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 1: SPIN paralogues differentially influence TCam-2 cell apoptosis. 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: Cells were washed with 1× PBS (Lonza), and then visualized using a Leica DMi8 IVD microscope under UV (for Hoechst 33258 detection) and visible light. siRNA knockdown For transient knockdowns, TCam-2 cells were transfected with one of the following specific siRNAs at 10 nM final concentration: SPIN1 (sc-92696), SPIN3 (sc91032), PUM1 (sc-62912), PUM2 (sc-44773) or control (sc-37007) (Santa Cruz Biotechnology, USA).

Techniques: Flow Cytometry, Western Blot, Over Expression, Expressing, Transfection, Plasmid Preparation, 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 2: SPIN1 and SPIN3 promote TCam-2 cell cycle progression. 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: Cells were washed with 1× PBS (Lonza), and then visualized using a Leica DMi8 IVD microscope under UV (for Hoechst 33258 detection) and visible light. siRNA knockdown For transient knockdowns, TCam-2 cells were transfected with one of the following specific siRNAs at 10 nM final concentration: SPIN1 (sc-92696), SPIN3 (sc91032), PUM1 (sc-62912), PUM2 (sc-44773) or control (sc-37007) (Santa Cruz Biotechnology, USA).

Techniques: Cell Cycle Assay, Knockdown, Transfection, Control, 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: 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: Cells were washed with 1× PBS (Lonza), and then visualized using a Leica DMi8 IVD microscope under UV (for Hoechst 33258 detection) and visible light. siRNA knockdown For transient knockdowns, TCam-2 cells were transfected with one of the following specific siRNAs at 10 nM final concentration: SPIN1 (sc-92696), SPIN3 (sc91032), PUM1 (sc-62912), PUM2 (sc-44773) or control (sc-37007) (Santa Cruz Biotechnology, USA).

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: Cells were washed with 1× PBS (Lonza), and then visualized using a Leica DMi8 IVD microscope under UV (for Hoechst 33258 detection) and visible light. siRNA knockdown For transient knockdowns, TCam-2 cells were transfected with one of the following specific siRNAs at 10 nM final concentration: SPIN1 (sc-92696), SPIN3 (sc91032), PUM1 (sc-62912), PUM2 (sc-44773) or control (sc-37007) (Santa Cruz Biotechnology, USA).

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