Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: Figure 3: PUM1 and PUM2 proteins bind and regulate SPIN1 and SPIN3 mRNAs. Schematic of full-length human SPIN1 and SPIN3 3ʹUTRs (A). PBE-like motifs responsible for PUF-domain binding are in red and UGUA core motifs are in black (SPIN3). Short 3ʹUTR fragments containing PBE motifs, which were used for luciferase reporter assays, and full-length 3ʹUTRs are indicated in brackets, with position within the 3ʹUTR starting from the end of the stop codon. Enrichment of SPIN1 and SPIN3 in RIP-PUM1 and RIP-PUM2 (indicated by RIP P1 and RIP P2, respectively) was measured via RT-qPCR and was compared to the negative control (nonimmune IgG, indicated by RIP IgG) (B). Influence of PUM1 and PUM2 proteins on endogenous SPIN mRNA level (C). PUM1 and PUM2 siRNA knockdown efficiencies are shown on the left. Total RNA was isolated from TCam-2 cells in the presence of actinomycin D. SPIN expression was measured via RT-qPCR and was compared to that in untransfected cells. PUM1 or PUM2 overexpression downregulated endogenous SPIN1 as measured by western blotting (D). Graphs represent average values with standard errors. P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005.

Article Snippet: Constructs encoding PUM1 (RC201219), PUM2 (RC211307), SPIN1 (RC201938), or SPIN3 (RC215063), in the pCMV6-entry vector system for protein overexpression were purchased from OriGene Technologies.

Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, Negative Control, Knockdown, Isolation, Expressing, Over Expression, Western Blot

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: Figure 4: Influence of PUM1 and PUM2 proteins on luciferase reporter constructs carrying SPIN1 or SPIN3 3ʹUTRs. The effects of PUM proteins on SPIN expression were assessed using a dual luciferase assay. Luciferase reporter constructs carrying full-length 3ʹUTRs for SPIN1 (upper panel) or SPIN3 (lower panel) were tested with PUM1 (P1) or PUM2 (P2) overexpression or empty pCMV6-entry vector (pC) (A). Effects of PUM overexpression on luciferase reporter construct carrying full-length GAPDH mRNA 3ʹUTR, which lacks PBE motifs (negative control) (B). Effects of siRNA-mediated PUM1 (P1 KD) or PUM2 (P2 KD) knockdown (KD) on luciferase reporter constructs carrying full-length SPIN 3ʹUTRs (C). Effects of PUM1 or PUM2 overexpression (D). or knockdown (E). on luciferase constructs containing short SPIN1 or SPIN3 3ʹUTR fragments. **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.00005.

Article Snippet: Constructs encoding PUM1 (RC201219), PUM2 (RC211307), SPIN1 (RC201938), or SPIN3 (RC215063), in the pCMV6-entry vector system for protein overexpression were purchased from OriGene Technologies.

Techniques: Luciferase, Construct, Expressing, Over Expression, Plasmid Preparation, Negative Control, Knockdown

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: Figure 5: PUM1 induces TCam-2 cell apoptosis. TCam-2 cells were transfected with constructs encoding PUM1 (P1), PUM2 (P2), or empty vector and cultured for 48 h. Apoptosis was measured as described for SPINs (A). Dot-plot showing quality of TCam-2 cell separation into living, necrotic, and early or late apoptotic populations after transfection with empty vector (B), PUM1 (C), or PUM2 (D) constructs.

Article Snippet: Constructs encoding PUM1 (RC201219), PUM2 (RC211307), SPIN1 (RC201938), or SPIN3 (RC215063), in the pCMV6-entry vector system for protein overexpression were purchased from OriGene Technologies.

Techniques: Transfection, Construct, Plasmid Preparation, Cell Culture

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: Figure 6: PUM1 and PUM2 slightly downregulate TCam-2 cell cycle progression. PUM1 and PUM2 were separately overexpressed and the effects on cell cycle progression were measured 72 h later.

Article Snippet: Constructs encoding PUM1 (RC201219), PUM2 (RC211307), SPIN1 (RC201938), or SPIN3 (RC215063), in the pCMV6-entry vector system for protein overexpression were purchased from OriGene Technologies.

Techniques:

Journal: Blood

Article Title: PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells

doi: 10.1182/blood-2016-10-747436

Figure Lengend Snippet: PUM1 and PUM2 positively regulate FOXP1 mRNA expression via two 3′UTR located PBE. (A) Schematic representation of human FOXP1 mRNA with the 2 PUM-binding elements (PBE), PBE1 and PBE2, boxed (red), and regions A, B, C delineated. The numbers indicate positions in 3′UTR, starting from translation stop codon. (B) RNA pull-down assay. MOLM-14 cell lysates were incubated with WT1 or MUT1 biotin-labeled FOXP1-PBE1 probes, and none or ×5 unlabeled PBE1 (WT1 or MUT1) or PBE2 (WT2 or MUT2) competitor riboprobes, as indicated. Pulled‐down PUM proteins were analyzed by immunoblotting. (C) RNA immunoprecipitation assays. Lysates from shRNA-transduced MOLM-14 cells were immunoprecipitated (IP) using control (IgG), PUM1 or PUM2 antibody, as indicated. Upper panel: Immunoblot analysis of the indicated proteins. Lower panel: RT-qPCR analysis of FOXP1 mRNA present in the IPs. The results are expressed as percent of input (n = 3). (D) Luciferase assay. MOLM-14 cells were transfected with control (siCtrl), PUM1 (siPUM1), or PUM2 (siPUM2) siRNA, and with the indicated luciferase reporter construct harboring the FL FOXP1-3′UTR. Relative luciferase activity was monitored 24 hours later and expressed as the ratio between Renilla luciferase (Rluc) activity and Firefly luciferase (Fluc) activity, used as transfection control. Data are expressed relative to expression in FOXP1-3′UTR FL and siCtrl transfected cells (n = 4). (E) Luciferase assays were performed as in panel D, with luciferase constructs harboring the indicated regions of the FOXP1-3′UTR (A-C) with WT, or doubly MUT1/2 PBEs. Data are expressed relative to expression in WT-FOXP1-3′UTR-C and siCtrl transfected cells (n = 4). (F) Luciferase assay. MOLM-14 cells were transfected with FOXP1-3′UTR-C luciferase reporter constructs harboring WT PBE1 and PBE2, mutated PBE1 (MUT1), mutated PBE2 (MUT2), or doubly mutated PBE1 and PBE2 (MUT1/2). Relative luciferase activity was monitored 24 hours later, expressed as the ratio Rluc/Fluc, and compared relative to expression in WT-FOXP1-3′UTR-C transfected cells (n = 4). Data are expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001 (Student t test).

Article Snippet: MOLM-14 cells (10 6 ) were transfected with 300 pmol of siPUM1 (SR306467B; OriGene), siPUM2 (SR308223C; OriGene), or scrambled control small interfering RNA (siRNA) (SR30004; OriGene) using Lipofectamine 2000.

Techniques: Expressing, Binding Assay, Pull Down Assay, Incubation, Labeling, Western Blot, RNA Immunoprecipitation, shRNA, Immunoprecipitation, Control, Quantitative RT-PCR, Luciferase, Transfection, Construct, Activity Assay

Journal: BMC Cancer

Article Title: Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

doi: 10.1186/1471-2407-8-20

Figure Lengend Snippet: Summary of candidate reference genes.

Article Snippet: PUM1 NM_001020658.1 , Pumilio homolog (Drosophila) (Hs00982765_m1) , RNA binding; translation factor. , 1p35.2 , 1757 (68) , - , (-/-).

Techniques: Binding Assay, RNA Binding Assay, Activity Assay

Journal: BMC Cancer

Article Title: Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

doi: 10.1186/1471-2407-8-20

Figure Lengend Snippet: Performance of the qPCR. Standard curve results are summarized and the amplification efficiency calculated.

Article Snippet: PUM1 NM_001020658.1 , Pumilio homolog (Drosophila) (Hs00982765_m1) , RNA binding; translation factor. , 1p35.2 , 1757 (68) , - , (-/-).

Techniques: Amplification

Journal: BMC Cancer

Article Title: Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

doi: 10.1186/1471-2407-8-20

Figure Lengend Snippet: Summary of top candidate reference genes identified by geNorm and NormFinder. For the geNorm data, the optimal number and identity of reference genes proven necessary by a Pairwise variation below 0.15, as recommended by Vandesompele et al. [12], is provided in the order given by the program. The stated genes should be viewed per program, and the combination of genes provided by geNorm cannot be used as single entities as optimal reference genes. The number of patients included was: ER+ (n = 11), ER- (n = 4), normal (n = 3) and ER+ cell lines (n = 3)

Article Snippet: PUM1 NM_001020658.1 , Pumilio homolog (Drosophila) (Hs00982765_m1) , RNA binding; translation factor. , 1p35.2 , 1757 (68) , - , (-/-).

Techniques: