Figure 4. KLF2 and statin mediated effects on WPB size.
(a) EGFP and EGFP-KLF2 overexpressing HUVECs. Scale bar, 10 μm; inset magnifications: 2 μm. (b) HTM analysis showing the fraction of the WPB area covered by long organelles in the subpopulations of EGFP or EGFP-KLF2 overexpressing HUVECs; ****P < 0.0001 (Mann-Whitney test). (c) HUVECs were treated with Luciferase or KLF2 targeting siRNAs and after 24 were incubated with DMSO (D), 2.5 μmol/L simvastatin (S) or 2.5 μmol/L fluvastatin (F) for 24 h before cell lysis. Equal protein amounts were analysed as described for Fig. 3d. Bars represent means ± SEM from 3 experiments. *P < 0.05; ***P < 0.001; ****P < 0.0001 (Student’s test). (d) HTM analysis of WPB size in Luciferase- and KLF2-siRNA treated HUVECs incubated with vehicle (DMSO) or 2.5 μmol/L simvastatin for 24 h. Cumulative frequency of WPB number as a function of size (note the right-shift of KLF2 curves in both conditions). The number of WPBs analysed was ~105 per condition. £, P < 10−15 for the indicated pairwise comparisons (Mann-Whitney test). (e) Left, the fraction of long WPBs (>2 μm) was expressed as % of the Luciferase-siRNA/DMSO controls. Bars, mean ± range for two replicate experiments. Right, the fraction of the WPB area covered by long organelles; ****P < 0.0001 (Mann-Whitney test); comparisons are with the Luciferase-siRNA/DMSO controls. (f) WPBs in Luciferase- and KLF2-siRNA treated cells after 24 h treatment with DMSO or 2.5 μmol/L simvastatin. Scale bar: 10 μm; inset magnifications: 2 μm. (g) HTM analysis of Golgi objects. The numbers of Golgi object analysed were similar to those in Fig. 2e,f. £, P < 10−15 (Mann-Whitney test). (h) Cells treated for 24 h with Luciferase- or KLF2-targeting siRNAs were then incubated for 24 h with 2.5 μmol/L simvastatin and processed for immunofluorescence. Scale bar: 20 μm.