Journal: Science translational medicine

Article Title: Hyaluronan synthase 2–mediated hyaluronan production mediates Notch1 activation and liver fibrosis

doi: 10.1126/scitranslmed.aat9284

Figure Lengend Snippet: (A and B) Heat maps of genes related to Notch pathway. (A) RNA-seq for HSCs from Has2ΔHSC or HAS2 Tg mice. (B) RNA-seq for HSCs from WT, Cd44−/−, or Tlr4−/− mice. (C to F) Effect of Has2 or CD44 modulation on Notch1. (C) Liver (n = 8 per group), (C and D) HSCs (n = 3 to 4 per group), and (E and F) human HSC line LX-2 (n = 5 to 6). (G) ChIP assay (n = 4). IgG, immunoglobulin G. (H) NOTCH1 promoter activity in human embryonic kidney 293 A (HEK293A) (three replicates in each of four experiments). **P < 0.01 versus −1239/+1684 (WT) and mock-transfected; ##P < 0.01 versus −1239/+1684 (WT) and CD44-transfected. (I) qRT-PCR for Timp1 and Ccl2 (n = 3). Mouse HSCs were treated with vehicle (Con), LMW-HA, or LPS for 12 hours. **P < 0.01 versus WT-Con; ##P < 0.01 versus WT-LMW; N.S. versus WT-LPS. (J) BrdU incorporation assay (n = 9). (K) Matrigel invasion assay (n = 3). (L) Effect of Notch1 depletion on expression of fibrogenic mRNAs in HSCs (n = 3). (M) Sirius red staining and quantification of collagen deposition in liver sections from mice with HSC-specific Notch1 deletion 3 weeks after BDL (sham-operated, n = 3 per group; BDL-operated, n = 7 per group). Scale bar, 100 μm. (N) Sirius red staining in liver sections 2 weeks after BDL (WT, n = 10; Notch1ΔHSC, n = 9; ASMA-HAS2 Tg, n = 6; ASMA-HAS2 Tg/Notch1ΔHSC, n = 7). (C to N) Data are means ± SEM. (O) qRT-PCR (means ± SD) and (P) Pearson correlation coefficient analysis in liver tissue from patients with fibrosis and chronic hepatitis B. Two-tailed Student’s t test (C to F and L) and one-way ANOVA with Tukey’s post hoc analysis (G to K and M to O). *P < 0.05 and **P < 0.01.

Article Snippet: The human NOTCH1 promoter (−1239/+1) and customized human NOTCH1 promoter (−1239/+1684) vectors were purchased from GeneCopoeia (catalog no. HPRM48887-PG04).

Techniques: RNA Sequencing, Activity Assay, Transfection, Quantitative RT-PCR, BrdU Incorporation Assay, Invasion Assay, Expressing, Staining, Two Tailed Test

Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: Osimertinib drug‐tolerant persister (DTP) cells show resistance to osimertinib and the Notch pathway was activated in DTP cells. (A) PC‐9 and H1975 cells were treated with osimertinib for 72 h. Cell viability was assessed using MTT assays ( n = 3; mean ± SD). (B) DTP cells were isolated from three cell lines after treatment with 3 μmol/L osimertinib for 9 days. (C) Parental cells and osimertinib DTP cells were treated with osimertinib for 72 h and cell viability was assessed using the MTT assay. Three independent experiments were performed ( n = 3; mean ± SD). * p < 0.05. (D) Differential gene expression between samples of parental PC‐9 cells or PC‐9 DTP cells were calculated. The results are shown as a volcano plot. Upregulated NOTCH1 ‐responsive genes (marked in orange) as observed in RNA‐seq analyses are shown.

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Isolation, MTT Assay, Expressing, RNA Sequencing Assay

Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: Combined osimertinib and γ‐secretase inhibitor (GSI) suppresses the Notch pathway in drug‐tolerant persister (DTP) cells. (A) PC‐9, H1975, and HCC827 cells were incubated with vehicle, 3 μmol/L osimertinib, 1 μmol/L GSI, or a combination for 9 days. The medium was replenished every 72 h. The cells were lysed and the indicated proteins were detected by western blotting ( n = 3). (B) Relative mRNA expression of Notch1 , HES1 , and HEY1 was determined by qRT‐PCR ( n = 3; mean ± SD). * p < 0.05.

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Incubation, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: Notch1 knockdown inhibits the emergence of osimertinib drug‐tolerant persister (DTP) cells and suppresses phospho‐ERK in osimertinib DTP cells. (A) Stable PC‐9 and H1975 cells were generated by introducing short hairpin RNA (shRNA) inhibiting Notch1 expression (shNotch1) and control non‐targeting shRNA (shControl). Notch1 knockdown cells and shControl cells were treated with vehicle or 3 μmol/L osimertinib. The medium was replenished every 72 h for 28 days. Tumor cell counts were visually checked every 72 h and plotted over time ( n = 3; mean ± SD). (B, C) Notch1 knockdown cells and shControl cells were incubated with vehicle or 3 μmol/L osimertinib for 9 days. The medium was replenished every 72 h. The cells were lysed and the indicated proteins were detected by western blotting ( n = 3).

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Generated, shRNA, Expressing, Incubation, Western Blot

Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: The combination of osimertinib and GSI suppresses phospho‐ERK and the Notch pathway in vivo. (A, B) After subcutaneous injection of PC‐9 or H1975 cells into nude mice, vehicle (control), 5 mg/kg osimertinib, 3.3 mg/kg γ‐secretase inhibitor (GSI), or osimertinib plus GSI was administered for 9 days. The cells were lysed and the indicated proteins were detected by western blotting ( n = 3). (C) Relative mRNA expression of Notch1 , HES1 , and HEY1 was determined by qRT‐PCR ( n = 3; mean ± SD). * p < 0.05.

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: In Vivo, Injection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Notch pathway regulates osimertinib drug‐tolerant persistence in EGFR ‐mutated non–small‐cell lung cancer

doi: 10.1111/cas.15674

Figure Lengend Snippet: Notch1 or HES1 expression is enhanced after EGFR tyrosine kinase inhibitor (TKI) treatment in human tumor tissues from EGFR ‐mutated patients with non–small‐cell lung cancer (NSCLC). (A, B) Immunohistochemical staining of Notch1 and HES1 in NSCLC tissues. Notch1 expression was detected in the cytoplasm and/or nucleus (scale bars, 50 μm). HES1 expression was observed in the nucleus (scale bars, 50 μm). Notch1 and HES1 levels before and after EGFR‐TKI treatment were compared using paired t ‐tests in patients with lung adenocarcinoma. Line graphs show changes in Notch1 and HES1 protein expression after EGFR‐TKI treatment. Each line represents one patient. Red lines represent patients who showed increased expression levels after therapy, whereas black lines represent patients who did not show increased expression levels after therapy.

Article Snippet: Plasmids encoding human Notch1 shRNA, empty vector (pGFP‐V‐RS), and TurboFectin 8.0 transfection reagent, were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: The Journal of Experimental Medicine

Article Title: Leukemia-specific delivery of mutant NOTCH1 targeted therapy

doi: 10.1084/jem.20151778

Figure Lengend Snippet: Thap-OH demonstrates anti-NOTCH1 and antileukemia properties in T-ALL in vitro. (A) Effect of Thap-OH on SERCA binding. Lysates from T-ALL cells (ALL/SIL) were cotreated with the indicated concentrations of biotinylated thapsigargin or Thap-OH for 6 h and subjected to streptavidin pull-down for 24 h. The immunoblot was stained with SERCA2 and SERCA3 antibodies. (B) Effect of 24 h of Thap-OH treatment on NOTCH1 cell-surface staining as assessed by flow cytometry. (C) Effect of Thap-OH treatment for 24 h on NOTCH1 (N1) processing and activation in T-ALL cell lines all with heterodimerization mutations (DND41 and ALL/SIL (L1594PΔPEST), PF382 (L1575PΔPEST), and RPMI 8402 (ins1584PVELMPPE). The blot was stained with an antibody against the C terminus of NOTCH1 that recognizes both the furin-processed NOTCH1 transmembrane subunit (TM) and the unprocessed NOTCH1 precursor (FL). The immunoblot was also stained with anti-ICN1 antibody (Val1744) and GAPDH as a loading control. (D) Effect of Thap-OH treatment on cell viability after 72 h of treatment in NOTCH1 mutated T-ALL cells (ALL/SIL, DND41, PF382, RPMI 8402) or wild type (LOUCY, MOLT16, SUPT11). Statistical significance for mutated versus wild type (*, P ≤ 0.05; **, P ≤ 0.01) was determined by one-way ANOVA with Bonferroni’s correction for multiple comparison testing. Error bars denote the mean ± SD of four NOTCH1 mutated T-ALL cell lines (ALL/SIL, DND41, PF382, and RPMI 8402) and three NOTCH1 wild-type T-ALL cell lines (LOUCY, MOLT16, and SUPT11). (E) Effect of Thap-OH treatment (24 h) on processing of NOTCH1 mutant (ALL/SIL) or wild-type (LOUCY, MOLT16) NOTCH1 receptors. NOTCH1 (N1) was detected with an antibody against the C terminus of NOTCH1 that recognizes the furin-processed NOTCH1 transmembrane subunit (TM) and the unprocessed NOTCH1 precursor (FL). GAPDH was used as loading control. (F) Effect of Thap-OH treatment (6 and 12 h) on wild-type NOTCH1 (Loucy, MOLT16) cell surface staining as assessed by flow cytometry. (G) Effect of Thap-OH treatment (6 and 12 h) on mutated NOTCH1 (PF382, RPMI 8402) cell surface staining as assessed by flow cytometry.

Article Snippet: Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described ( ).

Techniques: In Vitro, Binding Assay, Western Blot, Staining, Flow Cytometry, Activation Assay, Control, Comparison, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: Leukemia-specific delivery of mutant NOTCH1 targeted therapy

doi: 10.1084/jem.20151778

Figure Lengend Snippet: JQ-FT demonstrates anti-NOTCH1 properties. (A) Effect of JQ-FT treatment (24 h) on NOTCH1 (N1) processing and activation in T-ALL cell lines all with heterodimerization mutations. The blot was stained with an antibody against the C terminus of NOTCH1 that recognizes both the furin-processed NOTCH1 transmembrane subunit (TM) and the unprocessed NOTCH1 precursor (FL). The immunoblot was also stained with anti-ICN1 antibody. GAPDH was used as a loading control. (B) Effect of 24-h JQ-FT treatment on NOTCH1 cell surface staining as assessed by flow cytometry. (C) Mean expression of NOTCH1 target genes in T-ALL cells (PF382, RPMI 8402) treated for 24 h with the indicated concentrations of thapsigargin, JQ-FT, Thap-OH, FA, or the γ-secretase inhibitor compound E was determined by quantitative RT-PCR. Error bars indicate the mean ± SD of four replicates. Data were analyzed using the ΔΔCT method and plotted as a percentage relative to the control gene RPL13A . Statistical significance among groups for treated versus vehicle (DMSO) samples (****, P ≤ 0.0001) was determined by one-way ANOVA with Bonferroni’s correction for multiple comparison testing. (D) Effect of JQ-FT and Thap-OH treatment (24 h) on NOTCH1 (N1) processing and activation in T-ALL cell lines all with heterodimerization mutations in the presence or absence of excess of FA (10 µM). The blot was stained with an antibody against the C terminus of NOTCH1 that recognizes both TM and FL. The immunoblot was also stained with anti-ICN1 antibody. GAPDH was used as a loading control. (E) Effect of 24 h of JQ-FT and Thap-OH treatment in the presence or absence of excess of FA (10 µM) on NOTCH1 cell-surface staining as assessed by flow cytometry. (F) Effect of JQ-FT on the growth of MigR1 or MigR1-ICN1–transduced RPMI 8402 cells. Normalized data are plotted relative to vehicle. Error bars indicate mean ± SD of four replicates. Statistical significance for treated versus vehicle (DMSO; ****, P ≤ 0.0001) was determined by one-way ANOVA with Bonferroni’s correction for multiple comparison testing.

Article Snippet: Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described ( ).

Techniques: Activation Assay, Staining, Western Blot, Control, Flow Cytometry, Expressing, Quantitative RT-PCR, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Leukemia-specific delivery of mutant NOTCH1 targeted therapy

doi: 10.1084/jem.20151778

Figure Lengend Snippet: JQ-FT demonstrates anti-NOTCH1 properties in PDX cells. (A) Effect of JQ-FT treatment on cell viability in PDX T-ALL cells ex vivo. Errors bars denote mean ± SD of four replicates. (B) Effect of JQ-FT treatment (24 h) on NOTCH1 processing in PDX cells in vitro. The blot was stained with an antibody against the C terminus of NOTCH1 (N1) that recognizes both the furin-processed NOTCH1 transmembrane subunit (TM) and the unprocessed NOTCH1 precursor (FL). GAPDH was used as a loading control. (C) Immunofluorescence analysis of JQ-FT treatment (24 h) on NOTCH1 activation in permeabilized PDX cells in vitro. Cells were probed with an anti-NOTCH1 antibody (green), and nuclei were counterstained with DAPI. Images were acquired at 60× magnification. Bars, 10 µm. (D) Expression of indicated NOTCH1 target genes in T-ALL PDX cells treated with JQ-FT for 24 h was determined by quantitative RT-PCR. Error bars indicate the mean ± SD of four replicates. Data were analyzed using the ΔΔCT method and plotted as a percentage relative to the control gene RPL13A . Statistical significance (***, P ≤ 0.001; ****, P ≤ 0.0001) for treated versus vehicle (DMSO) was determined by one-way ANOVA with Bonferroni’s correction for multiple comparison testing.

Article Snippet: Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described ( ).

Techniques: Ex Vivo, In Vitro, Staining, Control, Immunofluorescence, Activation Assay, Expressing, Quantitative RT-PCR, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Leukemia-specific delivery of mutant NOTCH1 targeted therapy

doi: 10.1084/jem.20151778

Figure Lengend Snippet: JQ-FT demonstrates activity in T-ALL mouse model. (A) Effect of JQ-FT treatment on cell growth (72 h) in murine NOTCH1 L1601P ΔPEST–expressing leukemia cells. Viability data are represented as percentage relative to vehicle treatment and errors bars denote mean ± SD of three replicates. Statistical significance of treated versus vehicle (DMSO) samples (**, P ≤ 0.01; ***, P ≤ 0.001) was determined by one-way ANOVA with Bonferroni’s correction for multiple comparison testing. (B) Effect of JQ-FT on ICN1 levels in murine NOTCH1 L1601P ΔPEST–expressing leukemia cells. The immunoblot contains cell lysates stained with anti-ICN1 antibody (Val1744) after treatment with 10 µM JQ-FT for 24 h in vitro. GAPDH was used as a loading control. (C) ICN1 loss was quantified, and the bar graph corresponds to the results of the quantification of three independent experiments. Statistical significance of treated versus vehicle (DMSO) samples (*, P ≤ 0.05) was determined by Student’s t test. Error bars denote the mean ± SD of two indipendent experiments. (D and E) Expression of indicated NOTCH1 target genes Hes1 (D) and Dtx1 (E) in murine NOTCH1 L1601P ΔPEST–expressing leukemia cells treated with 10 µM JQ-FT for 24 h was determined by quantitative RT-PCR. Error bars indicate the mean ± SD of three replicates. Data were analyzed using the ΔΔCT method and plotted as a percentage relative to the control gene Gapdh . Statistical significance for treated versus vehicle (DMSO; *, P ≤ 0.05) was determined by Student’s t test. (F) Pharmacokinetic ratio analysis of JQ-FT to Thap-OH. Quantification expressed as hydrolysis ratio of JQ-FT/Thap-OH in bone marrow, liver, plasma, and spleen after i.p. injection of 60 mg/Kg JQ-FT at 50 min. Error bars indicate mean ± SD of three replicates (three mice/time point). (G) Histological analysis of the spleen and the liver in a NOTCH1 L1601P ΔPEST murine model treated with JQ-FT 60 mg/kg or vehicle for 5 d. The spleen and the liver of all mice were examined; representative results for one control animal and one JQ-FT–treated animal are shown. Formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylin and eosin. Growth suppression of leukemia cells (dark purple) was observed in JQ-FT–treated animals. Bars, 10 µm. (H) Effect of JQ-FT on T-ALL growth in a NOTCH1 L1601P ΔPEST murine model. Antileukemic activity of JQ-FT was assessed by measuring spleen weight after 5 d of treatment with JQ-FT (60 mg/kg i.p.) or vehicle (65% D5W + 30% PEG-400 + 5% Tween-80 only). The chart shows spleen weight for each animal (each dot), and the horizontal bar represents the mean of the four animals per group. Statistical significance for treated versus vehicle (*, P ≤ 0.05) was determined by nonparametric t test (Mann–Whitney). (I) Antileukemic activity of JQ-FT on bone marrow NOTCH1 L1601P ΔPEST GFP-positive leukemia cells after 5 d of treatment with JQ-FT (60 mg/kg i.p.) or vehicle (65% D5W + 30% PEG-400 + 5% Tween-80 only). Error bars indicate mean ± SD of four replicates (of the four animals of each group). Statistical significance for treated versus vehicle (*, P ≤ 0.05) was determined by nonparametric t test (Mann–Whitney). (J) Effect of JQ-FT on Notch activation in a NOTCH1 L1601P ΔPEST murine model. The immunoblot contains splenic cell lysates stained with anti-ICN1 antibody (Val1744) after treatment with 60 mg/kg JQ-FT for 5 d. GAPDH was used as a loading control. (K) ICN1 loss was quantified, and bar graph corresponds to the results of the quantification. Statistical significance for treated versus vehicle (*, P ≤ 0.05) was determined by Student’s t test. Error bars denote the mean ± SD of the four samples loaded in the immunoblot depicted in J.

Article Snippet: Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described ( ).

Techniques: Activity Assay, Expressing, Comparison, Western Blot, Staining, In Vitro, Control, Quantitative RT-PCR, Clinical Proteomics, Injection, Formalin-fixed Paraffin-Embedded, MANN-WHITNEY, Activation Assay

Journal: The American Journal of Pathology

Article Title: Epigenetic Silencing of miRNA-34a in Human Cholangiocarcinoma via EZH2 and DNA Methylation

doi: 10.1016/j.ajpath.2017.06.014

Figure Lengend Snippet: Primer Sequences

Article Snippet: Notch1 (SC215771), Jagged1 (HmiT004470), and Notch2 (HmiT011875) 3′-UTR reporter plasmids were obtained from Origene (Rockville, MD) and Genecopoeia (Rockville, MD).

Techniques: Methylation

Journal: The American Journal of Pathology

Article Title: Epigenetic Silencing of miRNA-34a in Human Cholangiocarcinoma via EZH2 and DNA Methylation

doi: 10.1016/j.ajpath.2017.06.014

Figure Lengend Snippet: MiR-34a inhibits the Notch pathway by targeting Notch1, Notch2, and Jag1. A: Putative miR-34a binding sites in the 3′untranslated region (UTR) of Notch1, Jag1, and Notch2 mRNA. 3′UTR fragments of Notch1, Jag1, and Notch2 containing wild-type or mutated (three mutated nucleotides are in bold) miR-34a binding site. B: Western blot analysis for Notch1 (transmembrane/intracellular region: notch transmembrane region), Notch2, and Jag1 in CCLP1 and SG231 cells transfected with miR-34a mimic or scramble control (Jag1 protein was not detectable in CCLP1 cells). C: Relative Notch1, Jag1, and Notch2 mRNA expression in CCLP1 and SG231 cells transfected with miR-34a mimic or scramble control. D: Western blot and quantitative RT-PCR analysis for Notch1, Jag1, and Notch 2 in control (pEZX-CN) and miR-34a–overexpressed (pEZX-miR34a) SG231 cells. E: Western blot analysis for Notch1, Jag1, and Notch2 in TFK1 cells transfected with anti–miR-34a and scramble control. F: Luciferase reporter activity in SG231 cells transfected with wild-type (WT) or mutant (Mut) Notch1, Jag1, or Notch2 3′UTR plasmids and treated with miR-34a mimic or scramble control miRNA. Data are expressed as means ± SEM (C and D); data are expressed as means ± SD (F). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus control mimic or plasmid transfected cells. Anti-NC, antisense miRNA negative control; ctrl, control.

Article Snippet: Notch1 (SC215771), Jagged1 (HmiT004470), and Notch2 (HmiT011875) 3′-UTR reporter plasmids were obtained from Origene (Rockville, MD) and Genecopoeia (Rockville, MD).

Techniques: Binding Assay, Western Blot, Transfection, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Negative Control

Journal: The American Journal of Pathology

Article Title: Epigenetic Silencing of miRNA-34a in Human Cholangiocarcinoma via EZH2 and DNA Methylation

doi: 10.1016/j.ajpath.2017.06.014

Figure Lengend Snippet: Overexpression of miR-34a inhibits cholangiocarcinoma growth in vivo. SG231 cells stably expressing miR-34a or scramble control miRNA (1.0 × 106) were mixed in Matrigel solution and inoculated directly into the livers of severe combined immunodeficiency (SCID) mice (intrahepatic injection). We initially started with six mice per group, but one mouse from the control group died during the injection. After 6 weeks, the mice were sacrificed and monitored for tumor growth. A: Representative images of xenograft tumors recovered from five mice for control and six mice for miR-34a–overexpressing cells. B: Tumor volume and liver weight. C: The levels of miR-34a in xenograft tumor tissues as determined by quantitative RT-PCR. Data are expressed as means ± SEM. D: Western blot analysis for Notch1, Jag1, and Notch2 in control and miR-34a–overexpressed tumors. ∗P < 0.05, ∗∗P < 0.01. CN, scramble control.

Article Snippet: Notch1 (SC215771), Jagged1 (HmiT004470), and Notch2 (HmiT011875) 3′-UTR reporter plasmids were obtained from Origene (Rockville, MD) and Genecopoeia (Rockville, MD).

Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Injection, Quantitative RT-PCR, Western Blot