To study the impact of
MELK knockdown on tumor growth, mice were randomly sorted into groups on the second day of injection, and were untreated or treated with doxycycline (2 mg / ml in 5 % dextrose in drinking water, refreshed twice a week) for the duration of the study.
Cell death events were dramatically increased upon
MELK knockdown (5 out of 235 cells in control, 151 out of 317 in doxycycline - treated cells during 10 hr of imaging).
(E)
MELK knockdown induces the accumulation of cells with 4n DNA content and G2 / M arrest.
(C) A caspase inhibitor prevents
MELK knockdown - induced cell death.
In contrast, in five luminal breast cell lines,
MELK knockdown with equivalent efficiency did not result in obvious inhibition on cell growth (Figure 5E, Figure 5 — figure supplement 1A, B).
(C and D) Effects of
MELK knockdown on established tumors arising from implantation of basal (C) or luminal (D) breast cancer cells.
Not exact matches
We found that the proliferation of MDA - MB - 468 cells expressing shMELK was restored, when the
MELK expression level in these cells was rescued by expression of a shMELK - resistant allele of
MELK (
MELK - R)(Figure 5F, Figure 5 — figure supplement 3), confirming that the effects of shRNA are due to the specific
knockdown of
MELK.
It could be that the degree of
knockdown of
MELK in the luminal cell lines did not go below a necessary threshold to cause a proliferation effect.
Reviewer # 1 also had concerns that the degree of
knockdown of
MELK by shMELK in the basal cell lines appears stronger than in the luminal cell lines, equivalent
knockdown needs to be demonstrated by both quantitative protein and mRNA analysis.