Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo

Abstract

As a transcription factor, NR2F1 regulates spatiotemporal gene expression in the nucleus particularly during development. Aberrant NR2F1 causes the rare neurodevelopmental disorder Bosch-Boonstra-Schaaf Optic Atrophy Syndrome. In addition, altered NR2F1 expression is frequently observed in various cancers and is considered a prognostic marker or potential therapeutic target. NR2F1 has been found in both the nucleus and nucleoli, suggesting a non-canonical and direct role in the latter compartment. Hence, we studied this phenomenon employing various in vitro and in vivo models using different antibody-dependent approaches. Examination of seven commonly used anti-NR2F1 antibodies in different human cancer and stem cells as well as in wild type and null mice revealed that NR2F1 nucleolar localization is artificial and has no functional role. Our subsequent comparative analysis demonstrated which anti-NR2F1 antibody best fits which approach. The data allow for correct data interpretation and underline the need to optimize any antibody-mediated technique.

Fig. 1. IF of endogenous NR2F1 in hiPSC-derived hNCC using Ab H8132.

Gary boxes indicate the selected zoom-in areas. A Co-IF of NR2F1 (green) either with HP1 (red) corresponding to inactive chromatin or H3K27ac (red) corresponding to active chromatin status. B Co-IF of NR2F1 (green) with ZSCAN1 (red) and their co-localization in nucleoli (gray arrowheads). C As indicated by gray arrowheads, co-IF of NR2F2 (green) with ZSCAN1 (red) showed no nucleolar-like localization. Nucleoplasm (blue) was stained with DAPI. Scale bars: 20 µm.

Fig. 2. Ab-dependent NR2F1 localization was tested in different cell types.

Gray boxes show the selected zoom-in areas. A IF of NR2F1 by Ab H8132 (green) or by Ab #6364 (red) was performed in HeLa cells, hiPSC-derived hNCC, and undifferentiated hiPSC. Nucleolar-like foci stained by Ab H8132 are indicated by arrowheads. Ab #6364 stains endogenous NR2F1 only in the nucleoplasm and specifically in HeLa and hNCC, but not in hiPSC, which do not express NR2F1. DAPI marks necleoplasm (blue). B IF of NR2F1 Ab H8132 (green), Hoechst (blue), and two different nucleolar markers, Fibrillarin (red) and NPM1 (red). C Live-cell imaging of hiPSC^NR2F1-GFP 24 h after transfection shows nuclear but not nucleolar NR2F1 localization. Zoom-in is marked with a white box and shown for the GFP channel below. Arrowheads point to NR2F1-GFP-negative nucleoli. Scale bars: 50 µm.

Fig. 3. Localization and quantification of NR2F1 levels by IF in untransfected HEK293 and HEK293^NR2F1 cells.

A and B Gray boxes in the first column show the selected zoom-in area placed next to it. Nucleoplasm (blue) was stained with Hoechst. Scale bars: 50 µm. A Negative control: untransfected HEK293 cells that do not express NR2F1 endogenously. Arrowheads indicate nucleolar-like foci exclusively in the Ab3 staining. B HEK293^NR2F1 cells 48 h after transfection with an NR2F1 plasmid. Arrowheads indicate examples of nucleoli typically not stained by Ab1 and Ab2 and nucleolar-like foci stained by Ab3, seen only in cells that were unlikely to have been successfully transfected. C MFI of histograms resulting from NR2F1 staining measured for each Ab used (x-axis) in untransfected control HEK293 cells (gray columns) and in HEK293^NR2F1 cells (pink columns). The data are shown in arbitrary units (AU) and represented as mean ± SEM; the MFI was measured in at least 330 and up to 800 cells per sample. D SNRs between the MFI of HEK293^NR2F1 and untransfected HEK293 cells are shown in AU as mean ± SEM for each Ab.

Fig. 4. Detection of endogenous NR2F1 in WT and hiPSC^−/−, NP/N^−/− by IF using seven Abs.

A and B Gray boxes show the selected zoom-in area placed next to it. (A-C) The neuronal marker TUJ1 (green) was used only for IF in differentiated cells. Nucleoplasm (blue) was stained with Hoechst. Scale bars: 50 µm. The corresponding zoom-in is next to each image. A Undifferentiated WT hiPSC: arrowheads point to unspecific IF signals in the cytoplasm after Ab1 and Ab2 staining and to nucleolar-like foci after Ab3 staining. B NP/N derived from WT hiPSC. C Undifferentiated hiPSC^−/− with arrowheads indicating nucleolar-like foci stained by Ab3 as well as NP/N^−/−, which showed no foci. D MFI of each NR2F1 staining (Abs1–7) in AU (mean ± SEM) measured in the nucleus of undifferentiated hiPSC (gray columns) and of hiPSC-derived NP/N (magenta columns) in at least 120 cells per sample (up to 500 cells per sample). E SNRs measured for each Ab (Abs1–7) in undifferentiated WT hiPSC and hiPSC-derived WT NP/N shown in AU as mean ± SEM. F MFI measured at least in 60 and up to 150 cells per sample of each NR2F1 staining (Ab1–Ab7) displayed as mean ± SEM in undifferentiated hiPSC^−/− (blue columns) and NP/N^−/− (green columns).

Fig. 5. Comparison of the anterior-posterior Nr2f1 gradient in WT and KO mouse brains at E13.5 in IF using seven Abs.

A–F Sagittal sections of whole WT and KO mouse brains. Nucleoplasm (blue) was stained with Hoechst. A–D The overview of IF staining with Ab1 and Ab3 in WT and KO shows a strong background signal in the case of Ab3 in KO with a schematic of the anterior–posterior and dorsoventral axes shown in (A). Scale bars: 500 µm. E Posterior images of Nr2f1 KO. Greay boxes indicate the area of zoom-in shown next to it. F Anterior areas and posterior areas in WT. Gray boxes in E and F indicate zoom-in areas. Only Abs1-4 can detect Nr2f1, but Ab3 leads to high background staining localized in the cytoplasm or extracellular matrix rather than in the nucleoli. Scale bars: 50 µm in the first column of E and first and second columns of F; 25 µm in the last column of (E) and (F). G MFI of each Nr2f1 staining measured in random fields from at least 6 and up to 12 different cortical regions per sample in Nr2f1 KO and WT brains represented as mean ± SEM in AU. H SNRs (WT vs. KO).

Fig. 6. Different fixation times and conditions for IF staining with Ab3 and nucleolar co-localization of signals resulting from Ab3 with fibrillarin.

Gray boxes show the selected zoom-in area placed next to it; arrowheads indicate nucleolar areas. A–D Co-staining of NR2F1 with Ab1 (green) and Ab3 (red) in untransfected HEK293 cells and 48 h post-transfection in HEK293^NR2F1 cells. Nucleoplasmi was stained by Hoechst. Only successfully transfected HEK293^NR2F1 cells display double-positive nuclei co-stained by Ab1 and Ab3. A PFA fixation for 15 min and B 2 h reduced the unspecific nucleolar-like foci resulting from Ab3 staining without affecting the Ab1 signal. C 15 min and D 2 h fixation with 70% ethanol completely prevented the occurrence of unspecific nucleolar-like foci in the Ab3 staining without affecting the Ab1 staining. Scale bars: 50 µm. E and F Scale bars: 59 µm. Co-staining of fibrillarin (red) with Ab1, Ab2, and Ab3 (red). E–F Nucleoplasm was stained by DAPI (blue). E WT hPSC shows a specific nucleolar pattern stained by Ab3 that overlaps with fibrillarin in DAPI-depleted regions. F In WT hNCC cells, all Abs stain the NR2F1 nucleus (DAPI-positive but fibrillarin-negative regions), but only Ab3 overlaps with fibrillarin in DAPI-depleted regions.

Fig. 7. Testing all of the seven anti-NR2F1 Abs in FC and WB.

A–D Quantification of NR2F1 levels by FC using all seven Abs in HEK293 and HEK293^NR2F1 cells. A and B Untransfected and C and D HEK293^NR2F1 cells were fixed with 70% ethanol 48 h after transfection, and stained with the primary Abs, followed by adequate AF488 as a secondary Ab. A and C FC data. For each sample, two batches with each 10,000 events were measured using the same settings and show that Ab1, Ab3, and Ab4 detect NR2F1 best under these conditions. B and D After FC measurement, the cell suspensions of the remaining samples were stained with Hoechst (blue), spotted on glass slides, and visualized by fluorescence microscopy. Scale bars: 50 µm. E and F Detection of NR2F1 in WB in samples from WT hiPSC and hNCC as well as hNCC^−/− and hNCC^+/−. 20 µg per sample of RIPA lysate was loaded. Red arrows mark the 44–46 kDa band corresponding to the specific size of NR2F1, blue arrows mark GAPDH (~36 kDa) used as loading control. E Undifferentiated WT hiPSC and hNCC. F hNCC^−/− and hNCC^+/−. All Abs recognized the 44–46 kDa band corresponding to NR2F1 hNCC^+/−, and, depending on the Ab, several other bands. Ab6 and Ab7 also recognized the 44–46 kDa band in hNCC^−/−, which was not detected by other Abs. M molecular size marker.