Fig. 1: Primary cilia inhibit necroptotic cell death in renal epithelial cells.

A Immunofluorescence staining of primary cilia in the mIMCD3 subclones Ckc (ciliated kidney cells) and Nckc (non-ciliated kidney cells; ARL13B (magenta), acetylated tubulin (green) and DAPI (blue); scale bar 20 µm). Quantification of cells carrying primary cilia (n = 3; total count of 404 cells for Ckc and 613 cells for Nckc). B Neutral-red assay in Ckc and Nckc cells after RCD induction with TNFα (TNF, 4 ng/100 µl) and birinapant (biri, 5 µM) for 16 h. Additionally, caspase-8 inhibitor emricasan (Em, 10 µM), Ripk1 inhibitor necrostatin-1s (Nec1s, 40 µM), and Ripk3 inhibitor GSK872 (GSK872, 5 µM) were used for 16 h (n = 4). C Neutral-red assay in Ckc and Nckc cells after RCD induction with TNFα (TNF, 4 ng/100 µl) and cycloheximide (CHX, 2 µg/100 µl) for 16 h. Additionally, caspase-8 inhibitor emricasan (Em, 10 µM) and necroptosis inhibitor necrostatin-1s (Nec1s, 40 µM) were used for 16 h (n = 4). D Immunoblot analysis of ciliated and non-ciliated cells using the apoptosis marker cleaved-Caspase-3 (~17 kDa) and the necroptosis marker phospho-Mlkl (~56 kDa). Either pan-actin (~44 kDa) or beta-tubulin (~55 kDa) were used as control (n = 3). E Live-cell imaging over the period of 24 h after treatment with TNF and CHX (TC), and TNF, CHX, and Em (TCE) or DMSO as control. Cells were stained with the dead cell marker DiYO-1. Images were captured every 2 h (n = 3).