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PNAS 110 (1): 157-162

Copyright © 2013 by the National Academy of Sciences.

Long-lived microRNA–Argonaute complexes in quiescent cells can be activated to regulate mitogenic responses

Scott H. Olejniczaka,1, Gaspare La Roccaa,1, Joshua J. Gruberb, and Craig B. Thompsona,2

aDepartment of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; and bAbramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104


Figure 01
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Fig. 1. Stable siRNAs repress Ars2 and proliferation upon growth-factor restimulation of quiescent cells. (A) Schematic depicting experiments performed to test Ars2 function over long-term growth-factor withdrawal and restimulation. (B) Proliferation of IL-3 DKO cells transfected with two independent Ars2 siRNAs or control siRNA and then withdrawn from IL-3 for 0, 1, 2, or 3 wk followed by 1–2 wk of IL-3 restimulation. Arrows indicate time points at which IL-3 was added to cells (circle, no withdrawal; diamond, 1 wk IL-3 withdrawal; square, 2 wk IL-3 withdrawal; and triangle, 3 wk IL-3 withdrawal). A representative experiment is shown and has been repeated more than three times. (C) Expression of Srrt mRNA, which codes for Ars2 protein, in IL-3 DKO cells transfected with control or Ars2 siRNAs and then withdrawn from IL-3 for 1, 2, or 3 wk followed by 3 d of IL-3 restimulation. Bars represent expression relative to IL-3 DKO cells immediately following transfection (RQ) measured by qPCR in quadruplicate using the {Delta}{Delta}Ct method ±95% confidence interval of the mean. Eif2c2 was used as an endogenous control. (D) Expression of Ars2 protein in IL-3 DKO cells transfected with control or Ars2 siRNAs and then withdrawn from IL-3 for 2 wk (0 d of restimulation) followed by 4 or 6 d of IL-3 restimulation. Quantification of Ars2 protein (normalized to actin) relative to cells maintained in complete medium is shown.

 

Figure 02
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Fig. 2. Stabilization of microRNAs in quiescent cells. (A) Expression of mature miR-23a and miR-26a (Left) or the primary transcript containing miR-23a or miR-26a (Right) in IL-3 DKO cells maintained in the absence of IL-3 for 1, 2, or 3 weeks. Bars represent change in expression (RQ) relative to day 0 of withdrawal measured in quadruplicate using the {Delta}{Delta}Ct method ±95% confidence interval of the mean. U6 snRNA was used as an endogenous control for mature microRNAs and Eif2c2 was used as an endogenous control for primary transcripts. (B) Northern blot for mature miR-26 and U6 snRNA using RNA isolated from IL-3 DKO cells in the presence of IL-3 (+) or following 2 wk of IL-3 withdrawal (–). Either 10 μg of RNA per lane (Left) or 2 x 105 cell equivalents of RNA per lane (Right) was loaded on 15% urea-PAGE gels for Northern blot analysis. Blots were quantified using ImageJ software and relative band intensities are displayed below each blot.

 

Figure 03
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Fig. 3. Mitogenic stimuli promote assembly of high molecular weight Ago2-containing complexes. (A) Western blot analysis of Ago2 over the course of 14 d of IL-3 withdrawal followed by 6 d of IL-3 restimulation. (B) Superose 6 fractionation followed by Western blot for Ago2 on day 0 (Top) or day 14 (Middle) as in A. (C) Western blot analysis for GW182 in IL-3 DKO cells maintained in IL-3–replete medium or IL-3–deficient medium for 2 wk. (D) Western blot analysis for GW182 in resting splenic T cells or splenic T cells stimulated for 3 d with beads coated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 (30 units/mL). (E) Superose 6 fractionation followed by Western blot for Ago2 in resting splenic T cells or splenic T cells stimulated as in D. For B and E, fractions 1–7 were consistently devoid of protein and fractions 8–21 consistently contained cellular proteins, with fraction 8 containing the void of the column. Ago2 expression was quantified using ImageJ software and the relative amount of Ago2 in each fraction was plotted (Bottom).

 

Figure 04
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Fig. 4. Glucose availability determines microRNA stability and association of Ago2 with GW182. (A) Expression of primary (blue line) and mature miR-21 (red line) in DKO MEFs over the course of 5 d of glucose withdrawal followed by 2 d of glucose restimulation. Change in expression ±95% confidence interval of the mean relative to day 0 was determined using the {Delta}{Delta}Ct method with U6 snRNA or 18S rRNA for mature or primary transcripts, respectively, as endogenous controls. (B) Northern blot analysis of miR-21 in DKO MEFs treated with actinomycin D (5 µg/mL) in complete medium (Upper) or medium lacking glucose (Lower) over the course of 5 d. (C) Quantification of Northern blots from three independent experiments as in B. Each point represents the mean miR-21 signal normalized to 5S rRNA ±SD. (D) Western blot analysis of GW182 and Ago2 in DKO MEFs over the course of 5 d of glucose withdrawal followed by 2 d of glucose restimulation. (E) Superose 6 fractionation followed by Western blot for Ago2 or GW182 from lysates of MEFs maintained in glucose-free medium overnight. Ago2 and GW182 expression was quantified using ImageJ software and the relative amount of Ago2 and GW182 in each fraction was plotted (Lower).

 

Figure 05
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Fig. 5. CXCR4 siRNA is activated to repress microRNA and siRNA reporters following mitogenic stimulation of quiescent cells. (A) Schematic depicting experiments performed to test CXCR4 siRNA function over the course of glucose withdrawal and restimulation. (B) Superose 6 fractionation followed by Northern blot for CXCR4 siRNA from DKO MEFs transfected with CXCR4 siRNA followed by 5 d of glucose withdrawal (Upper) and 1 or 2 d of subsequent glucose restimulation (Lower). (C) Dual luciferase (DLR) assays performed on DKO MEFs transfected with control or CXCR4 siRNA before 5 d of glucose withdrawal and subsequent transfection with renilla luciferase reporters (24) containing a 3' UTR with one perfectly complementary CXCR4 siRNA binding site (perfect), six CXCR4 siRNA binding sites with imperfect complementarity (bulge), or three let-7 binding sites (N/S). Following transfection of reporters DKO MEFs were restimulated with glucose for 4–24 h before determination of luciferase activity. (D) DLR assays performed using the CXCR4 siRNA system described in C transfected into IL-3 DKO cells following 0, 2, or 3 wk of IL-3 withdrawal. Following transfection of reporters, IL-3 DKO cells were restimulated for 4–24 h before determination of luciferase activity. For C and D, bars represent mean normalized luciferase activity obtained from four independent cultures ±SD. pGL3 control vectors were cotransfected and luciferase activity was calculated as renilla luciferase RLU ÷ firefly luciferase RLU and normalized to control siRNA transfection.

 

Figure 06
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Fig. 6. Stable miR-21 represses PDCD4 and enhances AP-1 activity upon growth-factor restimulation. (A) Schematic depicting experiments performed to test miR-21 function over long-term growth-factor withdrawal and restimulation of IL-3 DKO cells. (B) Level of miR-21 following 2 wk of IL-3 withdrawal from IL-3 DKO cells transfected with control microRNA (blue bar) or miR-21 (red bar) (Left). Bars represent relative quantification (RQ) of mature miR-21 using the {Delta}{Delta}Ct method normalized to control microRNA transfection ±95% confidence interval of the mean. U6 snRNA was used as an endogenous control. Western blot for the miR-21 target PDCD4 following 2 wk of IL-3 withdrawal from IL-3 DKO cells transfected with control microRNA or miR-21 (Right). ImageJ software was used to quantify PDCD4 expression normalized to actin. (C) Western blot for PDCD4 in cells transfected with a control microRNA or miR-21 followed by 2 wk of IL-3 withdrawal (0 h) or at the indicated times following IL-3 restimulation. (D) Quantification of PDCD4 protein expression from three independent experiments, as in C, ±SD. ANOVA analysis of the time course of IL-3 restimulation revealed a significant (P < 0.005) decrease in the magnitude of PDCD4 induction in miR-21 transfected cells. (E) AP-1 activity measured 12 h following IL-3 restimulation, as in C, using a reporter containing 6 tandem AP-1 binding sites upstream of firefly luciferase. Bars represent the average AP-1 activity (firefly luciferase RLU ÷ renilla luciferase RLU) from three independent experiments performed in triplicate ±SD. *P < 0.0005 as determined by paired T test.

 


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