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Fig. 8. Depletion of 4.1B by RNAi causes disruption of the F-actin cytoskeleton. (A) K2 cells injected with Cy3-labelled siRNA targeting rat Epb41l3 (R-epb41l3 siRNA) or control siRNA were fixed 48 hours later and labelled with Alexa Fluor-488–phalloidin. Cells with siRNA targeting rat Epb41l3 displayed a disrupted F-actin cytoskeleton, with fewer stress fibres and less total F-actin. Cells transfected with Cy3-labelled control siRNA were unchanged. (B) Similarly, HeLa cells transfected with three independent siRNAs targeting human EPB41L3 (H-epb41l3 siRNA1, H-epb41l3 siRNA2 and H-epb41l3 siRNA 3) exhibited fewer stress fibres after 48 hours of incubation. Some fine stress fibres remained, but the majority of cells had a disordered F-actin cytoskeleton and few F-actin; images are representative of five independent experiments. F-actin was labelled with Rhodamine-phalloidin. (C) Western blot confirming that HeLa cells transfected with three independent siRNAs targeting human EPB41L3 expressed reduced levels of 4.1B after 48 hours. Densitometric quantification showed that the actin-normalised presence of 4.1B was reduced to 44%, 18% and 28% when using H-epb41l3 siRNA1, H-epb41l3 siRNA2 and H-epb41l3 siRNA3, respectivley. (D) EPB41L3 siRNA-treated HeLa cells transfected with GFP cDNA as a control exhibited lack of stress fibres similarly to the experiments with siRNA on its own shown in B. EPB41L3 siRNA-treated HeLa cells subsequently transfected with an RNAi-resistant 4.1B cDNA recovered stress fibres and organised F-actin structures to similar levels observed in control siRNA treated cells shown in B. This rescue experiment confirms that the siRNAs were specific; images shown in D are representative of three independent experiments. F-actin was labelled with Rhodamine-phalloidin. (E) Quantitative analysis of HeLa cells with actin stress fibres showed that siRNA targeting human EPB41L3 resulted in a significant reduction, from half of the cells to less then a quarter on average (ANOVA ***P<0.001; the numbers of cells evaluated for control siRNA were 48 and 427 for siRNA targeting human EPB41L3). EPB41L3 siRNA-treated HeLa cells with and without GFP cDNA transfection had similar significantly reduced numbers of cells with actin stress fibres (ANOVA ***P<0.001; 216 siRNA-treated cells transfected with GFP cDNA were evaluated). EPB41L3 siRNA-treated HeLa cells transfected with an RNAi-resistant 4.1B cDNA did not have significantly changed number of cells with actin stress fibres compared with the control siRNA (n.s., no significance in ANOVA; 766 siRNA-treated cells transfected with RNAi-resistant 4.1B cDNA were evaluated). Cells that lost actin stress fibres exhibited disordered F-actin, with F-actin puncta and cortical F-actin organisation.
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