Figure 4.

SP-shRNAs reduce expression of endogenous and transfected SP in PC12 cells and hippocampal neurons. A, B, For knocking down SP expression, three different shRNA sequences containing 19 nt corresponding to the rat synaptopodin gene were cloned into the pSUPER vector. One shRNA (variant 1) was targeting to the cds, the other two shRNAs (variant 2 and 3) were targeting 3′ untranslated regions (3′-UTRs) of rat synaptopodin. For control, a 19 nt scrambled sequence with no significant homology to any other mammalian gene was used. C, All constructed shRNAs significantly reduced endogenous SP expression in PC12 cells as demonstrated by Western blot analysis. D, shRNA variant 1 was also effective in reducing transfected SP (expressing cds only), whereas the 3′UTR-shRNAs (variants 2 and 3) did not affect cotransfected SP. E, Similar results could be obtained in hippocampal neurons. All transfected shRNAs significantly reduced the number of SP(+) spines [p < 0.001; arrowhead point to SP(+) spines]. The attempt to rescue the effects of shRNAs with cotransfected SP demonstrated results comparable to those seen in PC12 cells; only shRNA variant 1 could not be rescued by cotransfected SP (p = 0.36). Scale bar, 2 μm. F, Spine head size (p > 0.31), spine length (p > 0.78), and spine density (p > 0.7) were not different [n = 10 cells transfected with a scrambled control sequence, n = 31 shRNA-transfected cells, n = 11 cells cotransfected with SP(cds)].