Skip to main content
. Author manuscript; available in PMC: 2015 Nov 1.
Published in final edited form as: Biochim Biophys Acta. 2014 Oct 2;1839(11):1330–1340. doi: 10.1016/j.bbagrm.2014.09.015

Fig. 4.

Fig. 4

NF-Y regulation of PRMT5 expression is required for prostate cancer cell growth. (A and B) Knockdown of NF-YA inhibits cell growth in LNCaP (A) and A549 (B). Stable cell lines SC and shYA#1 were induced with 1 μg/ml of doxycycline (Dox+) to express shRNAs or without treatment (Dox-) for the indicated times, and cell numbers were counted using haemocytometer. Results from 4 independent experiments in duplicate are presented as mean ± SEM. Statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001) was determined by two-way ANOVA followed by Tukey’s test. (C and D) Knockdown of NF-YA decreases BrdU-incorporated positive cells in LNCaP and A549 cells. SC and shYA#1 stable cell lines were induced with 1 μg/ml of Dox (Dox+) or without treatment (Dox-) for 84 hours, followed by BrdU treatment for another 8 hours. Number of BrdU-positive cells was determined using Image J software (total cell number > 1000, n=10). Results obtained from 4 independent experiments in duplicate are presented as mean ± SEM. Statistical significance (**, p<0.01; ***, p<0.001) when compared with SC was determined by two-way ANOVA followed by Dunnett’s test. (E and F) Effect of NF-YA knockdown on cell death. Stable and inducible cell lines targeting NF-YA (shYA#1) or the SC control were cultured in 6 cm dishes, and induced with 1 μg/ml of Dox (Dox+) or without treatment (Dox−) for 72 hours. Cells were trypsinized and counted to determine the percentage of dead cells by Trypan blue exclusion method. (G and H) Overexpression of PRMT5 rescues cell growth inhibition induced by NF-YA knockdown in LNCaP cells, but not in A549. LNCaP and A549 stable cell lines expressing shYA#1 were induced with 1 μg/ml of Dox (Dox+) or without induction (Dox−) for 48 hours, followed by transient transfection with pCMV-Myc (Myc-vector) or pCMV-Myc-PRMT5 (Myc-PRMT5) and incubation for another 48 hours. Top, results are presented as mean ± SEM from three independent experiments. Statistical significance was determined by two-way ANOVA followed by Tukey’s test. *, p<0.05; n.s., no significance. Bottom, the expression level of PRMT5 and NF-YA was determined by immunoblotting analysis. Shown are representative blots from three independent experiments. Note that the expression of both NF-YA longer isoform (NF-YA-L) and shorter isoform (NF-YA-S) was detectable in A549 cells whereas the expression of NF-YA-S was detectable in LNCaP cells only.