(
A) HCT116 cells were individually infected with lentiviruses expressing
LAST shRNA-1,–2 and non-targeting control shRNA. Ninety-six hours after infection, cells were stained with PI, followed by flow cytometric analysis of the cell cycle phase distribution. (
B) The percentage numbers of cells from (
A) in the G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are the mean ± SD (n = 3; *p<0.05, **p<0.01, two-tailed t-test). (
C) HCT116 cells were infected with lentiviruses expressing control shRNA,
LAST shRNA-1 or −2. Ninety-six hours after infection, total RNA was extracted and the transcript levels of
DHCR7 and
NADSYN1 were analyzed by real-time RT-PCR. Data shown are the mean ± SD (n = 3; two-tailed t-test). Cell lysates were analyzed by western blotting to examine actin, DHCR7 and NADSYN1 expression. (
D) HCT116 cells expressing control shRNA or
LAST shRNA-1 were separately transfected with either mock control or shRNA-resistant
LAST. Twenty-four hours after transfection, total extracted RNA was subjected to real-time RT-PCR to analyze the mRNA level of
CCND1. Data shown are the mean ± SD (n = 3; **p<0.01, two-tailed t-test). (
E) Cell lysates from cells treated as described in
Figure 2—figure supplement 1B were analyzed by western blotting to detect the cyclin D1 protein level.