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. 2015 Mar 16;125(4):1648–1664. doi: 10.1172/JCI74440

Figure 2. Validated high-confidence hits affect cell migration in association with modulated FA dynamics.

Figure 2

(A) Sixty-four selected genes were tested in a deconvolution PKT screen with 4 single siRNAs per gene. Validated high-confidence genes (confirmed effect in primary screen in at least 3 of 4 single siRNAs and SMARTpool) are highlighted in yellow (increased compared with control) or blue (decreased). (B) Hits for each parameter were selected and tested in a live cell imaging–based migration assay. Stills from a representative movie chosen from 2 independent experiments with each 3 replicates per siRNA are shown. Scale bar: 100 μm. (C) FA dynamics was studied using TIRF imaging of H1299 cells expressing GFP-paxillin (PXN). In each experiment (mock n = 12, siGFP n = 10, siMEK2 n = 3, siSRPK1 n = 12, and siIKBKE n = 2) at least 10 cells were imaged, and representative images are shown. Decreased cell migration after knockdown of SRPK1 and MAP2K2 was related to increased stabilization of FA as indicated by the presence of “white” FAs in the time color overlay. Knockdown of IKBKE, affecting minor axis, does not affect cell migration per se, but resulted in increased cell area, reflecting the increase in minor axis in the PKT screen. Scale bar: 20 μm.