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: SPIN1 and SPIN3 were overexpressed or silenced in TCam-2 cells and apoptosis was assessed using flow cytometry. Representative western blot showing SPIN overexpression compared to VINCULIN ( A ). Apoptosis was analyzed in TCam-2 cells overexpressing SPINs ( B ) and in cells in which SPINs were silenced ( C ) CYCD1 expression was measured via real-time qPCR in cells overexpressing SPIN1 and SPIN3 ( D ). Cells transfected with an empty vector (overexpression) or control siRNA (knockdown) were the baselines in (B) and (C). * 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: Flow Cytometry, Western Blot, Over Expression, Expressing, Transfection, Plasmid Preparation

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: TCam-2 cell cycle analysis was performed following siRNA-mediated SPIN1 or SPIN3 knockdown ( A ) Cells transfected with control siRNA represented the baseline. * P ≤ 0.05. TCam-2 cell cycle analysis was performed following SPIN1 or SPIN3 overexpression ( B ) Values higher than the baseline indicate an increase in a given cell population, while values below the baseline indicate a decrease. The p16 and p21 CDK inhibitors were used as positive controls ( C ).

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: Cell Cycle Assay, Transfection, Over Expression

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: Schematic of full-length human SPIN1 and SPIN3 3′UTRs ( A ). PBE-like motifs responsible for PUF-domain binding are in red and UGUA core motifs are in black ( SPIN3 ). Short 3′UTR fragments containing PBE motifs, which were used for luciferase reporter assays, and full-length 3′UTRs are indicated in brackets, with position within the 3′UTR 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, 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: The effects of PUM proteins on SPIN expression were assessed using a dual luciferase assay. Luciferase reporter constructs carrying full-length 3′UTRs 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 3′UTR, 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 3′UTRs ( C ). Effects of PUM1 or PUM2 overexpression ( D ). or knockdown ( E ). on luciferase constructs containing short SPIN1 or SPIN3 3′UTR 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: Expressing, Luciferase, Construct, Over Expression, Plasmid Preparation, Negative Control