Fig 1: RASSF1A promotes proliferation and glycolytic shift in hypoxic human PASMCs. Human PASMCs (a–c) were transfected with a RASSF1 siRNA (si-RASSF1) and control siRNA (si-Control) or c si-RASSF1 and HIF1α siRNA (si-HIF1A) in combination or b RASSF1A-FLAG and empty vector (EV). 24 h after transfection, cells were exposed to normoxia or hypoxia for 48 h and proliferation was measured by BrdU incorporation assay. Human PASMCs were transfected with d, e si-RASSF1 and si-Control or f, g RASSF1A-FLAG and EV. 24 h after transfection, cells were exposed to normoxia or hypoxia for 24 h. d, f Real time PCRs for indicated genes were performed. e, g Cell lysates were subjected to (left) western blotting, followed by (right) densitometric quantification of relative PDK1, LDHA and HK2 to ACTB expression. h HEK293 cells were transfected with RASSF1A-FLAG or EV and 24 h after transfection, exposed to normoxia or hypoxia for 24 h and intracellular lactate production was measured. *P < 0.05, **P < 0.01, ***P < 0.001 compared to a, c–e si-Control (hypoxia) or b, f–h EV (hypoxia), one-way ANOVA followed by SNK multiple comparison test. c §P < 0.01 compared to si-RASSF1, two-way ANOVA. n = 3 independent experiments from 3 biological replicates each; data represent mean ± s.e.m.
Fig 2: Pharmacological inhibition of YAP1 by dasatinib causes inhibition of ERα and FOXM1 expression similar to knockdown of YAP1. (A) Conditional RASSF1A MCF7 cells were cultured in the absence or presence of the indicated concentrations of dasatinib. After 16 h, cells were harvested and subsequent lysates were analyzed by immunoblotting using the indicated antibodies. (B) Dasatinib downregulates transcription of ERα and FOXM1 and causes decreased amounts of YAP1 protein. Mean values ± s.d. of three independent experiments are presented. p-Values < 0.05 are indicated by asterisks.
Fig 3: Expression and localization of RASSF1A during meiotic maturation in mouse oocytes. (A) Oocytes at GV, GVBD, MI, and MII stages were collected and subjected to immunoblot analysis with anti-RASSF1A antibody. β-actin was used as a loading control. Each lane contains 50 oocytes. Normalized expression of RASSF1A was quantified and expressed as the mean ± SEM from two independent experiments. (B) Immunostaining of RASSF1A during oocyte meiotic maturation. Oocytes at different stages were fixed and stained with anti-RASSF1A antibodies. DNA and spindle were stained with DAPI and anti-acetylated-α-tubulin antibody, respectively. Dashed line indicates oocyte cortex. Scale bar, 40 μM. (C) Oocytes at the MI stage were cultured in medium containing 10 μM taxol for 45 min or 20 μg/ml nocodazole for 10 min and then stained with anti-RASSF1A antibody. DNA and spindle were stained with DAPI and anti-acetylated-α-tubulin antibody, respectively. Dashed line indicates oocyte cortex. Scale bar, 40 μM.
Fig 4: ANRASSF1 interacts with PRC2 and affects its occupancy at the RASSF1A promoter.(A) Endogenous ANRASSF1 levels bound to PRC2 were measured in HeLa cells through RNA IP with anti-SUZ12 relative to the input. A control IP with non-specific IgG was performed in parallel. As a negative control, GAPDH mRNA, which was not expected to bind to PRC2, was used. The percent input in the IP fractions was shown as the ANRASSF1/GAPDH ratio. These data show the means ± SD from three independent experiments. (B) lincRNA SFPQ is a positive control that binds to PRC2; RNA IP was assayed as in (A). These data show the means ± SD from three independent experiments. (C) ChIP assay using an anti-SUZ12 antibody in HeLa cells overexpressing ANRASSF1 (pCEP4 ANRASSF1, black bars) or control cells (empty pCEP4, white bars). The promoter regions of the RASSF1A and RASSF1C isoforms and two other genes on either side of the RASSF1 locus on chr 3 were investigated (the promoters are indicated with vertical lines in the scheme shown at the bottom of the figure). Control GAPDH was included as a gene not expected to be regulated through SUZ12. Control HOXA9 is a gene regulated through SUZ12 [34] and encoded on chr 7. The amount of DNA in anti-SUZ12 samples at each promoter region detected through qPCR analysis was calculated in relation to the input. These data show the means ± SD from three independent experiments. *t-test p<0.02 relative to control at the RASSF1A locus. No significant changes were detected at other loci. (D) ChIP analysis using an anti-H3K27me3 antibody in an assay similar to that described in (C), except that the enrichment was calculated relative to anti-H3 ChIP. These data show the means ± SD from three independent experiments. *t-test p<0.02 relative to control at the RASSF1A locus. No significant changes were detected at the other loci. (E) ChIP analysis using an anti-DNMT3B antibody in an assay similar to that described in (C). These data show the means ± SD from three independent experiments. No significant change was observed. (F) DNA methylation at the RASSF1A promoter region was detected through qPCR with a methylation-dependent McrBC endonuclease assay in the ANRASSF1-overexpressing or control cells. The percentage of DNA remaining was calculated after comparing the amount of DNA amplified through qPCR following treatment with McrBC endonuclease with that following no-endonuclease treatment. These data show the means ± SD from three independent experiments. No significant change was observed.
Fig 5: Inverse correlation between ANRASSF1 and RASSF1A expression in non-tumor and tumor cell lines.Expression of ANRASSF1 and of RASSF1A in (A) breast and (B) prostate cell lines. Tumor cell lines (white bars) and non-tumor immortalized cell lines (hatched bars) were tested. These data show the means ± SD from two or three independent biological replicates for each cell line. The expression values in tumors are shown compared with the expression in the non-tumor cell line. These data are calculated relative to HPRT1 expression.
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