Figure 4

Knock-out of STIM1 expression by CRISPR/Cas9 D10A gene editing. Gene editing was performed in U2OS cells following the strategy presented in Supplementary Fig. 1. (a) STIM1 expression was assessed using antibodies against C-terminal and N-terminal epitopes. GAPDH was used as loading control. (b) Top panels. 1: wild-type cells were transfected for the transient expression of GFP-CTTN, and monitored for 10 min at 37 °C (image acquisition every 3 sec). Assay medium was Leibovitz’s L-15 + 10% FBS. 2: Orthogonal projections of the areas marked show the fluorescence intensity over the time (10 min) (kymograph). 3: GFP-CTTN fluorescence of the selected area in panel 2 was evaluated to assess membrane ruffling. 4: STICS analysis is shown as overlaid vectors on a pseudocolor scale. The figure is representative of 32 recordings from 4 independent assays. Full time-lapse is given in Supplementary Movie 3. Bottom panels. 1: STIM1-KO cells were transfected for the transient expression of GFP-CTTN, and monitored as indicated above (Panels 2–4). The figure is representative of 22 independent assays. Full time-lapse is given in Supplementary Movie 4. (c) Cell migration was evaluated by a wound-healing assay. Cell monolayer was scratched (time = 0), and the culture was extended for 14 hours in DMEM + 10% FBS. Quantitative image analysis was performed with ImageJ. Bar = 200 μm. Scatter plot depicts the mean of at least 28 independent images from 4 experiments. (d) STIM1-KO cells were transfected for transient expression of GFP-CTTN and STIM1-mCherry, and mCherry-positive cells were selected to monitor GFP fluorescence (CTTN) as in panel b. Assay medium was Leibovitz’s L-15 + 10% FBS. The figure is representative of 21 recordings from 3 independent assays. Full time-lapse is shown in the Supplementary Movie 5. (e) Cell migration of STIM1-KO cells transfected for the expression of STIM1-mCherry was assessed by a wound-healing assay. Control cells (wild-type or KO) were transfected for the expression of empty vector. The box-plot depicts data from 3 independent experiments (n = 70, wild-type; n = 60, STIM1-KO; n = 100, STIM1-KO + STIM1-mCherry). Statistical analysis was done using the unpaired t-test. ***p < 0.001.