1.5 polymer coverslip

Cells were grown in a 35 mm µ-Dish with #1.5 polymer coverslip (ibidi, cat. no. 81156) the day before transfection. Cells were transfected with plasmids expressing either PC1-CTT or mCherry-PKD1-eGFP, mito-BFP (Addgene 49151) for mitochondrial marker, or pEF.myc.ER-E2-Crimson for ER marker (Addgene 38770^{66 (link)}) by FuGENE® 6 Transfection Reagent (Promega Corporation E2691) following manufacturer’s instructions. For counterstaining of mitochondria, cells were incubated in 100 nM of MitoTracker® Deep Red FM (Thermo Fisher Scientific M22426) or MitoTracker® Green FM (Thermo Fisher Scientific M7514) for 30 min. For ER counterstaining, cells were incubated in 100 nM of ER-Tracker™ Blue-White DPX for 30 min. Images were captured with a Zeiss LSM700, LSM780 or LSM 880 with Airyscan confocal microscope using the Plan-Apochromat 63 ×/1.40 Oil DIC M27 lens. For mitochondrial network rescue experiments, Pkd1^ko/ko renal epithelial cells in suspension were transfected with plasmids expressing either hPC1-CTT-eGFP or hPC1-MTS-eGFP by FuGENE® HD Transfection Reagent (Promega Corporation E2311) the day before assay. Images were acquired with a Zeiss LSM780 using the Plan-Apochromat 63 ×/1.40 Oil DIC M27 lens. A total of two to four independent experiments analyzing three independent mutant cell lines were used.