Cellular localisation of Fl-HBP1 and ΔHBP1.

<p>Myc-tagged HBP1 variants were transfected into HEK293 cells and immunostained for MYC expression. Cells were counterstained with DAPI to identify the nuclei and were visualised using confocal microscopy. FL-HBP1 was localised to the nucleus with some cytoplasmic staining also detected. ΔHBP1 remained in the cytoplasm. The control represents cells transfected without DNA. Scale Bar = 10μm.</p

Detection of <i>Fl-Hbp1</i> and <i>ΔHbp1</i> transcripts in wildtype and <i>W</i>^<i>e</i><i>/W</i>^<i>e</i> gonads.

<p><b>(A)</b> Detection of <i>Fl-Hbp1</i> and <i>ΔHbp1</i> in 14.5 dpc XX and XY gonads using whole mount in situ hybridisation <b>(B)</b> qRT-PCR analysis detected lower levels of <i>Fl-Hbp1</i> and <i>ΔHbp1</i> gene expression in 13.5 dpc XY and XX <i>W</i>^<i>e</i><i>/W</i>^<i>e</i> mutant gonads which lack germ cells. Expression was normalised to <i>18S</i> RNA (mean ± S.E.M of three independent experiments, each performed in triplicate) and wildtype controls set to 1. *<i>P</i> < 0.05; *<i>*P</i> < 0.005. <b>(C)</b> qRT-PCR detected the <i>Fl</i>-<i>Hbp1</i> and <i>ΔHbp1</i> transcripts in gonad-only cDNA samples from 12.5–16.5 dpc in XX and XY gonads. Expression was normalised to <i>18S</i> RNA (mean ± S.E.M of three independent experiments, each performed in triplicate).</p

Immunohistochemical and gene expression analysis of <i>Hbp1</i>^<i>+/+</i> and <i>Hbp1</i>^<i>-/-</i> embryonic gonads.

<p><b>(A)</b> Proliferation marker Ki67 in germ cells (MVH-positive cells) was detected in both <i>Hbp1</i>^<i>+/+</i> and <i>Hbp1</i>^<i>-/-</i> gonads at 12.5 dpc, but was absent in germ cells of 14.5 and 16.5 dpc gonads of both genotypes. Pluripotency marker OCT3/4 was expressed similarly in germ cells (MVH-positive cells) in <i>Hbp1</i>^<i>+/+</i> and <i>Hbp1</i>^<i>-/-</i> gonads at 14.5 dpc and was undetectable at 16.5 dpc. Scale bar = 50μm. <b>(B)</b> qRT-PCR analysis of <i>Hbp1</i>^<i>+/+</i> and <i>Hbp1</i>^<i>-/-</i> 16.5 dpc gonad samples revealed comparable expression between of various germ cell and somatic cell markers. Germ cell markers: <i>Mvh</i>, <i>Oct3/4</i>and G₁/G₀ arrest indicator <i>p63</i>, somatic cell markers <i>Fgf9</i> and <i>Sox9</i>, Retinoblastoma family members <i>Rb1</i>, <i>p130</i> and <i>p107</i>, and cell cycle regulators <i>p21</i>, <i>Ccnd1-3</i> and <i>p53</i> displayed no significant difference between <i>Hbp1</i>^<i>+/-</i> <i>and Hbp1</i>^<i>-/-</i> samples. Samples normalised to <i>18S</i> RNA (mean ± S.E.M of three independent experiments, each performed in triplicate). *<i>P</i> < 0.05; *<i>*P</i> < 0.005; ns = not significant.</p

LacZ expression analysis of <i>Hbp1</i>^{<i>lacZ_1</i>}.

<p>LacZ expression, driven by the 2 kb <i>Hbp1</i> proximal promoter, was assessed at embryonic stages 11.5 <b>(A)</b> and 14.5 dpc <b>(B)</b>; wildtype (Wt); transgenic (Tg). Promoter activity was also detected in 14.5 dpc somatic tissues including the forebrain <b>(C),</b> hair follicles <b>(D)</b>, eye <b>(E)</b>, limb tips <b>(F)</b> and neural tube <b>(G,H)</b>. Expression was also detected at 14.5 dpc in the XY <b>(I)</b>, but not XX <b>(J)</b> gonad.</p

Nullbasic induces Rev redistribution without protein interaction.

<p>(A) HeLa cells expressing Myc-Rev alone (row 1) or with Nullbasic (NB)-mCherry (row 2) were fixed and immunostained with anti-Myc antibody before being analyzed by fluorescence microscopy for Myc-Rev (green) and NB-mCherry (red) subcellular localizations. Nuclei were stained with DAPI. The overlay panels show the merge of the Myc-Rev and NB-mCherry panels. The figure is representative of four fields each from four independent experiments. (B) HeLa cells were transfected to express HIV-eGFP alone (row 1) or with NB-mCherry (row 2). Fixed cells were immunostained with anti-Rev antibody before the subcellular localizations of Rev (cyan) and NB-mCherry (red) were visualized by fluorescence microscopy. Expression of HIV-eGFP was confirmed in the FITC channel (green). Nuclei were stained with DAPI. The overlay panels show the merge of the Nullbasic-mCherry and Rev panels. Figures are representative of five fields each from four independent experiments. (C) HEK293T cells were transfected with either empty vector (pcDNA3.1) or plasmids expressing Nullbasic (NB)-FLAG alone, Tat-FLAG alone, or Myc-Rev with either NB-FLAG or Tat-FLAG, as indicated. The FLAG-tagged proteins and their interacting factors were immunoprecipitated using anti-FLAG beads. Total cell lysates (left) and immunoprecipitated proteins (right) were subjected to western blot analysis using anti-FLAG and anti-Myc antibodies. The endogenous protein CDK9, detected using anti-CDK9 antibody, was used as a positive control for Tat interaction. The figure is representative of four independent experiments. The white bar in last panel is equal to 10 µm.</p

Morphological and protein expression analysis of <i>Hbp1</i>^<i>+/+</i> and <i>Hbp1</i>^<i>-/-</i> mutant embryos.

<p><b>(A)</b> Gene structure of the two <i>Hbp1</i> splice variants and the <i>Hbp1</i> gene-trap variant which contains a ß-galactosidase cassette inserted directly downstream of exon 5. <b>(B)</b> Whole 14.5 dpc <i>Hbp1</i>^<i>+/+</i> and <i>Hbp1</i>^<i>-/-</i> embryos were stained for LacZ expression. LacZ was detected in <i>Hbp1</i>^<i>-/-</i> embryos in the eye, hair follicles, limbs, ears and mesonephros, but not testes at 14.5 or 16.5 dpc. <b>(C)</b> Western blot analysis was performed on whole 14.5 dpc <i>Hbp1</i>^<i>+/+</i>, <i>Hbp1</i>^<i>+/-</i> and <i>Hbp1</i>^<i>-/-</i> embryo lysates. Anti-HBP1 detected reduced levels of HBP1 protein in the <i>Hbp1</i>^<i>+/-</i> and <i>Hbp1</i>^<i>-/-</i> samples compared to <i>Hbp1</i>^<i>+/+</i> control. TUBA1A was used as the loading control.</p

<i>Hbp1</i> expression in <i>Rb</i>^<i>+/+</i> and <i>Rb</i>^<i>-/-</i> mutant XY gonads.

<p>Using qRT-PCR analysis, normalizing gene expression to <i>18S</i>RNA, both <i>Fl-Hbp1</i> <b>(A)</b> and <i>ΔHbp1</i> <b>(B)</b> gene expression was significantly increased in <i>Rb</i>^<i>-/-</i> mutant XY gonads at 14.5 dpc. In 16.5 dpc <i>Rb</i>^<i>-/-</i> cultured XY gonads, there was no significant difference in either <i>Fl-Hbp1</i> or <i>ΔHbp1</i> gene expression, although germ cell marker <i>Mvh</i> was significantly increased at this timepoint (<b>C</b>). When gene expression was normalized to germ cell marker <i>Mvh</i>, both <i>Fl-Hbp1</i> <b>(D)</b> and <i>ΔHbp1</i> <b>(E)</b> gene expression was significantly decreased in 16.5 dpc <i>Rb</i>^<i>-/-</i> cultured XY gonads. (mean ± S.E.M of three independent experiments, each performed in triplicate; wildtype controls (<i>Rb</i>^<i>+/+</i>) set to 1). *<i>P</i> < 0.05; *<i>*P</i> < 0.005.</p

Alternative splicing of <i>Hbp1</i> transcripts.

<p><b>(A)</b> Gene structure of the two <i>Hbp1</i> splice variants. The full-length transcript comprises 11 exons and the truncated transcript is composed of the first 9 exons. Both transcripts contain identical 5’UTRs in addition to distinct 3’UTRs (open boxes), the specific riboprobes and real time PCR probes are depicted. <b>(B)</b> Location of HBP1 protein domains and nuclear localisation signals (NLS).</p

Coexpression of Nullbasic and Rev alters the subcellular localization of C23 but not fibrillarin.

<p>HeLa cells were transfected to express Myc-Rev alone (rows 1 and 4), Nullbasic (NB)-mCherry alone (rows 2 and 5) or cotransfected Myc-Rev with NB-mCherry (rows 3 and 6). Fixed cells were immunostained with anti-Myc (green) and either anti-C23 (magenta, upper panels) or anti-fibrillarin (magenta, lower panels) antibodies before the subcellular localizations of Myc-Rev, endogenous C23 or endogenous fibrillarin, and NB-mCherry (red) were analyzed by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the Myc-Rev panel with either the C23 or fibrillarin panels (rows 1, 3, 4 and 6) and the NB-mCherry panel with either the C23 or fibrillarin panels (rows 2 and 4). All figures are representative of five fields each from four independent experiments. The white bar in last panel is equal to 10 µm.</p

Nullbasic does not affect the nuclear import of importin β and transportin 1 cargoes.

<p>HeLa cells were transfected to express the importin β-dependent nuclear import reporter GFP₂-cNLS, or the transportin 1-dependent nuclear import reporter GFP₂-M9core, either alone (rows 1 and 3, respectively) or with Nullbasic (NB)-mCherry (rows 2 and 4, respectively). The subcellular localizations of the reporter proteins (green) and NB-mCherry (red) were analyzed in fixed cells by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the GFP reporter and DAPI panels. Plasmid expressing GFP₂ was also transfected into cells either alone or with NB-mCherry as a vector control (rows 5 and 6, respectively). Figures are representative of five fields each from four independent experiments. The white bar in last panel is equal to 10 µm.</p