans blue were purchased from Sigma-Aldrich (St. Louis, MO, USA), while advanced glycosylation end products (AGEs) were acquired from BioVision (Palo Alto, CA, USA). Tunicamycin (TUN) was obtained from Abcam (Cambridge, CA, USA), while 4-phenylbutyric acid (4-PBA), BAPTA-AM, and Protein A/G magnetic beads were purchased from MedChemExpress (Lowell, NJ, USA). Cell counting kit-8 (CCK8), JC-1 assay kit, Calcein/PI assay kit, MPTP assay kit, Mito-Tracker Green, ER-Tracker Red, and a mitochondria isolation kit were purchased from Beyotime (Nantong, China). On the other hand, the TUNEL assay kits were purchased from Roche (Basel, Switzerland). MitoSOX™ Red, DAPI, ECL kits, and goat anti-mouse/rabbit IgG (H+L, Alexa Fluor 555/488) were acquired from Thermo Fisher (Waltham, CA, USA). We used primary antibodies including GRP75, Cyt c, Bcl-xl, Bax, cleaved caspase-3, and CHOP (Cell Signaling Technology, Boston, MA, USA); 4-HNE and Brn3a (Abcam, Cambridge, CA, USA); IP3R1, VDAC1, VEGF, 8-OHDG, and 3-NT (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and COX IV, Bcl-2, and β-actin (Proteintech, Wuhan, China). More detailed information about the materials and reagents is offered in Table S1.

For the quantification of protein expression, we utilized a flow cytometry-based technique that incorporates the use of monoclonal primary antibodies and fluorescently conjugated secondary antibodies for protein detection. This technique has been validated for intracellular and cell surface proteins and is relatively high-throughput [42 (link), 44 ]. Moreover, it has comparative sensitivity and specificity to other methods of protein quantification [45 (link)]. Monoclonal primary antibodies; IP3R1 (Ms, Santa Cruz), IP3R3 (Rb, Santa Cruz), RYR1 (Rb, Millipore), RYR3 (Rb, Millipore) and S100A6 (Rb, Ab Cam) detected protein expression in SH-SY5Y and IMR-32 cells fluorescently stained with alexa-488 conjugated secondary Ab (Life Technologies). This was analyzed by using the BD LSR Fortessa (BD Medical Technology). Fluorescence signals were acquired using a 488 Laser (530/30) FITC filter and 640 laser with APC 660/40. To exclude duplets, aggregates and debris, FSC/SSC gating was applied to an unstained control sample and single stained control samples were used to adjust the voltage of the photomultiplier tubes (PMTs) to generate a signal free from background fluorescence. Finally, fluorescence intensity was measured with respect to the unstained and isotype control and the data were analyzed using BD FACS Diva 6.3.