Figure 1.
(A) Schematic representation of 5′-Bromo-uridine immunoprecipitation chase (BRIC). BrU is incorporated into newly transcribed RNA in place of uridine. Cells are collected and total RNA extracted at multiple time points after BrU washout. BrU-labeled RNAs are immunopurified using anti-BrdU beads. Non-labeled RNAs are discarded. Then, BrU-labeled RNAs are quantified by RT-qPCR. (B) The decay rate of GAS5 as determined by BRIC in HEK293T cells. Relative quantitative values at time 0 h were set to 100%. Values represent mean ± errors obtained from two independent experiments.
Figure 2.
Knockdown of UPF1 increases the expression level and prolongs the decay rate of GAS5, and decreases the expression of glucocorticoid-responsive genes.
HEK293T cells were treated with a control siRNA or with a siRNA targeting UPF1. (A) The expression levels of UPF1 in control cells (black bar) and in UPF1-depleted cells (grey bar) were determined by RT-qPCR. (B) The expression levels of GAS5 and MALAT1 in control cells (black bar) and in UPF1-depleted cells (grey bar) were determined by RT-qPCR. (C) The decay rates of GAS5 and MALAT1 were determined by BRIC in control cells (solid circle and black bar) and in UPF1-depleted cells (open circle and grey bar). Relative quantitative values at 0 h were set to 100%. (D) The expression levels of glucocorticoid-responsive genes, cIAP2 and SGK1, and the PPARδ-responsive gene, ADRP, in control cells (black bar) and in UPF1-depleted cells (grey bar) were determined by RT-qPCR. (A–D) The levels of GAPDH were used for normalization. Values represent mean±SD obtained from three independent experiments (*P<0.05, **P<0.01, Student's t test).
Figure 3.
Over-expression of GAS5 decreased the expression levels of glucocorticoid-responsive genes.
HEK293 cells were treated with plasmid vectors as indicated. The expression levels of the indicated RNAs in control cells with mock vector (black bar) and in cells over-expressing GAS5 (grey bar) were determined by RT-qPCR. The levels of GAPDH and ACTB were used for normalization. Values represent mean±SD obtained from three independent experiments (*P<0.05, **P<0.01, Student's t test).
Figure 4.
Serum starvation increases the expression level and prolongs the decay rate of GAS5, and decreases the expression of glucocorticoid-responsive genes.
HEK293 cells were cultured with 0, 0.5, and 10% serum as indicated. (A) The relative expression levels of GAS5 in cells with 0 and 0.5% serum relative to control cells (cultured with 10% serum) were determined by RT-qPCR. (B) The decay rates of GAS5 were determined using the Click-iT Nascent RNA Capture Kit (Invitrogen) in control cells (solid circle and black bar) and in serum-starved cells (open circle and grey bar). Relative quantitative values at 0 h were set to 100%. (C) The relative expression levels of glucocorticoid-responsive genes, cIAP2 and SGK1, and the PPARδ-responsive gene, ADRP, in cells with 0 and 0.5% serum relative to control cells (cultured with 10% serum) were determined by RT-qPCR. (A–C) The levels of GAPDH and ACTB were used for normalization. Values represent mean±SD obtained from two (in B) or three (in A and C) independent experiments (*P<0.05, **P<0.01, Student's t test).
Figure 5.
Knockdown of GAS5 suppresses the effects of serum withdrawal on the expression of glucocorticoid-responsive genes.
HEK293 cells were treated with a control siRNA or with a siRNA targeting GAS5, and cultured either with or without serum for 48 h. (A, B) The relative expression levels of the indicated RNAs in cells with serum (black bar) or without serum (grey bar) relative to control (cells cultured at 0 h) were determined by RT-qPCR. The levels of GAPDH and ACTB were used for normalization. Values represent mean±SD obtained from three independent experiments (*P<0.05, **P<0.01, Student's t test).