Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A ) Representative images of immunohistochemical staining of Hrd1 in the brains of 7-week-old C57BL/6J mice on LFD ( n = 2 each group). Zoomed-in images of ARC and thalamus are shown on the right. 3V, third ventricle. Representative images of negative control IgG are shown in . ( B ) Representative images of Hrd1 staining in the ARC of 7-week-old POMC-eGFP reporter mice after an overnight fast with or without 6-hour refeeding. Quantitation of Hrd1 signals in POMC neurons (green arrows) and non-POMC neurons (white arrows) shown on the right ( n = 2 mice each group, 70 neurons each mouse). ( C ) Representative images of Hrd1 staining in the ARC of 8-week-old Sel1L POMC ;POMC-eGFP and control Sel1L POMC/+ ; POMC-eGFP mice on LFD ( n = 3–4 each group). Green arrows point to POMC neurons; white arrows point to non-POMC neurons. ( D ) Quantitation of Hrd1 level shown in C in POMC and non-POMC neurons in the ARC ( n = 70 and n = 100 neurons per mouse, n = 3–4 mice each). ( E and F ) Growth curve of Sel1L fl/fl ( n = 5), heterozygous Sel1L POMC/+ ( Sel1L fl/+ ;Pomc-Cre , n = 3), and Sel1L POMC mice ( n = 7) on LFD. In E , a green dotted line marks the age at which Sel1L POMC mice became significantly more obese. ( G ) Body weight of 10- and 40-week-old mice on LFD. ( H ) Representative image of 40-week-old mice on LFD. ( I ) Body composition of 10-week-old ( n = 3 each) and 40-week-old ( n = 6–7 each) male mice on LFD. ( J and K ) Representative images of peripheral tissues ( J ) and H&E images of peripheral tissues ( K ) from 40-week-old mice ( n = 3 each group). gWAT, gonadal WAT. ( L and M ) Serum leptin ( L ) and insulin ( M ) levels of 8- and 40-week-old mice of both sexes fed ad libitum LFD ( n = approximately 4–6 each group). Values are shown as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, 2-way ANOVA.

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Immunohistochemical staining, Staining, Negative Control, Quantitation Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A ) Schematic diagram showing specific domains and processing derivatives of POMC recognized by various antibodies. SP, signal peptide; β-endo, β-endorphin. ( B – E ) Representative immunofluorescence images of ( B ) POMC as a prohormone ( n = 5–6 each) in the ARC of mice approximately 5 to 10 weeks of age, ( C ) α-MSH in the axons of POMC neurons in the PVN of mice approximately 5 to 10 weeks of age ( n = 4 each), ( D ) β-endorphin ( n = 4–5 in each), and ( E ) ACTH ( n = 2-3 each) in the ARC and DMH of mice approximately 5 to 8 weeks of age fed ad libitum LFD. White arrows indicate staining in the cell bodies of POMC neurons. Quantitation of POMC and neuropeptide signal intensity in cell bodies and axons are shown in . ( F ) Pomc mRNA level in the ARC of mice approximately 5 to 8 weeks of age fed ad libitum LFD ( n = 4 each). ( G ) α-MSH levels in the hypothalamus of mice approximately 6 to 7 weeks of age, measured by ELISA ( n = 3 each group). ( H ) Cumulative food intake in 8-week-old mice injected daily i.p. with α-MSH (1 mg/kg body weight) for 3 days ( n = approximately 5–7 each group). Vehicle, saline. Values are shown as mean ± SEM. * P < 0.05, Student’s t test ( F and G ) or 2-way ANOVA ( H ).

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Immunofluorescence, Staining, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Injection, Saline

Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A and B ) Steady-state protein levels of POMC in WT and ERAD-deficient N2a cells transfected with POMC-WT-Flag. In B , proteasomal inhibitor MG132 was added for the last 2 hours. mRNA levels of Pomc in A and B are shown in . ( C and D ) Ubiquitination of POMC by HRD1 in gain- ( C ) and loss-of-function ( D ) systems: Western blot of ubiquitin in POMC-Flag immunoprecipitates of HEK293T cells transfected with POMC-WT-Flag with or without HRD1. MG132 ( C ) or bortezomib ( D ) was added for the last 2 hours. In D , quantitation of the ratio of ubiquitination (Ub) signal intensity to POMC band intensity is shown in the lower panels. ( E ) Western blot analysis of POMC-Flag immunoprecipitates in transfected WT and Hrd1 –/– N2a cells under nonreducing (-β-ME) or reducing (+β-ME) SDS-PAGE. ( F ) Western blot analysis of endogenous POMC processing using ACTH antibody in POMC-expressing mouse pituitary tumor line AtT20 with or without Sel1L. ( G ) Representative confocal images of POMC in POMC-transfected WT and Sel1L –/– N2a cells. White arrows point to POMC-containing secretory granules, while yellow arrows point to perinuclear POMC. KDEL marks the ER. Representative data from at least 2 independent experiments are shown.

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Transfection, Ubiquitin Proteomics, Western Blot, Quantitation Assay, SDS Page, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A ) Schematic diagram showing amino acid sequence of POMC 26–50 and the positions of 2 disulfide bonds and a free thiol in POMC- C28F . ( B ) Western blot analysis of steady-state levels of POMC proteins in WT and Hrd1 –/– N2a cells transfected with POMC-WT and - C28F . mRNA levels of each sample are shown below. ( C ) Western blot analyses of ubiquitination following immunoprecipitation of POMC in HEK293T cells transfected with POMC-WT-Flag or POMC- C28F -Flag construct with or without HA-Ub, Myc-tagged HRD1-WT, or HRD1 E3 ligase-dead C2A mutant. ( D ) Representative confocal images of POMC in POMC-transfected WT and Sel1L –/– N2a cells. White arrows point to secreted POMC in granules, while yellow arrows point to perinuclear POMC, possibly in the form of aggregates. KDEL marks the ER. ( E ) Sucrose gradient fractionation (fractions 1–7 from top to bottom of gradient) of HEK293T cells expressing POMC-WT or - C28F under nonreducing (–β-ME) and reducing (+β-ME) SDS-PAGE. ( F ) Western blot analysis of Myc-tagged POMC in WT and Hrd1 –/– N2a cells transfected with POMC-WT or - C28F under reducing and nonreducing SDS-PAGE. Red box marks HMW aggregates, while green box marks monomers and dimers. HMW, high molecular weight. (POMC) 2 , POMC dimers. Representative data from at least 2 independent experiments are shown.

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Sequencing, Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Construct, Mutagenesis, Fractionation, Expressing, SDS Page, High Molecular Weight

Journal: The Journal of Clinical Investigation

Article Title: Hypothalamic ER–associated degradation regulates POMC maturation, feeding, and age-associated obesity

doi: 10.1172/JCI96420

Figure Lengend Snippet: ( A ) Under physiological conditions, Sel1L-Hrd1 ERAD plays an important role in promoting a conducive environment for the maturation of nascent POMC in the ER by degrading, likely misfolded, POMC. ( B ) In the absence of ERAD, misfolded POMC accumulates and interferes with the maturation of nascent POMC in the ER in a disulfide bond–dependent manner. ( C ) Under pathological conditions, POMC- C28F is consistently misfolded and forms aggregates via intermolecular C50-mediated disulfide bonds. This model explains the dominant-negative effect of POMC- C28F . ( D ) The maturation defects of POMC- C28F can be completely rescued by an intragenic suppressor mutation, C50S .

Article Snippet: Antibodies for immunostaining were as follows: HA (mouse, 1:500; catalog H9658, Sigma-Aldrich), XTP3-B (goat, 1:500; catalog sc-161409, Santa Cruz Biotechnology Inc.) and Hrd1 (rabbit, 1:500), POMC (goat, 1:250; catalog NB100-1533, Novus Biologicals), ACTH (rabbit, 1:100; catalog G-001-06, Phoenix Pharmaceuticals), α-MSH (sheep, 1:2,000; catalog AB5087, Millipore), β-endorphin (rabbit, 1:2,000; catalog H-022-33, Phoenix Pharmaceuticals, provided by Carol Elisa), and GFP (chicken, 1:500; catalog GFP-1020, Aves Labs).

Techniques: Dominant Negative Mutation, Mutagenesis

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: Genetically engineered human pituitary corticotroph tumor organoids exhibit divergent responses to glucocorticoid receptor modulators

doi: 10.1016/j.trsl.2023.01.002

Figure Lengend Snippet: Expression of cell linages and transcription factors in iPSC generated pituitary tumor organoids. (A) , Immunohistochemistry using FFPE sections prepared from iPSC ctrl , iPSC USP48MV , iPSC USP48MI , and iPSC USP8 organoids stained with antibodies specific for CAM5.2, T-Pit, Synaptophysin and ACTH. High magnification images are shown in insets. (B) , Differential expression of transcription factors TPit, PIT1 and SF1 and POMC were measured by qRT-PCR. ( C ), Plot comparing the ACTH secretion (pg/mL), measured by an ELISA using conditioned media of iPSC ctrl , iPSC USP48MV , iPSC USP48MI , and iPSC USP8 throughout the directed differentiation schedule. (D) , viSNE maps showing concatenated flow cytometry standard files for iPSC ctrl , iPSC USP48MV , iPSC USP48MI , and iPSC USP8 organoids 30 days post-directed differentiation. Maps define spatially distinct cell populations using pituitary specific cell lineage, stem cell and transcription factor markers between iPSC ctrl organoids and mutant lines. ( E ), viSNE heatmaps showing the number of cells positive for proliferation marker Ki67. ( F ), Violin plots comparing the TPit lineage expression, through proliferation marker Ki67 between control and mutated iPSC lines. * P < 0.05 compared to iPSC ctrl organoids, n = 3 individual experimental replicates.

Article Snippet: Pre-designed real-time polymerase chain reaction assays were purchased for the following genes: TPit (Thermo Fisher Scientific 4331182 Hs00193027), SF1 (Thermo Fisher Scientific 4331182 Hs00610436), SSTR2 (Thermo Fisher Scientific 4331182 Hs00265624_s1), SSTR5 (Thermo Fisher Scientific 4331182 Hs00990407_s1), POMC (Thermo Fisher Scientific 4331182 Hs01596743_m1), hPRT (Thermo Fisher Scientific 4331182 Hs02800695_m1), and FKBP5 (Thermo Fisher Scientific 4331182 Hs01561006_m1).

Techniques: Expressing, Generated, Immunohistochemistry, Staining, Quantitative Proteomics, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Mutagenesis, Marker, Control

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: Genetically engineered human pituitary corticotroph tumor organoids exhibit divergent responses to glucocorticoid receptor modulators

doi: 10.1016/j.trsl.2023.01.002

Figure Lengend Snippet: Differential expression of SSTR2 and SSTR5 in iPSC generated pituitary tumor organoids in response to mifepristone and relacorilant. Differential expression of (A) , SSTR2 and (B) , SSTR5 in iPSC ctrl , iPSC USP48MI , iPSC USP48MV and iPSC USP8 organoids in response to vehicle (Veh), mifepristone (Mife, 500nM) or relacorilant (Rela, 500nM). * P < 0.05 compared to vehicle-treated organoids, # P < 0.05 compared to mifepristone treated organoids, n = 3 experimental replicates/organoid line. (C) , Differential expression of SSTR2 and SSTR5 in iPSC ctrl organoids treated with vehicle (Veh), mifepristone (Mife), GANT61 (GANT, 5 μ M), Mife+GANT, ketoconazole (Keto, 10 μ M), Mife+Keto, or dexamethasone (Dexa, 100nM). * P < 0.05 compared to iPSC ctrl organoids treated with Veh, n = 3 experimental replicates/organoid line. (D) , Differential expression of SSTR2 and SSTR5 in iPSC ctrl organoids treated with vehicle (Veh), relacorilant (Rela), GANT61 (GANT), Rela+GANT, ketoconazole (Keto), Rela+Keto, or dexamethasone (Dexa). * P < 0.05 compared to iPSC ctrl organoids treated with Veh, n = 3 experimental replicates/organoid line. (E) , Mutations in USP48 and USP8 in PitNETs are believed to enhance corticotropin releasing hormone (CRH)-induced production coherent with the Hh signaling pathway. Hh ligand, Sonic Hedgehog (Shh), binds to Patched (Ptch1) that relieves suppression of Smoothened (SMO) and subsequently Gli1 activation. Crosstalk between Shh and CRH at the Gli1 level stimulates POMC transcription and ACTH secretion. (F) , Differential expression of POMC in iPSC ctrl , iPSC USP48MI , iPSC USP48MV and iPSC USP8 organoids. * P < 0.05 compared to iPSC ctrl organoids, n = 3 experimental replicates/organoid line. (G) , Representative western blots of the expression of Gli1 relative to GAPDH in iPSC ctrl , iPSC USP48MI , iPSC USP48MV and iPSC USP8 organoids. (H) , Quantification of western blots shown in G . * P < 0.05 compared to iPSC ctrl organoids, n = 3 experimental replicates/organoid line. (I) , Mutations in USP8 and USP48 detected in hPITO cultures (hPITO1 and 7) were also expressed in the patient’s matched PitNET tissue. (J) , Human PITO cultures expressing USP8 and USP48 mutations were used for gene ChIP analysis after treatment with vehicle or GANT61. * P < 0.05 compared to iPSC ctrl organoids, n = 4 experimental replicates/organoid line.

Article Snippet: Pre-designed real-time polymerase chain reaction assays were purchased for the following genes: TPit (Thermo Fisher Scientific 4331182 Hs00193027), SF1 (Thermo Fisher Scientific 4331182 Hs00610436), SSTR2 (Thermo Fisher Scientific 4331182 Hs00265624_s1), SSTR5 (Thermo Fisher Scientific 4331182 Hs00990407_s1), POMC (Thermo Fisher Scientific 4331182 Hs01596743_m1), hPRT (Thermo Fisher Scientific 4331182 Hs02800695_m1), and FKBP5 (Thermo Fisher Scientific 4331182 Hs01561006_m1).

Techniques: Quantitative Proteomics, Generated, Activation Assay, Western Blot, Expressing