To further exclude the possibility of "off-target" effects associated with RNAi, we recapitulated this result using a siRNA distinct from the Qiagen library siRNA (Fig. 1B) and two pharmacologic PARP1 inhibitors, 3-aminobenzamide (3-AB) and NU1025 (Fig. 1C).
(b) MDA-MB-231 cells previously silenced for calpain-1, using two distinct siRNA, and/or for calpain-2, and transiently expressing MYO10-GFP, were plated on FN for 2 h, fixed and the number of MYO10-positive filopodia per cell was quantified (n>62 cells, three biological repeats; ***P value<4.01 × 10−11).
Thus, when we set out to inhibit MMP-10 in the xenograft experiments, we targeted both human and mouse MMP-10 using distinct siRNA reagents.
These results are distinct from those observed using a similar siRNA approach in MDA-MB-231 or MCF-7 cells, which express low levels of PRLR LF.
(d) MDA-MB-231 cells previously silenced for talin-1 using two distinct siRNAs and transiently expressing MYO10-GFP were plated on FN for 2 h, fixed and the number of MYO10-positive filopodia per cell was quantified (n>65 cells, three biological repeats, ***P value<1.8 × 10−24).
Means and standard deviations from five independent experiments using two distinct siRNAs targeting SETD2 are shown.
Results were confirmed using second, distinct siRNAs or by Western immunoanalysis (Fig. S2A, B, & F).
Immunoblotting for Beclin-1 and α-tubulin (loading control) was provided in cell extracts silenced using a random siRNA (Randsi) or Beclin siRNA (Beclinsi).
Immunoblotting for Apaf1, caspase-9 and α-tubulin (loading control) was provided in cell extracts silenced using a random siRNA (Randsi) or Apaf1 siRNA (Apafsi).
siRNA-mediated downregulation of Ctnnb1, Eno1 or Pdia3 (ERp57) was performed using an siRNA pool of three target-specific siRNAs (Ctnnb1 siRNA, α-Enolase siRNA, ERp57 siRNA, Santa Cruz Biotechnology, Dallas, TX).
