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Pou5f1

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POU5F1 is a protein-coding gene that plays a crucial role in the regulation of embryonic development and the maintenance of embryonic stem cell pluripotency. It is a member of the POU family of transcription factors and is widely used as a marker for undifferentiated stem cells.

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Lab products found in correlation

Tnnt2, by Thermo Fisher Scientific (3 mentions) Ab25388, by Novus Biologicals (2 mentions) Ab11174, by Fortis Life Sciences (2 mentions) Hexim1, by Abcam (2 mentions) Sc 8629, by Abcam (2 mentions) Nb100 1781, by Abcam (2 mentions) γ h2ax, by Abcam (2 mentions) Mesp1, by Abcam (2 mentions) Nanog, by Cell Signaling Technology (2 mentions) Desmin, by Santa Cruz Biotechnology (1 mentions) Tnnt2, by Santa Cruz Biotechnology (1 mentions) Sox17, by R&D Systems (1 mentions) Nestin, by Abcam (1 mentions) Triton x 100, by Thermo Fisher Scientific (1 mentions) Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions) Ix70 inverted microscope, by Olympus (1 mentions) Ab150404, by Abcam (1 mentions) Ab110175, by Abcam (1 mentions) Cxcr4, by Abcam (1 mentions) Smad4, by Abcam (1 mentions)

6 protocols using pou5f1

1

Immunofluorescence Analysis of Stem Cell Markers

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Thawed cells were allowed to establish colonies, passaged and grown on coverslips. Specimens were fixed in 4% v/v paraformaldehyde in PBS for 15 min at room temperature, washed, and exposed to either 5% v/v goat serum or 5% v/v donkey serum, 1% w/v bovine serum albumin, and 0.1% v/v Triton X-100 (Fisher) in PBS for 30 min. The fixed specimens were then incubated with primary antibodies at 4 °C overnight. After washing, they were exposed to secondary antibodies. Colonies exposed only to secondary antibody served as controls. VECTASHIELD mounting medium with DAPI (Vector Laboratories) was used to mount the coverslips. Primary antibodies were: POU5F1 (1:100; Santa Cruz Biotechnology, catalog no. sc-9081), SOX2 (1:1000; R&D Systems, catalog no. MAB2018), NANOG (1:200; Abcam, catalog no. ab109250), SSEA1 (1:50; Developmental Studies Hybridoma Bank [DSHB], Hybridoma Product MC-480 deposited by Solter, D./Knowles, B.B), SSEA4 (1:50; DSHB, Hybridoma Product MC-813-70 deposited by Solter, D./Knowles, B.B), KRT7(1:100; DAKO, catalog no. M701801-2), DESMIN (1:100; Santa Cruz Biotechnology, catalog no. sc-14026), NESTIN (1:100; Abcam, catalog no. ab6320), SOX17 (1:100; R&D Systems, catalog no. AF1924), TNNT2(1:100; Santa Cruz Biotechnology, catalog no. sc-8121). Images were taken under an Olympus IX70 inverted microscope (www.biotech.missouri.edu/mcc/Olympus.html).
Yuan Y., Yang Y., Tian Y., Park J., Dai A., Roberts R.M., Liu Y, & Han X. (2016). Efficient long-term cryopreservation of pluripotent stem cells at −80 °C. Scientific Reports, 6, 34476.
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2

ChIP-seq analysis of transcription factors

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ChIP-seq was performed as described previously33 . DNA was purified for subsequent qPCR analysis or deep sequencing library construction (as above for ChIRP-seq). The following antibodies were used for ChIP studies: Pou5f1 (Santa Cruz, sc-8629), Hexim1 (Abcam, ab25388), Ddx21 (Novus, NB100-1781), and Baf155 (Gift from G. Crabtree), γ-H2AX (Abcam, ab11174), and Brd4 (Bethyl, A301-985A).
Flynn R.A., Do B.T., Rubin A.J., Calo E., Lee B., Kuchelmeister H., Rale M., Chu C., Kool E.T., Wysocka J., Khavari P.A, & Chang H.Y. (2016). 7SK-BAF axis controls pervasive transcription at enhancers. Nature structural & molecular biology, 23(3), 231-238.
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3

Pluripotency and Lineage Markers Quantification

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POU5F1 (sc‐5279, Santa Cruz, Heidelberg, Germany); NANOG (4903S, Cell Signaling, Leiden, Netherlands); SOX2 (3579, Cell Signaling); PAX6 (DSHB, Iowa, Iowa); TBXT (AF2085, R&D system, Minneapolis, Minnesota); MESP1 (ab77013, Abcam, Cambridge, UK); ISL1 (39.4D5, DSHB); NKX2‐5 (sc‐14 033, Santa Cruz); TNNT2 (MS‐295‐P1, ThermoFisher Scientific); V5 (R960‐25, ThermoFisher Scientific); ZIC2 (ab150404, Abcam).
Xu J., Zhou C., Foo K.S., Yang R., Xiao Y., Bylund K., Sahara M, & Chien K.R. (2020). Genome‐wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors. Stem Cells (Dayton, Ohio), 38(6), 741-755.
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4

ChIP-seq analysis of transcription factors

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ChIP-seq was performed as described previously33 . DNA was purified for subsequent qPCR analysis or deep sequencing library construction (as above for ChIRP-seq). The following antibodies were used for ChIP studies: Pou5f1 (Santa Cruz, sc-8629), Hexim1 (Abcam, ab25388), Ddx21 (Novus, NB100-1781), and Baf155 (Gift from G. Crabtree), γ-H2AX (Abcam, ab11174), and Brd4 (Bethyl, A301-985A).
Flynn R.A., Do B.T., Rubin A.J., Calo E., Lee B., Kuchelmeister H., Rale M., Chu C., Kool E.T., Wysocka J., Khavari P.A, & Chang H.Y. (2016). 7SK-BAF axis controls pervasive transcription at enhancers. Nature structural & molecular biology, 23(3), 231-238.
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5

Pluripotency and Cardiac Differentiation

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POU5F1 (sc‐5279, Santa Cruz Biotechnology Inc., Santa Cruz, CA); NANOG (4903S, Cell Signaling Technology, Beverly, MA); SOX2 (3579, Cell Signaling); PAX6 (Developmental Studies Hybridoma Bank [DSHB], Iowa City, IA); TBXT (AF2085, R&D Systems Inc.); MESP1 (ab77013, Abcam, Cambridge, U.K.); ISL1 (39.4D5, DSHB); NKX2‐5 (sc‐14033, Santa Cruz Biotechnology Inc.); TNNT2 (MS‐295‐P1, Thermo Fisher Scientific); V5 (R960‐25, Thermo Fisher Scientific); SMAD4 (ab110175, Abcam; sc‐7966, Santa Cruz Biotechnology Inc.); GAPDH (8884S, Cell Signaling); CXCR4 (ab1670, Abcam); APLNR (702069, Thermo Fisher Scientific); CD13 (ab7417, Abcam).
Xu J., Gruber P.J, & Chien K.R. (2018). SMAD4 Is Essential for Human Cardiac Mesodermal Precursor Cell Formation. Stem Cells (Dayton, Ohio), 37(2), 216-225.
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6

Flow Cytometry Analysis of Stem Cell Markers

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Specific markers were probed with antibodies using flow cytometry. Cells were dissociated and centrifuged into pellet in a microcentrifuge tube. The cells were fixed with reagent A of the Fix and Perm kit (ThermoFisher, Cat.#GAS004) for 20 mins at room temperature, followed by a wash with 1 mL of 1% BSA (Sigma, Cat.#A7906) in PBS and spun at 10,000 rpm for 30 s. Subsequently, cells were permeabilized with reagent B in the Fix and Perm kit and incubated together with primary antibodies: POU5F1 (Santa Cruz, Cat.#sc-5279, 1:20), Tra1-60 (Millipore, Cat.#MAB4360, 1:50), TNNT2 (Thermo Scientific, Cat.#MS-295-p1, 1:200), MYH1 (DSHB, Cat.#MF-20, 1:20) or appropriate isotype control, for 20 mins at room temperature. After incubation, the cells were washed with 1% BSA, spun and incubated in the dark for 15 mins with the appropriate Alexa-conjugated secondary antibody (ThermoFisher) at 1:1000 dilutions in 1% BSA. Finally, the cells were washed and resuspended in 300 mL of 1% BSA prior to analysis in FACS Fortessa (Becton Dickinson). Data were analyzed using FlowJo software (Version 8). Cell gating (% expression) was done at the intersection between the isotype control and the marker expression (Yap et al., 2011) . There were 10 replicates in each group (n = 10).
Yap L., Wang J.W., Moreno-Moral A., Chong L.Y., Sun Y., Harmston N., Wang X., Chong S.Y., Vanezis K., Öhman M.K., Wei H., Bunte R., Gosh S., Cook S., Hovatta O., de Kleijn D.P.V., Petretto E, & Tryggvason K. (2019). In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors. Cell reports, 26(12).
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