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. 2021 Nov 25;12:6839. doi: 10.1038/s41467-021-27172-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Immunofluorescence of NOTCH1 co-stained with DDX4 in E11.5–E16.5 mouse male gonads. Hollow and solid yellow arrowheads indicate NOTCH1^Negative and NOTCH1^Positive subtypes in DDX4⁺ cells, respectively. Scale bar, 10 μm. b Immunofluorescence of HES1 co-stained with DDX4 in E11.5–E16.5 mouse male gonads. White and yellow arrowheads indicate HES1^Dim and HES1^Bright subtypes in DDX4⁺ cells, respectively. Scale bar, 10 μm. c Proportion of NOTCH1^Negative and NOTCH1^Positive subtypes in DDX4⁺ cells in E11.5–E16.5 mouse male gonads. Mean ± SEM, n = at least 2 biologically independent samples for each time-point. d Proportion of HES1^Dim and HES1^Bright subtypes in DDX4⁺ cells in E11.5–E16.5 mouse male gonads. Mean ± SEM, n = at least three biologically independent samples for each time-point. e Schematic showing the workflow of treatment of γ-Secretase inhibitor–DAPT or LY411575 for Notch signaling pathway blocking. f Immunofluorescence of NOTCH1 co-stained with DDX4 and the quantification of relative fluorescence intensity in DAPT-treatment and control mouse male gonads at E15.5. Scale bar, 10 μm. Mean ± SEM, n = 3 per group, **** P < 0.0001, P = 9.5E–13, unpaired two-tailed t test. g Immunofluorescence of HES1 co-stained with DDX4 and the quantification of relative fluorescence intensity in DAPT-treatment and control mouse male gonads at E15.5. Scale bar, 10 μm. Mean ± SEM, n = 3 per group, **** P < 0.0001, P = 4.0E–25, unpaired two-tailed t test. h Immunofluorescence of MKI67 co-stained with DDX4 and proportion of MKI67⁺ cells in DDX4⁺ cells in DAPT-treatment and control mouse male gonads at E15.5. Solid yellow arrowheads indicate MKI67^Positive subtypes in DDX4⁺ cells. Scale bar, 10 μm. Mean ± SEM, n = 3 per group, **** P < 0.0001, P = 8.8E–07, unpaired two-tailed t test. i Immunofluorescence of ProSPG and SPG marker ZBTB16 co-stained with DDX4 and proportion of ZBTB16⁺ cells in DDX4⁺ cells in DAPT-treatment and control mouse male gonads at PND0. Hollow and solid yellow arrowheads indicate ZBTB16^Negative and ZBTB16^Positive subtypes in DDX4⁺ cells, respectively. Scale bar, 10 μm. Mean ± SEM, n = 3 per group, **** P < 0.0001, P = 1.2E–18, unpaired two-tailed t test.