Effect of xanthohumol on serum cholesterol and CETP activity.

<p>Serum HDL-C concentration in the control group (closed circle), xanthohumol group (opened circle) and Chow group (closed triangle) of CETP-Tg mice (A), and in the control group (closed square) and xanthohumol group (opened square) of wild-type mice (B) over time. (C) Serum CETP activity after 18 weeks of treatment. (D) Correlation of serum CETP activity and HDL-C/T-Cho (%) in CETP-Tg mice fed HCD after 18 weeks. CE content of serum (E) and HDL-fraction (F) after 18 weeks. (N = 15; CETP-Tg mice control, N = 16; CETP-Tg mice xanthohumol, N = 10; CETP-Tg mice Chow, N = 3; wild-type mice control, N = 8; wild-type mice xanthohumol) Means±SEM. *<i>P</i><0.05, **<i>P</i><0.01.</p

c-Myc overexpression partially rescues RhoA suppression by miR-34a.

<p>(A) PC-3 cells were transiently transfected with pCMV6-ENTRY only or pCMV6-ENTRY-c-Myc for 8 h followed by transient transfection with pre-miR negative control (NC) or pre–miR-34a for 72 h. Protein level was analyzed by Western blot. (B) c-Myc expression partially rescues inhibition of invasion induced by miR-34a. PC3 cells were transiently transfected with pCMV6-ENTRY only (control) or pCMV6-ENTRY-c-Myc for 8 h followed by transfection with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and subjected to transwell invasion assay. *, P<0.05 compared with control. (C) Representative images of invaded cells.</p

Xanthohumol increased apoE protein expression in CETP-Tg mice after 18 weeks.

<p>(A) Serum (upper) and HDL-fraction (lower), and (B) liver tissue. Data are represented as relative expression: the CETP-Tg mice control group was set at 1.0. (N = 8; CETP-Tg mice control, N = 8; CETP-Tg mice xanthohumol, N = 3; CETP-Tg mice Chow, N = 6; wild-type mice control, N = 7; wild-type mice xanthohumol) Means±SEM. *<i>P</i><0.05, **<i>P</i><0.01.</p

Changes in cholesterol accumulation over 18 weeks.

<p>Data are presented as cholesterol amount in the aortic arch (A) and liver (B). (N = 15; CETP-Tg mice control, N = 18; CETP-Tg mice xanthohumol, N = 12; CETP-Tg mice Chow, N = 10; wild-type mice control, N = 7; wild-type mice xanthohumol) Means±SEM. *<i>P</i><0.05, **<i>P</i><0.01.</p

miR-34a inhibits cell growth.

<p>(A) Relative miR-34a expression in laser capture microdissected (LCM) prostate cancer tissues (C) and matched adjacent normal regions (N). (B) miR-34a overexpression suppresses soft agar colony formation of a stably transfected miR-34a PC-3 cell line. After 14 days incubation, colonies with over 50 cells were counted. The values are normalized to those of control. (C) Representative images of the colonies. (D) miR-34a inhibits <i>in vivo</i> tumor growth. Time course of tumor growth in nude mice after subcutaneous injection of a stably transfected miR-34a PC-3 cell line or control cell line. *, P<0.05 compared with control. (E) Representative images of tumors in nude mice at 5 weeks after subcutaneous injection of a stably transfected miR-34a PC-3 cell line or control cell line. (F) miR-34a induces apoptosis in PC-3 cells. PC-3 cells were transfected with pre-miR negative control (NC) or pre-miR-34a for 3 days. PC-3 cells were stained with AnnexinV-FITC/7-AAD and apoptosis was analyzed by flow cytometry. The data shows the percentage of early apoptotic and apoptotic cells out of the total cell population of PC-3 cells. *, P<0.05 compared with control.</p

Expression analyses in mice liver and ab. aorta.

<p>(A) SR-B1 and (B) LCAT protein expression in liver. Data was standardized for β-actin expression. (N = 15; CETP-Tg mice control, N = 18; CETP-Tg mice xanthohumol, N = 11; CETP-Tg mice Chow, N = 6; wild-type mice control, N = 8; wild-type mice xanthohumol) (C, D) Transcript analyses of liver (upper) and ab. aorta (lower) in CETP-Tg mice (C) and wild-type mice (D). (N = 12; CETP-Tg mice control, N = 13; CETP-Tg mice xanthohumol, N = 9 to 10; CETP-Tg mice Chow, N = 5; wild-type mice control, N = 7 to 8; wild-type mice xanthohumol) All data were standardized for GAPDH expression. Expression levels of control group (without xanthohumol) were set at 1.0. Means±SEM. *<i>P</i><0.05, **<i>P</i><0.01.</p

miR-34a suppresses the c-Myc-P-TEFb complex.

<p>(A) miR-34a suppresses the formation of the endogenous c-Myc-P-TEFb complex. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 72 h and total cell lysates were immunoprecipitated with c-Myc antibody or IgG (control), followed by Western blot analysis. (B) miR-34a suppresses CAD and NUC mRNA expression and DRB revereses the suppression. PC-3 cells were treated with 20 µM of DRB for 12 h and were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 48 h. CAD and NUC mRNA level was analyzed by real-time PCR.</p

c-Met overexpression partially rescues suppression of cell invasion by miR-34a.

<p>(A) PC-3 cells were transiently transfected with pCMV6-ENTRY only or pCMV6-ENTRY-c-Met for 8 h followed by transient transfection with pre-miR negative control (NC) or pre–miR-34a for 72 h. Protein level was analyzed by Western blot. (B) c-Met expression partially rescues inhibition of invasion induced by miR-34a. PC3 cells were transiently transfected with pCMV6-ENTRY only (control) or pCMV6-ENTRY-c-Met for 8 h followed by transfection with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and subjected to transwell invasion assay. *, P<0.05 compared with control. (C) Representative images of invaded cells.</p

miR-34a inhibits invasion and migration of PC-3 cells.

<p>(A) miR-34a inhibits invasion of PC-3 cells. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and subjected to transwell invasion assay. The values are normalized to those of NC. *, P<0.05 compared with control. (B) Representative images of the invaded cells. (C) miR-34a inhibits wound-healing of PC-3 cells. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and subjected to wound-healing assay. The width of the remaining open wound calculated in relation to separation at time 0 h. *, P<0.05 compared with control. (D) Representative images of the wound healing assay.</p

miR-34a suppresses the c-Myc transcriptional complex of RhoA.

<p>PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre-miR-34a for 72 h. (A) RhoA mRNA level was analyzed by real-time PCR. (B) RhoA protein expression was analyzed by Western blot. (C) c-Myc pulls down endogenous Miz1, Skp2 and Max in PC-3 cells. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 72 h and total cell lysates were immunoprecipitated with c-Myc antibody or IgG (control), followed by Western blot analysis. (D) miR-34a decreases the recruitment of c-Myc to the RhoA promoter. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 72 h and ChIP assays were performed. *, P<0.05 compared with control.</p