1. Reciprocal Regulation of HIF-1α and LincRNA-p21 Modulates the Warburg Effect 
Fan Yang, Huafeng Zhang, Yide Mei, Mian Wu 
Molecular Cell 
Volume 53, Issue 1, Pages (January 2014)
DOI: /j.molcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2014 53, 88-100DOI: (10.1016/j.molcel.2013.11.004)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Human lncRNA LincRNA-p21 Mediates the Promoting Effect of Hypoxia on Glycolysis
(A) Real-time RT-PCR analysis of the indicated lncRNA levels in the presence or absence of hypoxia. HeLa cells were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions for 24 hr. Total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ±SD (n = 3). Hypoxic condition (hypoxia), if without specification, was hereafter defined as the condition of 1% O2.
(B) HeLa, MCF7, H1299, and IMR90 cells were cultured under normoxic or hypoxic conditions for 24 hr. Total RNA was then subjected to real-time RT-PCR analysis to examine lincRNA-p21 expression levels. Data shown are mean ±SD (n = 3).
(C) HeLa cells were cultured under the indicated conditions for 24 hr. Total RNA was then subjected to real-time RT-PCR analysis. Data shown are mean ±SD (n = 3). Cell lysates were also analyzed by western blot to examine expression levels of HIF-1α.
(D) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions for 24 hr. Acidification of the culture medium was evaluated by visually inspecting the color of the medium.
(E–G) HeLa (E), MCF7 (F), and H1299 (G) cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions for 24 hr. Levels of lactate in the culture medium were then measured and normalized to cell number. Data shown are mean ±SD (n = 3).
(H–J) HeLa (H), MCF7 (I), and H1299 (J) cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions for 24 hr. Intracellular glucose levels were then measured and normalized based on protein concentration. Data shown are mean ±SD (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
The Effect of LincRNA-p21 on Hypoxia-Increased Glycolysis Is HIF-1α Dependent
(A) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions for 24 hr. Enzymatic activity of LDHA was measured and normalized to total protein content. Shown data are mean ±SD (n = 3).
(B) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions for 24 hr. Cells were then subjected to immunofluorescence analysis.
(C) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions for 24 hr. Total RNA was extracted and subjected to real-time RT-PCR analysis to examine GLUT1 and LDHA mRNA levels. Data shown are mean ±SD (n = 3).
(D) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cotransfected with the indicated reporter constructs and Renilla luciferase plasmid. Twelve hours later, cells were cultured under normoxic or hypoxic conditions for an additional 24 hr. Reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Shown data are mean ±SD (n = 3).
(E) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cotransfected with the HRE reporter construct and Renilla luciferase plasmid. Twelve hours later, cells were cultured under normoxic or hypoxic conditions in the presence or absence of digoxin for an additional 24 hr. Reporter activity was then measured and plotted after normalizing with respect to Renilla luciferase activity. Shown data are mean ±SD (n = 3). The expression levels of HIF-1α are shown in Figure S2A.
(F) HeLa cells expressing control shRNA, lincRNA-p21 shRNA, HIF-1α shRNA, or both lincRNA-p21 and HIF-1α shRNAs were cotransfected with the HRE reporter construct and Renilla luciferase plasmid. Twelve hours later, cells were cultured under normoxic or hypoxic conditions for an additional 24 hr. Reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Shown data are mean ±SD (n = 3). The expression levels of HIF-1α are shown in Figure S2B.
(G) HeLa, H1299, and MCF7 cells expressing either control shRNA or lincRNA-p21-2 shRNA were cultured under normoxic or hypoxic conditions for 24 hr. Cell lysates were then analyzed by western blotting.
(H) HeLa cells expressing control shRNA, lincRNA-p21 shRNA, or lincRNA-p21 shRNA plus shRNA-resistant lincRNA-p21 were cultured under normoxic or hypoxic conditions for 24 hr before cell lysates were analyzed by western blotting.
(I and J) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were transfected with or without p3×Flag-myc-cmv-24-HIF-1α, as indicated. Twenty-four hours later, cells were cultured under normoxic or hypoxic conditions for an additional 24 hr. Lactate levels in the culture medium (I) and intracellular glucose levels (J) were then measured. Data shown are mean ±SD (n = 3).
(K) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were transfected with or without p3×Flag-myc-cmv-24-HIF-1α, as indicated. Twenty-four hours later, cells were cultured under normoxic or hypoxic conditions for an additional 24 hr. Total RNA and cell lysates were subjected to real-time RT-PCR and western blot analyses, respectively. Data shown are mean ±SD (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Hypoxia-Induced LincRNA-p21 Stabilizes HIF-1α
(A) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions for 24 hr. Total RNA was then subjected to real-time RT-PCR analysis. Data shown are mean ±SD (n = 3). The term “ns” indicates no significance.
(B) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were transfected with pcDNA-V5/His-HIF-1α. Twelve hours later, cells were cultured under normoxic or hypoxic conditions for an additional 24 hr before they were treated with cycloheximide (20 μg/ml) for the indicated periods of time. Cell lysates were then analyzed by western blotting.
(C) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions in the presence or absence of MG132 (5 μM) for 24 hr. Cell lysates were then analyzed by western blotting.
(D) HeLa cells expressing control shRNA, lincRNA-p21 shRNA, or both VHL and lincRNA-p21 shRNAs were cultured under normoxic or hypoxic conditions for 24 hr. Cell lysates were then analyzed by western blotting.
(E) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions in the presence or absence of MG132 (5 μM) for 24 hr. Cell lysates were then analyzed by western blotting.
(F and G) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under normoxic or hypoxic conditions in the presence of MG132 (5 μM) for 24 hr.
(F) Cell lysates were immunoprecipitated with either control IgG or anti-HIF-1α antibody. The immunoprecipitates and input were analyzed by western blotting.
(G) Cell lysates were immunoprecipitated with control IgG, anti-VHL, or anti-HIF-1α antibody. The immunoprecipitates and input were analyzed by western blotting.
(H) GST-VHL proteins bound with glutathione agarose beads were incubated with purified recombinant Flag-HIF-1α in the presence or absence of in vitro-transcribed lincRNA-p21 for 3 hr. The beads were then analyzed by western blotting.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 LincRNA-p21 Associates with HIF-1α and VHL
(A and B) HeLa cells were cultured under hypoxic conditions for 24 hr. Cells were then crosslinked with or without 1% formaldehyde before IP real-time RT-PCR assays were carried out.
(A) An isotype-matched IgG and anti-VHL antibody were used in IP real-time RT-PCR assays.
(B) Isotype-matched IgG and anti-HIF-1α antibody were used in IP real-time RT-PCR assays. Data shown are mean ±SD (n = 3).
(C) HeLa cells were cultured under normoxic or hypoxic conditions for 24 hr before IP real-time RT-PCR assays were carried out. Shown data are mean ±SD (n = 3).
(D) HeLa cells were cultured under normoxic or hypoxic conditions for 24 hr. Cell lysates were then incubated with in vitro-synthesized biotin-labeled sense or antisense lincRNA-p21 probes for biotin pull-down assay, followed by western blot analysis.
(E) HeLa cells were transfected with p3×Flag-myc-cmv-24-HIF-1α and pEGFP-C1-VHL. Twelve hours after transfection, cells were cultured under hypoxic conditions in the presence of MG132 (5 μM) for another 24 hr. Cell lysates were first immunoprecipitated with either anti-Flag antibody or an isotype-matched control IgG. Proteins in the immunoprecipitates were then eluted with 3× Flag peptides. Ten percent of the eluent was boiled and analyzed by western blotting. Ten percent of the eluent was subjected to real-time RT-PCR analysis. Eighty percent of the eluent was further immunoprecipitated with either control IgG or anti-GFP antibody. The immunoprecipitates were then washed. After elution, 10% of the eluent was analyzed by western blotting. Ninety percent of the eluent was used for real-time RT-PCR analysis. Shown data are mean ±SD (n = 3). ND indicates that lincRNA-p21 in the second immunoprecipitates was not detectable.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 LincRNA-p21 Is a Direct Target of HIF-1α
(A and B) HeLa cells expressing control shRNA, HIF-1α shRNA, HIF-2α shRNA, or p53 shRNA were cultured under normoxic or hypoxic conditions for 24 hr.
(A) Total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ±SD (n = 3).
(B) Cell lysates were analyzed by western blotting.
(C) HeLa cells were transfected with 0.1 μg, 0.5 μg, or 2 μg pcDNA-V5/His-HIF-1α. Twenty-four hours after transfection, cell lysates and total RNA were subjected to western blot and real-time RT-PCR analyses, respectively. Data shown are mean ±SD (n = 3).
(D) HeLa were treated with hypoxia, CoCl2 (100 μM), or DMOG (100 μM) for 24 hr. Cells were then harvested for real-time RT-PCR and western blot analyses. Data shown are mean ±SD (n = 3).
(E) Schematic illustration of consensus HIF-1α responsive element in lincRNA-p21 gene promoter.
(F) HeLa cells were cultured under normoxic or hypoxic conditions for 24 hr. Lysates were then subjected to ChIP assay. ChIP products were amplified by PCR reaction.
(G and H) HeLa cells were cotransfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, cells were cultured under normoxic or hypoxic conditions for 24 hr. Reporter activity was then measured and plotted after normalizing with respect to Renilla luciferase activity (mean ±SD).
(I and J) HeLa cells expressing control shRNA, HIF-1α shRNA-1, or HIF-1α shRNA-2 were cotransfected with the HRE-WT reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, cells were cultured under normoxic or hypoxic conditions for 24 hr. Reporter activity was then measured and plotted after normalizing with respect to Renilla luciferase activity (mean ±SD).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
LincRNA-p21 Functions as an Oncogene to Promote Tumor Formation
(A and B) MEF cells expressing either control shRNA or mlincRNA-p21 shRNA were treated with the indicated concentration of doxorubicin for 24 hr.
(A) Cell lysates were then analyzed by western blotting.
(B) Viability of cells was measured by the MTT assay. Shown data are mean ±SD (n = 3). The expression levels of mlincRNA-p21 are shown in Figure S5A.
(C) MEF cells expressing either control shRNA or mlincRNA-p21 shRNA were treated with 1 μg/ml doxorubicin for 24 hr. Cells were then stained with annexin-FITC. Annexin V-FITC-positive cells were counted as apoptotic cells. Shown data are mean ±SD (n = 3).
(D–F) HeLa cells expressing either control shRNA or the indicated lincRNA-p21 shRNA were treated with 1 μg/ml doxorubicin for 24 hr.
(D) Cell lysates were then analyzed by western blotting with the indicated antibodies.
(E) Viability of cells was measured by the MTT assay. Shown data are mean ±SD (n = 3).
(F) Cells were stained with annexin-FITC. Annexin V-FITC-positive cells were counted as apoptotic cells. Shown data are mean ±SD (n = 3). The expression levels of lincRNA-p21 are shown in Figure S5B.
(G and H) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were cultured under hypoxic condition for 36 hr. A total of 2 × 106 cells were then injected subcutaneously into nude mice (n = 7 for each group).
(G) Representative photographs of xenografts were taken 3 weeks after injection.
(H) Weights of the xenografts harvested 3 weeks after injection were also shown (mean ±SD) (n = 7).
(I) Lysates from tumors excised from the indicated mice 3 weeks after injection were analyzed by western blotting.
(J) A schematic illustration of the proposed model depicting a positive feedback loop between HIF-1α and lincRNA-p21 that promotes glycolysis under hypoxia.
Molecular Cell  , DOI: ( /j.molcel )
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