hhex  (R&D Systems)

Journal: Nature Communications

Article Title: The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion

doi: 10.1038/s41467-025-68257-4

Figure Lengend Snippet: A Sf1-Cre high mice were bred with Rosa mT/mG mice to label steroidogenic lineage with membrane Green Fluorescent Protein (mG). Non-recombined cells express Tomato (mT). Representative image from a cryosection of an adult male Sf1-Cre high mT/mG adrenal gland depicting endogenous mGFP (steroidogenic cells) and mTomato fluorescence. Hoechst was used to stain the nuclei. B Enzymatic and mechanical single-cell dissociation at low temperature, followed by FACS sorting. The image depicts GFP-positive cells after sorting. C UMAP representation of scRNA-seq dataset depicting steroidogenic cell populations in the adult male adrenal ( n = 2 replicates, 34 adrenals). D , E Visualization of C yp11b2 ( D ) and Cyp11b1 ( E ) expression in the scRNA-seq dataset. F Cyp11b2 and Cyp11b1 RNAscope on adrenal serial sections of 15-week-old WT male ( n = 5) and WT female ( n = 5) mice. Nuclei were stained in blue with hematoxylin. Blue and red stars represent capsular arterioles used to image the same area for both transcripts. G Volcano Plot representing differentially expressed genes (DEGs) between zG and zF. p -values ( p ) were calculated using a non-parametric Wilcoxon rank sum test. Cyp11b1 and Cyp11b2 are highlighted in yellow, and Hhex is highlighted in red. Created in BioRender. Dumontet, T. (2026) https://BioRender.com/fdp7ezq . FACS Fluorescent-Activated Cell Sorting, GFP Green Fluorescent Protein, UMAP Uniform Manifold Approximation and Projection, NS non-significant, FC Fold change, ZG zona glomerulosa, ZF zona fasciculata, CYC cycling, WT Wild Type.

Article Snippet: Hhex flox/flox mice were purchased at Jackson Laboratory (Stock 025396; B6N.129S1(Cg)-Hhex /J; https://www.jax.org/strain/025396 ) and initially donated by Dr. Clifford Bogue, M.D., Yale University, USA .

Techniques: Membrane, Fluorescence, Staining, Single Cell, Expressing, RNAscope, FACS

Journal: Nature Communications

Article Title: The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion

doi: 10.1038/s41467-025-68257-4

Figure Lengend Snippet: A Visualization of Hhex expression in the scRNA-seq dataset. B , C Expression of HHEX in the male mouse ( n = 5) ( B ), and male rat ( n = 2) adrenal glands in brown by immunohistochemistry. Nuclei were stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. Dotted rectangles depict the insets. D Immunofluorescence for HHEX in yellow in the human adrenocortical cell line H295R ( n = 2). Nuclear counterstain in blue. E Expression of HHEX in the human adrenal ( n = 3). Dotted rectangles depict the insets. IHC immunohistochemistry, Cap. Capsule, zG zona glomerulosa, zF zona fasciculata, zR zona reticularis, Med. medulla, w weeks, neg. negative.

Article Snippet: Hhex flox/flox mice were purchased at Jackson Laboratory (Stock 025396; B6N.129S1(Cg)-Hhex /J; https://www.jax.org/strain/025396 ) and initially donated by Dr. Clifford Bogue, M.D., Yale University, USA .

Techniques: Expressing, Immunohistochemistry, Staining, Immunofluorescence

Journal: Nature Communications

Article Title: The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion

doi: 10.1038/s41467-025-68257-4

Figure Lengend Snippet: A Schematic representation of the transgenic mouse models used to inactivate HHEX in adrenocortical cells using Sf1-Cre high -mediated recombination of exon 2 and 3 of Hhex gene. B Quantification of Hhex transcripts by RT-qPCR in 6-week-old WT (n = 12) and Sf1-Cre high Hhex KO ( n = 8) male adrenals. p -value ( p ) was calculated using a two-tailed Mann–Whitney test. C Expression of HHEX in 6-week-old WT ( n = 4) and Sf1-Cre high Hhex KO ( n = 4) male adrenals in brown by immunohistochemistry. Nuclei were stained in blue with hematoxylin. D Corticosterone plasma levels of 15–19-week-old WT ( n = 21) and Sf1-Cre high Hhex KO males ( n = 12). p -value ( p ) was calculated using a two-tailed Mann–Whitney test. ( E ) Ex vivo corticosterone production, released in culture media by adrenal explants after 2.5 h incubation with 2.5 mM of Bt2-cAMP. WT ( n = 6) and Sf1-Cre high Hhex KO males ( n = 7). p -values ( p ) were calculated using a 2-way ANOVA followed by a Šídák’s multiple comparisons test. F , G Quantification of Cyp21a1 ( F ) and Cyp11b1 ( G ) transcripts by RT-qPCR in 15-week-old WT ( n = 12) and Sf1-Cre high Hhex KO ( n = 7) male adrenals. p -value ( p ) were calculated using a two-tailed unpaired t-test with Welch’s correction. ( H ) CYP21A1 immunohistochemistry in adrenals of 15-week-old WT ( n = 6) and Sf1-Cre high Hhex KO ( n = 6) male mice. Nuclei were stained in blue with hematoxylin. I Cyp11b1 RNAscope in adrenals of 15-week-old WT ( n = 7) and Sf1-Cre high Hhex KO ( n = 6) male mice. Nuclei were stained in blue with hematoxylin. J Oil Red O staining of 15-week-old WT ( n = 3) and Sf1-Cre high Hhex KO ( n = 3) male adrenals. Dotted rectangles depict the insets. K Cholesterol esters quantification in adrenals of 45-week-old WT ( n = 7) and Sf1-Cre high Hhex KO ( n = 5) male mice. p -value ( p ) was calculated using a two-tailed unpaired t-test with Welch’s correction. B , D–G , K Graph represents box plots with individual biological replicates, the range (whiskers) and the median (line) within the upper (75%) and lower (25%) quartiles. Source data and exact p -values are provided as a Source data file. C , H , I , J Dotted lines represent the corticomedullary junction. Created in BioRender. Dumontet, T. (2026) https://BioRender.com/fdp7ezq . IHC immunohistochemistry, Cap. Capsule, zG zona glomerulosa, zF zona fasciculata, izF inner zF, Med. medulla, w weeks, WT Wild Type, KO Knockout, Bt2-cAMP dibutyryl cyclic adenosine monophosphate, ns non-significant.

Article Snippet: Hhex flox/flox mice were purchased at Jackson Laboratory (Stock 025396; B6N.129S1(Cg)-Hhex /J; https://www.jax.org/strain/025396 ) and initially donated by Dr. Clifford Bogue, M.D., Yale University, USA .

Techniques: Transgenic Assay, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Expressing, Immunohistochemistry, Staining, Clinical Proteomics, Ex Vivo, Incubation, RNAscope, Knock-Out

Journal: Nature Communications

Article Title: The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion

doi: 10.1038/s41467-025-68257-4

Figure Lengend Snippet: A Schematic of lipophagy also known as LAL-mediated lipolysis. B PLIN1 immunohistochemistry in adrenals of 15-week-old WT ( n = 3) and Sf1-Cre high Hhex KO ( n = 5) male mice. Nuclei were stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. C p62/SQSTM1 immunohistochemistry in adrenals of 15-week-old WT ( n = 5) and Sf1-Cre high Hhex KO ( n = 6) male mice. Nuclei were stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. D Quantification of PLIN1 immunohistochemistry in the upper cortex and inner zF of 15-week-old WT ( n = 3) and Sf1-Cre high Hhex KO ( n = 5) male mice using Fiji. Scatter dot plot represents the mean with 95% confidence interval, and individual biological replicates. p -values ( p ) were calculated using a two-tailed unpaired t -test with Welch’s correction. E–G Quantification of Map1lc3a ( E ) ( n = 6 WT and 8 Sf1-Cre high Hhex KO ), Lipa ( F ) ( n = 12 WT and 7 Sf1-Cre high Hhex KO ), and Abca1 ( G ) ( n = 12 WT and 7 Sf1-Cre high Hhex KO ) transcripts by RT-qPCR in 15-week-old WT and Sf1-Cre high Hhex KO male adrenals. p -values ( p ) were calculated using a two-tailed Mann–Whitney test ( Map1lc3a ) and two-tailed unpaired t -tests with Welch’s correction ( Lipa and Abca1 ). E–G Graph represents box plots with individual biological replicates, the range (whiskers) and the median (line) within the upper (75%) and lower (25%) quartiles. H PLIN1 immunohistochemistry in adrenals of Sf1-Cre high Hhex KO , vehicle ( n = 7), and hydroxychloroquine sulfate-treated ( n = 5) 15-week-old male mice. Nuclei were stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. Dotted rectangles depict the insets. I Quantification of PLIN1 immunohistochemistry in the inner zF using Fiji (Vehicle: n = 7, HCQ-S: n = 5). Scatter dot plot represents the mean with 95% confidence interval, and individual biological replicates. p -value ( p ) was calculated using a two-tailed Mann–Whitney test. Source data and exact p -values are provided as a Source data file. Created in BioRender. Dumontet, T. (2026) https://BioRender.com/fdp7ezq . LD lipid droplet, LAL lysosomal acid lipase, IHC immunohistochemistry, Cap. Capsule, zG zona glomerulosa, zF zona fasciculata, Med. medulla, WT Wild Type, KO Knockout, Vhl vehicle, HCQ-S hydroxychloroquine sulfate, ns non-significant, w weeks.

Article Snippet: Hhex flox/flox mice were purchased at Jackson Laboratory (Stock 025396; B6N.129S1(Cg)-Hhex /J; https://www.jax.org/strain/025396 ) and initially donated by Dr. Clifford Bogue, M.D., Yale University, USA .

Techniques: Immunohistochemistry, Staining, Two Tailed Test, Quantitative RT-PCR, MANN-WHITNEY, Knock-Out

Journal: Nature Communications

Article Title: The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion

doi: 10.1038/s41467-025-68257-4

Figure Lengend Snippet: A PLIN1 immunohistochemistry in adrenals of WT ( n = 3) and Sf1-Cre high Hhex KO ( n = 3) adrenals at 2, 6, 15-week-old, and over 1-year-old male and female mice. Nuclei were stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. B PLIN1 immunohistochemistry in adrenals of 15-week-old gonadectomized WT ( n = 5), and Sf1-Cre high Hhex KO ( n = 4) male mice. Nuclei were stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. C p62/SQSTM1 (lipophagy marker) immunohistochemistry in adrenals of 15-week-old gonadectomized WT ( n = 5) and Sf1-Cre high Hhex KO ( n = 4) male mice. Nuclei were stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. D Time course of Hhex expression by RT-qPCR in male (circle) and female (triangle) adrenal glands from birth to over 1-year-old. p -values ( p ) were calculated using a 2-way ANOVA followed by a Šídák’s multiple comparisons test. Graph represents box plots with individual biological replicates, the range (whiskers) and the median (line) within the upper (75%) and lower (25%) quartiles. E HHEX expression by immunohistochemistry in the adrenals of 15-week-old WT intact ( n = 3) and gonadectomized ( n = 3) male adrenals. Nuclei were stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. F HHEX expression by immunohistochemistry in the adrenals of 15-week-old WT ( n = 3) and AS-Cre ARKO ( n = 3) male adrenals. Nuclei were stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. G Histogram depicting the accumulation of androgen receptor (AR) CUT&Tag products obtained from adult male adrenal glands matching the Hhex promoter region. H3K27ac is used to identify the open chromatin regions of the genome. Source data and exact p -values are provided as a Source data file. Created in BioRender. Dumontet, T. (2026) https://BioRender.com/fdp7ezq . IHC immunohistochemistry, Cap. Capsule, zG zona glomerulosa, zF zona fasciculata, Med. medulla, GDX gonadectomy, Sec. X-zone gonadectomy-induced secondary X-zone, w weeks, yo year-old, P postnatal day, WT Wild Type, KO Knockout, AS aldosterone synthase, bp base pair, ns non-significant.

Article Snippet: Hhex flox/flox mice were purchased at Jackson Laboratory (Stock 025396; B6N.129S1(Cg)-Hhex /J; https://www.jax.org/strain/025396 ) and initially donated by Dr. Clifford Bogue, M.D., Yale University, USA .

Techniques: Immunohistochemistry, Staining, Marker, Expressing, Quantitative RT-PCR, Knock-Out

Journal: Nature Communications

Article Title: The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion

doi: 10.1038/s41467-025-68257-4

Figure Lengend Snippet: A Principal Component Analysis (PCA) plots from bulk RNAseq analysis of 6-week-old WT and Sf1-Cre high Hhex KO male and female adrenal glands ( n = 4 for each group). B , C Nr0b1 ( B ) and Frzb ( C ) expression by RT-qPCR in the adrenal gland of 6-week-old female ( n = 17 WT and 6 Sf1-Cre high Hhex KO ) and male ( n = 17 WT and 8 Sf1-Cre high Hhex KO ) mice. p -values ( p ) were calculated using a 2-way ANOVA followed by a Šídák’s multiple comparisons test. D , E Expression of Nr0b1 ( D ) and Frzb ( E ) by RNAscope in adrenal glands of 6-week-old WT female ( n = 4) and male ( n = 5) and Sf1-Cre high Hhex KO male ( n = 4). F , G Time course of Nr0b1 ( F ) and Frzb ( G ) expression by RT-qPCR in male (circle) and female (triangle) adrenal gland from birth to over 1-year-old. p -values ( p ) were calculated using a 2-way ANOVA followed by a Šídák’s multiple comparisons test. B , C , F , G Graph represents box plots with individual biological replicates, the range (whiskers) and the median (line) within the upper (75%) and lower (25%) quartiles. H AR expression by immunohistochemistry in 15-week-old WT ( n = 5) and ARKO ( n = 3) male adrenals. Insets are focusing on capsule/zG cells (top) and zF cells (bottom). Nuclei are counterstained with hematoxylin. Dotted lines represent the corticomedullary junction. Dotted rectangles depict the insets. Asterisk is pointing out AR-positive capsular cells in ARKO . I Venn diagram of differentially expressed genes in 6-week-old Sf1-Cre high Hhex KO and 25-week-old AS-Cre ARKO male adrenals compared to their respective WT . Source data and exact p -values are provided as a Source data file. Created in BioRender. Dumontet, T. (2026) https://BioRender.com/fdp7ezq . PC principal component, WT Wild Type, KO Knockout, ns non-significant, w weeks, yo year-old, P postnatal day, zG zona glomerulosa, zF zona fasciculata, IHC immunohistochemistry, AS aldosterone synthase, AR androgen receptor.

Article Snippet: Hhex flox/flox mice were purchased at Jackson Laboratory (Stock 025396; B6N.129S1(Cg)-Hhex /J; https://www.jax.org/strain/025396 ) and initially donated by Dr. Clifford Bogue, M.D., Yale University, USA .

Techniques: RNA sequencing, Expressing, Quantitative RT-PCR, RNAscope, Immunohistochemistry, Knock-Out

Journal: Nature Communications

Article Title: The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion

doi: 10.1038/s41467-025-68257-4

Figure Lengend Snippet: A HHEX expression in adult 15-week-old female ( n = 3) and male ( n = 3) adrenals by immunohistochemistry. Nuclei are counterstained with hematoxylin. Dotted squares depict the insets. B Quantification of Hhex transcripts by RT-qPCR in 6-week-old WT ( n = 10) and Sf1-Cre high Hhex KO ( n = 6) female adrenals. C Venn diagram representing the differentially expressed genes in 6-week-old Sf1-Cre high Hhex KO adrenals compared to their respective WT (males in blue circles and females in orange triangles) and heatmap of commonly differentially expressed genes in males and females Sf1-Cre high Hhex KO . D Quantification of Abcb1b transcripts by RT-qPCR in 6-week-old WT ( n = 17 males and 17 females) and Sf1-Cre high Hhex KO ( n = 8 males and 6 females) adrenals. p -values ( p ) were calculated using a two-tailed unpaired t -test with Welch’s correction. E Abcb1 expression in 15-week-old WT and Sf1-Cre high Hhex KO male adrenals by RNAscope, at baseline ( n = 4 WT and 4 Sf1-Cre high Hhex KO ) and after chronic stress ( n = 5 WT and 5 Sf1-Cre high Hhex KO ). Nuclei are stained in blue with hematoxylin. Dotted lines represent the corticomedullary junction. F Quantification of Abcb1b transcripts by RT-qPCR in 15-week-old WT and Sf1-Cre high Hhex KO at baseline ( n = 14 WT and 14 Sf1-Cre high Hhex KO) and after chronic stress ( n = 12 WT and 8 Sf1-Cre high Hhex KO) adrenals. p -values ( p ) were calculated using a 2-way ANOVA followed by a Šídák’s multiple comparisons test. B , D , F Graph represents box plots with individual biological replicates, the range (whiskers) and the median (line) within the upper (75%) and lower (25%) quartiles. Source data and exact p -values are provided as a Source data file. IHC immunohistochemistry, Cap. capsule, zG zona glomerulosa, zF zona fasciculata, Med. medulla, WT Wild Type, KO Knockout, w weeks, FC Fold change, ACTH adrenocorticotropic hormone.

Article Snippet: Hhex flox/flox mice were purchased at Jackson Laboratory (Stock 025396; B6N.129S1(Cg)-Hhex /J; https://www.jax.org/strain/025396 ) and initially donated by Dr. Clifford Bogue, M.D., Yale University, USA .

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test, RNAscope, Staining, Knock-Out