Fig 1: Spindlin1 represses HBV transcription in HepAD38 cells.(A) HepAD38 cells cultured without tetracycline were transduced with an empty lentiviral vector (Ctrl) or a lentiviral vector encoding His-Myc-Spindlin1 (Myc-Spin1). Cells were then harvested 48 h after transduction for total RNA extraction and HBV transcription was analyzed by RT-qPCR (left graph). Transcript level in cells transduced with the empty lentiviral vector was set to 1. P values were determined by Wilcoxon test (**, P = 0.007). Error bars represent SD of eight independent experiments. Alternatively, cells were harvested 72 h after transduction and capsid-associated DNA was extracted. The replication intermediates were then analyzed by southern blot. RC: relaxed circular DNA, SS: single-stranded DNA, RI: replication intermediates. The relative intensities of RC DNA (1) and SS DNA (2) were quantified. The results are presented on the right graphs. The intensity in the control condition was set at 1. (B) Spindlin1 does not affect HBV RNA stability. HepAD38 cultured without tetracycline were transduced as in (A). After 48 h, the cells were treated with actinomycin D (5 µg/ml) that blocks polymerase II. Cells were collected at the indicated times after treatment and total RNA was prepared. HBV RNAs were then quantified by RT-qPCR. The relative level of HBV transcription was normalized with Roth2 gene. The amount of RNA at time zero was set at 100%. (C) Spindlin1 modulates the rate of HBV transcription. Nuclear run-on assays were performed on isolated nuclei from HepaAD38 cells transduced as in (A). Transcripts generated during run-on were purified using anti-BrdU beads and HBV RNAs were quantified by RT-qPCR. Transcript level in cells transduced with the empty lentiviral vector was set to 1. Error bars represent SD of three independent experiments. The expression of His-Myc-Spin1 was analyzed by immunoblot with anti-Myc antibodies. Tubulin was used as loading control (Right panel). (D) Spindlin1 knock-down increases HBV transcription. HepAD38 cells were transduced with a lentiviral vector allowing the expression of a control shRNA (shCtrl) or shRNAs directed against Spindlin1 (shSpindlin1). After selection with puromycin (4 µg/ml), cells stably expressing shCtrl or shSpin1 were transfected with 25 nM of control siRNA (siCtrl) or directed against Spindlin1 (siSpin1) respectively. 48 h post-transfection, cells were harvested for total RNA extraction and, HBV (left panel) or pre-rRNA transcription (right panel) were analyzed by RT-qPCR. Spindlin1 expression in shCtrl+siCtrl or shSpin1+siSpin1 HepAD38 cells was analyzed by RT-qPCR (bottom left graph) and by immunoblotting with anti-Spin1 antibodies. Anti-tubulin immunostaining was used as loading control (bottom right panel). Transcript level in shCtrl HepAD38 cells transfected with siCtrl was set to 1. P values were determined by Wilcoxon test (*, P<0.05). Error bars represent SD of six independent experiments.
Fig 2: Spindlin1 interacts with HBx.(A) Coimmunoprecipitation of His-myc-Spindlin1 (His-myc-Spin1) with HA-HBx using anti-Myc antibodies in HEK293 cells. Proteins in the immune complexes were revealed by Western blotting with anti-His and anti-HA antibodies. The expression of ß-catenin was used as loading control. (B) Whole-cell extracts, prepared from HepG2 cells transduced with a lentiviral vector encoding Flag-HA-HBx, were immunoprecipitated with anti-Spindlin1 antibodies (IP Spin1). Proteins were detected by Western blotting using anti-HA or anti-Spin1 antibodies. The expression of tubulin was used as loading control. (C) Schematic representation of the HBx clustered alanine substitution mutants (left panel). HEK293 cells were co-transfected with Flag-tagged HBx wt construct or the HBx alanine substitution mutants (F-HBx Cm5, F-HBx Cm7, F-HBx Cm8, F-HBx Cm9, F-HBx Cm13) and the His-myc-Spin1 plasmid. Cell extracts were immunoprecipitated with anti-Flag antibodies and analyzed by anti-Myc and anti-Flag immunoblot using the Odyssey system (right panel). The expression of tubulin was used as loading control. Signal strengths of the co-immunoprecipitated His-myc-Spindlin1 proteins were normalized to the ratio of HBx in the IP/HBx in the input. Immunoprecipitated His-myc-Spindlin1 level in cells expressing His-myc-Spindlin1 and Flag-HBx wt was set to 100% (lower right graph) (D) Extracts from HEK293 cells transfected with HA-HBx in combination with His-Myc-Spin1 or His-myc-Spindlin1 mutant containing a deletion of the Tudor-like domain II (His-myc-Spin1 mut) were immunoprecipitated with anti-Myc antibodies. Proteins were detected by Western blotting using anti-HA or anti-Myc antibodies. The expression of tubulin was used as loading control.
Fig 3: HBx expression correlates with the decrease of Spindlin1 recruitment to the cccDNA.(A) Differentiated HepaRG cells were infected with normalized amount of HBV WT or HBV X- viruses. 8 days after infection, cells were harvested and analyzed by ChIP using antibodies against Spin1. As a control, immunoprecipitation was performed with purified rabbit IgG. Input and immunoprecipitated DNA were analyzed in triplicate by qPCR with primers specific for HBV DNA or for rDNA and are displayed as percent input. Error bars represent SD of three independent experiments. (B). Spindlin1 mutants deficient for methyllysine binding interact with HBx. HEK293 cells were transfected with a plasmid coding for HA-HBx protein alone or in combination with plasmids coding for wild type Flag-Spindlin1 protein (F-Spin1) or for the Flag-tagged Spindlin1 mutants Y170A and F141A (F-Spin1 Y170A and F-Spin1 F141A respectively). After anti-Flag immunoprecipitation, proteins were analyzed by anti-HA and anti-Flag immunoblotting. Tubulin was used as a loading control. (C) Repression of cccDNA transcription by Spindlin1 is dependent of its ability to interact with methyllysine. HepAD38 cells were transfected by electroporation with plasmids encoding F-Spin1, F-Spin1 Y170A or F-Spin1 F141A. 48 h after transfection, cells were harvested and total RNA was extracted. HBV transcription was evaluated by RT-qPCR. Transcript level in cells transfected with the Ctrl plasmid was set to 1. Error bars represent SD of four independent experiments. (D) The level of H3K4me3 associated to the cccDNA or to the rDNA was studied by ChIP-qPCR with anti-H3K4me3 antibodies. As a control, immunoprecipitation was performed with purified rabbit IgG. Input and immunoprecipitated DNA were analyzed in triplicate by qPCR with primers specific for HBV DNA or for rDNA and are displayed as percent input. Error bars represent SD of three independent experiments. (E) Differentiated shCtrl or shSpin1 HepaRG cells were infected with normalized amount of HBV WT or HBV X- virus. 8 days after infection, cells were harvested and analyzed by ChIP assay using antibodies against H3K4me3. As a control, immunoprecipitation was performed with purified rabbit IgG. Input and immunoprecipitated DNA were analyzed in triplicate by qPCR with primers specific for HBV DNA and are displayed as percent input. Error bars represent SD of three independent experiments.
Fig 4: PUM1 and PUM2 proteins bind and regulate SPIN1 and SPIN3 mRNAsSchematic 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.
Fig 5: SPIN paralogues differentially influence TCam-2 cell apoptosisSPIN1 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.
Supplier Page from Abcam for Anti-spindlin 1 antibody