Fig. 4

TMIGD1 localizes to mitochondria and cell-cell contacts in HK-2 cells. a Sparsely grown HK-2 cells were fixed and stained with polyclonal antibodies against TMIGD1 (#HPA021946) and markers for mitochondria (MitoTracker), the trans-Golgi network (p230TG) and early endosomes (EEA1). Note that TMIGD1 localizes specifically to mitochondria. Scale bars: 5 μm. b HK-2 cells were grown as single cells or were grown and maintained at confluency for different periods of time, as indicated. Cells were fixed and stained with antibodies against TMIGD1 (#HPA021946) and MitoTracker (sparsely grown cells), or with antibodies against TMIGD1 (#HPA021946) and the cell-cell contact marker ZO-1 (cells grown to confluency). Right panels: Quantification of TMIGD1 signal intensities at cell-cell contacts (top bar graph) and of fraction of cells with TMIGD1-positive cell-cell contacts at different days of confluency (bottom bar graph). Quantification of TMIGD1 signal intensities at cell-cell contacts (top bar graph) was performed as described in the Methods section. Statistical analysis was performed using unpaired Student’s t-test. Data were obtained from three independent experiments and are presented as arithmetic means ± SEM; **P < 0.01, ***P < 0.001. Scale bars: 10 μm. c HK-2 cells stably transfected with the pInducer10-mir-RUP-PheS plasmid vector that allows expression of both a TMIGD1-specific shRNA and turboRFP (tRFP) under a doxycycline-regulated promoter were grown to low confluency (sparse) or high confluency (confluent), fixed with PFA and methanol, respectively, and analyzed by fluorescence microscopy with antibodies against TMIGD1 (#HPA021946) and the cell contact marker β-catenin, as indicated. In sparsely grown cells, the tRFP signal encoded by the pInducer10 vector was used as marker for shRNA-expressing cells. Note that the pInducer10 vector is based on a TetON system, i.e. shRNA expression is induced by doxycycline. Scale bars: 10 μm