Journal: Gene Therapy

Article Title: First use of gene therapy to treat growth hormone resistant dwarfism in a mouse model

doi: 10.1038/s41434-022-00313-w

Figure Lengend Snippet: a RT-PCR gel of mGHR mRNA expression. 18S rRNA housekeeping gene was used as loading control. b Western blot of mGHR protein expression. Actin housekeeping gene was used as loading control. c Immunofluorescence staining of mGHR expression, scale bars are 50 µm and nuclei were stained with DAPI. HepG2 cells were transfected with pAAV-HLP-mGHR consisted of hybrid liver-specific promoter (HLP) driving the expression of mGHR. Untransfected control and cells transfected with pAAV-HLP-Luc expressing luciferase gene were used as negative control. Analyses were performed 48 h post-transfection.

Article Snippet: The mGHR PCR fragment was amplified from cloning vector MG50043-M (Sino Biological, Beijing, China) containing mouse growth hormone receptor/GHR/GHBP transcript variant 1 gene ORF cDNA clone using forward primer with introducing NotI site: 5’-ATAAGAATGCGGCCGCACCATGGATCTTTGTCAGGTCTTC and reverse primer with introducing XhoI site: 5’-CCGCGCTCGAGCTACTGCATGATTTTGTTCAGTTG.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Negative Control

Journal: The FASEB Journal

Article Title: Leptin regulates parathyroid hormone secretion through CaSR ‐ ERK1 /2 signaling

doi: 10.1096/fj.202403141R

Figure Lengend Snippet: Reduced serum PTH levels in db/db mice, with no change in PTH and CasR mRNA levels. (A, B) Serum PTH levels in db/− and db/db mice at 4 (A) and 7 (B) months of age. (C, D) Serum calcium and blood urea nitrogen (BUN) levels at 7 months of age. (E, F) PTH and CaSR mRNA levels analyzed by qRT‐PCR of RNA extracted from thyroparathyroid glands of db/− and db/db mice at 4 (E) and 7 (F) months of age. * p < .05; *** p < .001.

Article Snippet: Immunostaining was conducted overnight at 4°C using the following primary antibodies diluted in Cas block (Zymed Laboratories, San Francisco, CA): PTH at a dilution of 1:300 (Bio‐Rad, Hercules, CA, RRID:AB 7170-6216); CaSR at a dilution of 1:1500 (Novus Biologicals, Littleton, CO, RRID:AB 7170-6216), klotho at a dilution of 1:100 (Labome, Lambertville, NJ, RRID:AB KLO001); FGFR1 at a dilution of 1:200 (Santa Cruz Biotechnology Dallas, TX, RRID:AB Flg (C-15): sc-121).

Techniques: Quantitative RT-PCR

Journal: The FASEB Journal

Article Title: Leptin regulates parathyroid hormone secretion through CaSR ‐ ERK1 /2 signaling

doi: 10.1096/fj.202403141R

Figure Lengend Snippet: Reduced PTH content in parathyroid sections from db/db mice at 4 months of age. Parathyroid glands form db/− and db/db mice were extracted at 4 months of age, paraffin embedded and analyzed by IF microscopy. (A) Representative staining for PTH (blue), nuclear sytox (green) and the merged image. (B) Quantification of staining intensity for PTH. (C) Representative staining for CaSR (blue), klotoho (red) and nuclear sytox (green) and their quantification (D, E). (F) Reprisentative staining for FGFR1 (red), nuclear sytox (green) and the merged image and quantification (G). Quantification was performed using Image J quantification program. * p < .05.

Article Snippet: Immunostaining was conducted overnight at 4°C using the following primary antibodies diluted in Cas block (Zymed Laboratories, San Francisco, CA): PTH at a dilution of 1:300 (Bio‐Rad, Hercules, CA, RRID:AB 7170-6216); CaSR at a dilution of 1:1500 (Novus Biologicals, Littleton, CO, RRID:AB 7170-6216), klotho at a dilution of 1:100 (Labome, Lambertville, NJ, RRID:AB KLO001); FGFR1 at a dilution of 1:200 (Santa Cruz Biotechnology Dallas, TX, RRID:AB Flg (C-15): sc-121).

Techniques: Microscopy, Staining

Journal: The FASEB Journal

Article Title: Leptin regulates parathyroid hormone secretion through CaSR ‐ ERK1 /2 signaling

doi: 10.1096/fj.202403141R

Figure Lengend Snippet: Schematic representation of interactions between adipose tissue, parathyroid glands, and kidneys mediated by leptin, PTH, and 1,25(OH) 2 D 3 . Leptin, predominantly secreted by adipocytes, directly stimulates PTH secretion by inhibiting the CasR/pERK1/2 signaling pathway, thereby lifting the CasR‐mediated suppression of PTH secretion by the parathyroid glands. Elevated PTH levels, in turn, may affect leptin production by adipocytes. ⁷ , ¹⁷ Additionally, the parathyroid glands themselves produce leptin, further integrating these pathways. PTH stimulates renal synthesis of 1,25(OH) 2 D 3 (1,25D) which suppresses PTH secretion and contributes to calcium and phosphate homeostasis. Leptin reduces 1,25D, thus promoting PTH secretion indirectly by decreasing the inhibitory feedback exerted by 1,25D on the parathyroid glands. This intricate regulatory network links leptin to mineral homeostasis through PTH, CaSR, and 1,25D.

Article Snippet: Immunostaining was conducted overnight at 4°C using the following primary antibodies diluted in Cas block (Zymed Laboratories, San Francisco, CA): PTH at a dilution of 1:300 (Bio‐Rad, Hercules, CA, RRID:AB 7170-6216); CaSR at a dilution of 1:1500 (Novus Biologicals, Littleton, CO, RRID:AB 7170-6216), klotho at a dilution of 1:100 (Labome, Lambertville, NJ, RRID:AB KLO001); FGFR1 at a dilution of 1:200 (Santa Cruz Biotechnology Dallas, TX, RRID:AB Flg (C-15): sc-121).

Techniques:

Journal: The FASEB Journal

Article Title: Leptin regulates parathyroid hormone secretion through CaSR ‐ ERK1 /2 signaling

doi: 10.1096/fj.202403141R

Figure Lengend Snippet: Leptin increases PTH secretion in microdissected parathyroid glands in culture while decreasing CasR and c‐fos gene expression. (A) Parathyroid glands from control mice with intact leptin regeptor and parathyroid‐specific td‐tomato ‐expression were identified (dotted lines) and excised using fluorescent‐guided microdissection. Glands were cultured in growth medium supplemented with leptin (1 μg/mL) or vehicle. (B) PTH accumulated in the growth medium at 3 h. (C) qRT‐PCR analysis of PTH and CaSR mRNA levels performed on RNA extracted from the glands in A, after 24 h incubation. (D) qRT‐PCR analysis of c‐fos mRNA levels performed on RNA extracted after 3 h incubation. (E) Parathyroid glands were incubated with the calcimimatic R568 (1 μM) in the presence or absence of leptin, and PTH accumulated in the growth medium measured at 3 h. * p < .05, ** p < .01.

Article Snippet: Immunostaining was conducted overnight at 4°C using the following primary antibodies diluted in Cas block (Zymed Laboratories, San Francisco, CA): PTH at a dilution of 1:300 (Bio‐Rad, Hercules, CA, RRID:AB 7170-6216); CaSR at a dilution of 1:1500 (Novus Biologicals, Littleton, CO, RRID:AB 7170-6216), klotho at a dilution of 1:100 (Labome, Lambertville, NJ, RRID:AB KLO001); FGFR1 at a dilution of 1:200 (Santa Cruz Biotechnology Dallas, TX, RRID:AB Flg (C-15): sc-121).

Techniques: Gene Expression, Control, Expressing, Laser Capture Microdissection, Cell Culture, Quantitative RT-PCR, Incubation