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: Oncotarget

Article Title: Targeting growth hormone receptor in human melanoma cells attenuates tumor progression and epithelial mesenchymal transition via suppression of multiple oncogenic pathways.

doi: 10.18632/oncotarget.15375

Figure Lengend Snippet: Figure 1: Growth hormone receptor knock-down (GHRKD) attenuates invasion, migration and clonogenicity in human melanoma cells. a-c. SK-MEL-28 cells transfected with scramble (scr) (a1-a4) or GHR-siRNA (b1-b4) were seeded onto U-bottom 96-well plates at 5000 cells/well and allowed to form a spheroid. A hydrogel invasion matrix was added above the spheroid and cells were monitored for up to 72 hr. in presence of 50 ng/mL hGH. Total pixels representing structural extensions from the spheroid were calculated using ImageJ software and reflected the invasive ability of the melanoma cells (c). A significant decrease in spheroid invasion was noted following GHRKD. d-g. SK-MEL-28 cells transfected with scr- (e1-e3) or GHR-siRNA (f1-f3) as well as un-transfected controls (d1-d3) were allowed to migrate into a 0.68 mm circular spot at the center of the well, in presence of 50 ng/mL hGH for up to 48 hr. The percentage free area was calculated using ImageJ software and reflected the decrease/inhibition in migration (g). A significant decrease in migration was noted following GHRKD. Similar results for migration and invasion assays with MALME-3M, MDA-MB-435 and SK-MEL-5 cells are presented in Supplementary Figure 2. h. SK-MEL-5, SK-MEL-28 and MDA-MB-435 cells transfected with 20 nM scramble or GHR-siRNA were allowed to form colonies on soft agar for 7 days in presence of 50 ng/mL hGH. The cells were lysed at the end time point and total DNA was quantified using a fluorescent readout. A significant decrease in total number of colonies was noted following GHRKD. [*, p < 0.05, Students t-test, n = 3].

Article Snippet: Pre-designed siRNA duplexes against human GHR (Origene, Rockville, MD) at different concentrations were evaluated and 20 nM was found to be optimum for decreasing the GHR RNA by >85%.

Techniques: Knockdown, Migration, Transfection, Software, Inhibition

Journal: Oncotarget

Article Title: Targeting growth hormone receptor in human melanoma cells attenuates tumor progression and epithelial mesenchymal transition via suppression of multiple oncogenic pathways.

doi: 10.18632/oncotarget.15375

Figure Lengend Snippet: Figure 2: GH-excess promotes and GHRKD attenuates human melanoma cell proliferation. a1. SK-MEL-28, a2. MALME- 3M, a3. MDA-MB-435 and a4. SK-MEL-5 cells were treated with increasing doses of hGH for 48 hr. and cell proliferation was estimated using a resazurin-based metabolic assay. A significant increase in cell proliferation was noted at and above 50 ng/mL hGH treatment. b1. SK-MEL-28, b2. MALME-3M, b3. MDA-MB-435 and b4. SK-MEL-5 cells were transfected with 20 nM scramble or GHR-siRNA for 24 hr. and grown for 48 hr. in presence or absence of 50 ng/mL hGH. Cell proliferation was estimated using resazurin-based metabolic assay. A significant decrease in cell proliferation was noted following GHRKD. Averages of at least four independent experiments performed in quadruplicate were taken. [*, p < 0.05, Students t-test].

Article Snippet: Pre-designed siRNA duplexes against human GHR (Origene, Rockville, MD) at different concentrations were evaluated and 20 nM was found to be optimum for decreasing the GHR RNA by >85%.

Techniques: Metabolic Assay, Transfection

Journal: Oncotarget

Article Title: Targeting growth hormone receptor in human melanoma cells attenuates tumor progression and epithelial mesenchymal transition via suppression of multiple oncogenic pathways.

doi: 10.18632/oncotarget.15375

Figure Lengend Snippet: Figure 3: GH-excess promotes and GHRKD attenuates multiple critical intracellular signaling pathways in human melanoma cells. a. Representative images of western blot (WB) analyses of phosphorylation levels b. JAK2, c. STAT5, d. STAT1, e. STAT3, f. SRC, g. AKT, h. mTOR and i. ERK1/2, in excess human-GH treated or GHRKD human melanoma cell lysates. SK-MEL-28 cells, 24 hr post-transfection with either scramble (scr)-siRNA or GHR-siRNA were treated for ten mins with GH and lysed as described. WB was performed using appropriate antibodies. Densitometry analyses of individual blots was performed using ImageJ software and the ratio of phosphorylated vs. total protein levels against untreated scr-siRNA transfected controls. Overall, excess GH increased while GHRKD decreased phosphorylation states. Similar results for MALME-3M, MDA-MB-435 and SK-MEL-5 human melanoma cells are presented in Supplementary Figure 3. Blots from individual experiments were quantified and the mean of three blots per antibody was taken. Protein levels were normalized against expression of β-actin. [*, p < 0.05, Students t test, n = 3].

Article Snippet: Pre-designed siRNA duplexes against human GHR (Origene, Rockville, MD) at different concentrations were evaluated and 20 nM was found to be optimum for decreasing the GHR RNA by >85%.

Techniques: Protein-Protein interactions, Western Blot, Phospho-proteomics, Transfection, Software, Expressing

Journal: Oncotarget

Article Title: Targeting growth hormone receptor in human melanoma cells attenuates tumor progression and epithelial mesenchymal transition via suppression of multiple oncogenic pathways.

doi: 10.18632/oncotarget.15375

Figure Lengend Snippet: Figure 4: GH-excess promotes and GHRKD attenuates markers of epithelial mesenchymal transition (EMT) in human melanoma cells. a-c. Relative RNA expression was quantified for N-cadherin (a), vimentin (b) and E-cadherin (c) in SK-MEL-28 melanoma cells following addition of 5, 50 and 150 ng/mL hGH or 24 hr following GHRKD, in presence or absence of 0 and 50 ng/mL hGH. Similar results for MALME-3M, MDA-MB-435 and SK-MEL-5 human melanoma cells are presented in Supplementary Figure 4. In all cases, RNA expressions were normalized against β-actin and GAPDH values as reference genes and compared against untreated control. [*, p < 0.05, Wilcoxon sign rank test, n = 4] d-f. Densitometry analyses of relative protein expressions of N-cadherin (d), vimentin (e), and E-cadherin (f) as estimated by western blot (WB) of lysates of SK-MEL-28, MALME-3M, MDA-MB-435 and SK-MEL-5 cells, collected 60 hr post-transfection with either scramble (scr)-siRNA or GHR-siRNA in presence of GH. g. Representative images of WB analyses of phosphorylation levels of N-cadherin, vimentin and E-cadherin in four melanoma cell lines. WB was performed using appropriate antibodies. Densitometry analyses of individual blots was performed using ImageJ software and the ratio of phosphorylated vs. total protein levels against untreated scr-siRNA transfected controls. Overall, excess GH promoted while GHRKD reversed EMT in human melanoma cells. Blots from individual experiments were quantified and the mean of three blots per antibody was taken. Protein levels were normalized against expression of β-actin. [*, p < 0.05, Students t test, n = 3].

Article Snippet: Pre-designed siRNA duplexes against human GHR (Origene, Rockville, MD) at different concentrations were evaluated and 20 nM was found to be optimum for decreasing the GHR RNA by >85%.

Techniques: RNA Expression, Control, Western Blot, Transfection, Phospho-proteomics, Software, Expressing

Journal: Oncotarget

Article Title: Targeting growth hormone receptor in human melanoma cells attenuates tumor progression and epithelial mesenchymal transition via suppression of multiple oncogenic pathways.

doi: 10.18632/oncotarget.15375

Figure Lengend Snippet: Figure 7: Model of GH regulation in melanoma. Endocrine/paracrine/autocrine GH binds to GHR expressed at high levels in human malignant melanoma cells and activates JAK2 and SRC kinases. This leads to downstream activation of STAT1, STAT3, STAT5, ERK1/2, AKT and mTOR. Subsequent transcription of target genes lead to aggressive phenotypes of tumor cell migration, invasion and proliferation and upregulate autocrine HGF-MET loop, ERBB3 and also drives epithelial-mesenchymal-transition. In our study excess GH upregulated (in green) while siRNA mediated GHR knock-down (GHRKD) downregulated (in red) these effects.

Article Snippet: Pre-designed siRNA duplexes against human GHR (Origene, Rockville, MD) at different concentrations were evaluated and 20 nM was found to be optimum for decreasing the GHR RNA by >85%.

Techniques: Activation Assay, Migration, Knockdown

Journal: Journal of Ovarian Research

Article Title: Positive effects of prolonged caloric restriction on the population of very small embryonic-like stem cells – hematopoietic and ovarian implications

doi: 10.1186/1757-2215-7-68

Figure Lengend Snippet: Effect of CR on morphology of ovaries and testes. Panel A . Ovaries of 10-month-old C57B1/6 mice fed AL (a, c, and e) or on CR (b, d, and f). a and b. In both groups, primary (PF), preantral (PAF), and antral (AF) follicles and corpus luteum (CL) are visible. We also observed primordial follicles and a greater number of atretic follicles (red arrowheads) in the CR group (b) than in the AL-fed group (a). Hematoxylin + eosin staining. Magnification: x100. Inset in panel b represents primordial follicles in an ovary from a mouse on CR. Magnification, x400. c-f. Immunohistochemical reaction for growth hormone receptor (GHR) expression in ovaries of mice fed AL (c and e) or on CR (d and f). High expression of GHR in ad libidum (AL)-fed mice was noted in granulosa cells (red arrow), preantral and antral follicles and zona pellucida (asterisk), ovarian surface epithelium (black arrowhead), and blood vessel endothelium (BV, c and e). By contrast, expression of GHR in CR mice was found only in a few granulosa cells (red arrow) of Graffian follicles (GF), and no expression of GHR was observed in granulosa cells of preantral (PAF), primary (PF), and primordial (PRF) follicles (d and f). Magnification: panels c and d, x100; panels e and f, x200. Representative images are shown. Panel B . The number of follicles at different stages of development in ovaries of 10 month-old female C57B/6 mice on CR compared with controls fed AL. The higher number of primordial, primary, preantral and atretic follicles in ovaries of mice on CR than in ovaries of mice fed ad libidum. P < 0.01. Panel C . Testes of 10-month-old C57B1/6 mice fed AL (panels a and c) or on CR (panels b and d). Magnification: panels a and b, x100; panels c and d, x200. PAS staining. Representative images are shown.

Article Snippet: After antigen retrieval in a water bath containing citrate buffer pH = 6 and incubation with blocking serum, anti-growth hormone receptor (GHR) antibody was added (rabbit polyclonal anti-GHR antibody, Acris Antibodies, GmbH) at 1:250 dilution for 1 hour at room temperature.

Techniques: Staining, Immunohistochemical staining, Expressing