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. 2017 Jul 25;6:e28131. doi: 10.7554/eLife.28131

Figure 3. Aire is essential for spindle assembly in ES cells.

(A) Scheme of the AireFl allele and genotyping strategy before and after cre-induced deletion. Genotyping results from Aire+/+R26CreERT+/+ and AireFl/Fl R26CreERT+/+ ES cells without tamoxifen (-) and with 5 ng/ml tamoxifen for 24 hr (+). (B) qPCR analysis of gene expression at passage one after tamoxifen treatment in Aire+/+R26CreERT+/+ and AireFl/Fl R26CreERT+/+ ES cells. Aire del: primers complementary to deleted region of Aire. Aire c-term: primers complementary to c-terminal region of Aire. Data presented as mean ± sd of 3 biological replicates. p-values were calculated with Wilson’s t-test. (C) Validation of Aire knockout by immunofluorescence imaging in Aire+/+R26CreERT+/+ and AireFl/Fl R26CreERT+/+ ES cells without tamoxifen (-) and with 5 ng/ml tamoxifen for 24 hr (+). bar: 16 μm. (D) Proliferation over two passages and colony formation assay showing total cell number (top panel) and ALP positive colony number (bottom panel) in tamoxifen treated over non-treated Aire+/+R26CreERT+/+ and AireFl/Fl R26CreERT+/+ ES cells. Data in each panel presented as mean ± sd of 3 biological replicates. p-values were calculated with Wilson’s t-test. (E) Representative immunofluorescence images of γ-tubulin (TUBG) foci on mitotic spindles in passage 1 Aire+/+R26CreERT+/+ and AireFl/Fl R26CreERT +/+ ES cells without tamoxifen (-) and with 5 ng/ml tamoxifen for 24 hr (+). Scale bar: 4 μm. Right column shows magnified views of spindle poles. Scale bar: 1 μm. (F) Quantitation of γ-tubulin (TUBG) foci on mitotic spindles in passage 1 Aire+/+R26CreERT+/+ and AireFl/Fl R26CreERT+/+ ES cells without tamoxifen (Con) and with 5 ng/ml tamoxifen for 24 hr (TAM). γ-tubulin (TUBG) foci were counted from 30 mitotic cells from three biological replicates for each group. Each point represents one mitotic cell. Error bars: s.d. of mean. p-values were calculated with Mann–Whitney–Wilcoxon test.

DOI: http://dx.doi.org/10.7554/eLife.28131.010

Figure 3.

Figure 3—figure supplement 1. Complete diagram of Aire floxed (Fl) allele and cre induced depletion of critical exons.

Figure 3—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.28131.011
Figure 3—figure supplement 2. Aire knockout abrogated proliferation and spindle assembly in Pair 2 ES cells.

Figure 3—figure supplement 2.

(A) Genotyping results from Aire Fl/Fl R26CreERT ± and AireFl/+R26CreERT ± ES cells without tamoxifen (-) and with 20 ng/ml tamoxifen for 24 hr (+). (B) qPCR analysis of gene expression at passage one after tamoxifen (TAM) treatment in Aire Fl/Fl R26CreERT ± and AireFl/+R26CreERT ± ES cells. Aire del: primers complementary to deleted region of Aire. Aire c-term: primers complementary to c-terminal region of Aire. Data presented as mean ± sd of 3 biological replicates. p-values were calculated with Wilson’s t-test. (C) Validation of Aire knockout by immunofluorescence imaging in Aire Fl/Fl R26CreERT ± and AireFl/+R26CreERT ± ES cells without tamoxifen (-) and with 20 ng/ml tamoxifen for 24 hr (+). Scale bar: 16 μm. (D) Proliferation over two passages and colony formation assay showing total cell number (top panel) and ALP positive colony number (bottom panel) in tamoxifen treated over non-treated (TAM/Con) Aire Fl/Fl R26CreERT ± and AireFl/+R26CreERT ± ES cells. Data in each panel presented as mean ± sd of 3 biological replicates. p-values were calculated with Wilson’s t-test. (E) Representative immunofluorescence images of γ-tubulin (TUBG) foci on mitotic spindles in passage 1 Aire Fl/Fl R26CreERT ± and AireFl/+R26CreERT ± ES cells without tamoxifen (-) and with 20 ng/ml tamoxifen for 24 hr (+). Scale bar: 10 μm. Right column shows magnified view of spindle poles. Scale bar: 0.5 μm. (F) Quantitation of γ-tubulin (TUBG) foci on mitotic spindles in passage 1 Aire Fl/Fl R26CreERT ± and AireFl/+R26CreERT ± ES cells without tamoxifen (Con) and with 20 ng/ml tamoxifen for 24 hr (TAM). γ-tubulin (TUBG) foci were counted from 30 mitotic cells from three biological replicates for each group. Each point represents one mitotic cell. Error bars: s.d. of mean. p-values were calculated with Mann–Whitney–Wilcoxon test.
DOI: http://dx.doi.org/10.7554/eLife.28131.012
Figure 3—figure supplement 3. Immunofluorescence images of pluripotency markers in Aire Floxed;R26CreERT cells.

Figure 3—figure supplement 3.

Scale bar: 1 μm. (A) Aire+/+R26CreERT+/+: Upper: Con; Bottom: TAM. (B) AireFl/Fl R26CreERT +/+: Upper: Con; Bottom: TAM. (C) AireFl/+R26CreERT+/-: Upper: Con; Bottom: TAM. (D) Aire Fl/Fl R26CreERT+/-: Upper: Con; Bottom: TAM.
DOI: http://dx.doi.org/10.7554/eLife.28131.013