Rab18 binding is required for KNT1 localization at the peripheral tubular ER. (a) U2OS cells transfected with siRNA control or siRNA against Rab18, or depleted for Rab18 and subsequently transfected with GFP-Rab18, were fixed and stained with DAPI and anti-KNT1 antibody. The insets show magnifications of the boxed areas and illustrate examples of KNT1 distribution under the different conditions. The inset shows the GFP channel for the sample transfected with GFP-Rab18. Images represent maximum-intensity projections from Z-stacks. Scale bar: 20 µm; inset: 10 µm. (b) Quantification of KNT1 intracellular distribution in control cells, cells knocked down for Rab18, and cells knocked down for Rab18 and transfected with GFP-Rab18. The graph represents the percentage of perinuclear KNT1 over the total. Data represent the mean ± SEM of three independent experiments (n > 60). *, P < 0.05; **, P < 0.01. (c) U2OS cells were transfected with control siRNA, siRNA against Rab18, or siRNA depleted for Rab18 and subsequently transfected with GFP-Rab18 or stably transfected with GFP-KNT1 FL or GFP-KNT1 Δ201–445. Cells were fixed and stained with anti-KNT1 and CLIMP-63 or RTN-3 antibody. Left: ER sheets area labeled by CLIMP-63 (top) and ER tubular area labeled by RTN-3 (bottom). Insets: GFP channel for the cells transfected with GFP-Rab18. Scale bar: 10 µm. (d and e) Quantification of KNT1 intracellular distribution in sheet (CLIMP-63; d) and tubule (RTN-3; e) area over the total. Data represent the mean ± SEM of three independent experiments (n > 60). *, P < 0.05; **, P < 0.01; ***, P < 0.001.