G cepia1er

Measurements of [Ca²⁺]_mito and [Ca²⁺]_ER in HeLa single cells were performed as described previously^{51 (link),52 (link)}, using the genetically-encoded Ca²⁺ indicators CEPIA3mt (Addgene ID 58219) and G-CEPIA1er (Addgene ID 58215), respectively, which were developed by Dr. M. Iino (The University of Tokyo, Japan)^{53 (link)}. The constructs were introduced into HeLa cells utilising the X-tremeGENE HP DNA transfection reagent (Roche, Mannheim, Germany) according to the manufacturer’s protocol. The [Ca²⁺] measurements were performed 48 h after transfection using a Zeiss Axio Observer Z1 Inverted Microscope equipped with a 20 × air objective and a high-speed digital camera (Axiocam Hsm, Zeiss, Jena, Germany). Changes in fluorescence were monitored in the GFP channel (Ex 480 nm, Em 520 nm). Extracellular Ca²⁺ was chelated with 3 mM EGTA, and PXA (10 μM) or thapsigargin (1 μM) were added as indicated on the figures. All traces were normalised (F/F₀) where F₀ is the starting fluorescence of each trace.

To monitor ER [Ca²⁺] ([Ca²⁺]_ER) or mitochondrial [Ca²⁺] ([Ca²⁺]_MITO) simultaneously with cytosolic [Ca²⁺]_i, mpkCCD cells were transfected with either ER Ca²⁺ biosensor R-CEPIA1er (Addgene Plasmid #58216, λ_ex: 543 nm, λ_em: 560–600 nm) or mitochondrial Ca²⁺ biosensor mito-RCaMP1h (Addgene Plasmid #105013, λ_ex: 543 nm, λ_em: 560–600 nm) (Suzuki et al., 2014 (link)). Cells were seeded at 6 × 10⁴ cells/cm² on collagen coated glass bottom dishes for 24 h before transfection. Cells were transfected with Lipofectamine (0.5 µg DNA/1 × 10⁵ cells) for 24 h according to manufacturer’s instruction. Studies were performed in transfected cells from 48 to 72 h after transfection. Cytosolic [Ca²⁺]_i was monitored simultaneously with cell permeant Ca²⁺ sensitive fluorescence probe (Cal-520/AM) in the transfected cells incubating with phenol red free medium (1:1 mixture of DMEM/Ham’s F12 medium, Gibco). To monitor ER-mitochondrial Ca²⁺ transfer in mpkCCD cells, cells were co-transfected with the ER Ca²⁺ biosensor (G-CEPIA1er, Addgene Plasmid #58215, λ_ex: 488 nm, λ_em: 510–540 nm) and mitochondrial biosensor mito-RCaMP1h. Fluorescent images were collected with the respective laser lines for excitation and spectral windows for emission using the Lecia TCS SP5 imaging system.

GCaMP5G (Addgene plasmid #31788), GCaMP6s (Addgene plasmid #40753), and G-CEPIA1er (Addgene plasmid #105012) were cloned into pLVX-Puro. RGECO1.2 (Addgene plasmid #45494), GCaMP6s was cloned into pLVX-IRES-Hygro. Lentivirus vectors for the GECI constructs were packaged in HEK293T cells as previously described^{23 (link)} or produced commercially (Cyagen Biosciences, Inc.). Production of the MA104-GCaMP5G cell line was previously described and similar methods were used to generate MA104-GCaMP6s, MA104-RGECO1/GCEPIAer and the MA104-GCaMP6s-shRNA lines^{23 (link)}. J3-HIEs stably expressing GCaMP6s (G6S-HIEs) were created using lentivirus transduction as described previously and grown in high Wnt3a CMGF+ with 1 µg/mL puromycin for selection³⁶ . Proper GECI functionality was validated by responses to 50 μM ATP.