recombinant rat lep  (R&D Systems)

Journal: Experimental cell research

Article Title: Leptin promotes tendon stem/progenitor cell senescence through the AKT-mTOR signaling pathway.

doi: 10.1016/j.yexcr.2024.114274

Figure Lengend Snippet: Fig. 1. The effect of leptin on TSPCs. TSPCs were exposed to different concentrations of leptin (0, 10, 50, 100 and 200 ng/mL) for 24 and 48 h. (a) The cell viability was examined by CCK8 assay. (b) Cell cycle was determined by flow cytometric analysis after 48 h of leptin exposure. (c) Proportions of apoptotic cells under leptin treatment for 48 h were analyzed by Annexin V-7 AAD staining. Data were representatives of at least 3 biological replicates, shown as mean ± SD. Significance threshold determined by One-way ANOVA: **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: After 24 h, culture media were refreshed containing various concentrations (1, 10, 50, 100 and 200 ng/mL) of recombinant rat leptin (R&D Systems, USA) and the cells were incubated for 24 and 48 h. The cells were subjected to CCK-8 (Bioss, CHN) analysis according to the product guide.

Techniques: CCK-8 Assay, Staining

Journal: Experimental cell research

Article Title: Leptin promotes tendon stem/progenitor cell senescence through the AKT-mTOR signaling pathway.

doi: 10.1016/j.yexcr.2024.114274

Figure Lengend Snippet: Fig. 2. The effects of leptin on TSPCs transdifferentiation and ECM modulators gene expressions. Post induction, the differentiation potential of TSPCs in exposure to 100 ng/mL leptin were examined, together with corresponding marker gene expression of each type of differentiation: (a) cartilage-like pellet formation (white frame); (b,e) chondrogenesis of TSPCs; (c,f) osteogenesis of TSPCs and (d,g) adipogenesis of TSPCs. The qRT-PCR analysis results of ECM modulators gene expressions in TSPCs treated with different concentrations of leptin (0, 10, 50, 100 and 200 ng/mL): (h) Collagen I mRNA expression; (i) IL-1α, IL-6 and TNF-α mRNA expressions; (j) MMP2, MMP3, MMP13, ADAMTS1, ADAMTS4 and ADAMTS5 mRNA expressions. Bar: 250 μm. Data were representatives of at least 3 biological replicates, shown as mean ± SD. Significant differences were indicated as: *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: After 24 h, culture media were refreshed containing various concentrations (1, 10, 50, 100 and 200 ng/mL) of recombinant rat leptin (R&D Systems, USA) and the cells were incubated for 24 and 48 h. The cells were subjected to CCK-8 (Bioss, CHN) analysis according to the product guide.

Techniques: Marker, Gene Expression, Quantitative RT-PCR, Expressing

Journal: Experimental cell research

Article Title: Leptin promotes tendon stem/progenitor cell senescence through the AKT-mTOR signaling pathway.

doi: 10.1016/j.yexcr.2024.114274

Figure Lengend Snippet: Fig. 3. Leptin induced TSPC senescence. (a) Representative images of SA-β-Gal staining results showing TSPCs treated with different concentrations of leptin (0, 10, 50, 100 and 200 ng/mL) for 6 days. (b) Semi-quantitative analysis of the proportions of SA-β-Gal positive cells in exposure to leptin for 6 days. (c) Western blot analysis result of p16 and p21 protein expression in leptin-treated TSPCs for 48 h. Bar: 100 μm. Data were representatives of at least 3 biological replicates, shown as mean ± SD. Significance was determined as: *: P < 0.05; ****: P < 0.0001.

Article Snippet: After 24 h, culture media were refreshed containing various concentrations (1, 10, 50, 100 and 200 ng/mL) of recombinant rat leptin (R&D Systems, USA) and the cells were incubated for 24 and 48 h. The cells were subjected to CCK-8 (Bioss, CHN) analysis according to the product guide.

Techniques: Staining, Western Blot, Expressing

Journal: Experimental cell research

Article Title: Leptin promotes tendon stem/progenitor cell senescence through the AKT-mTOR signaling pathway.

doi: 10.1016/j.yexcr.2024.114274

Figure Lengend Snippet: Fig. 4. Leptin promoted the AKT-mTOR signaling activities. (a) Western blot analysis result of (a) TSPCs treated with different concentrations of leptin (0, 10, 50, 100 and 200 ng/mL) for 48 h; (b) a time course of LC3-I/II protein expression in TSPCs treated with 100 ng/mL leptin. Results were representatives of at least 3 biological replicates.

Article Snippet: After 24 h, culture media were refreshed containing various concentrations (1, 10, 50, 100 and 200 ng/mL) of recombinant rat leptin (R&D Systems, USA) and the cells were incubated for 24 and 48 h. The cells were subjected to CCK-8 (Bioss, CHN) analysis according to the product guide.

Techniques: Western Blot, Expressing

Journal: Experimental cell research

Article Title: Leptin promotes tendon stem/progenitor cell senescence through the AKT-mTOR signaling pathway.

doi: 10.1016/j.yexcr.2024.114274

Figure Lengend Snippet: Fig. 5. Rapamycin could effectively rescue leptin-promoted TSPCs senescence. (a) Representative images of SA-β-Gal staining results of TSPCs in exposure to 100 ng/ mL leptin for 6 days, in the presence of either MK2206 (15 μM) and rapamycin (25 nM). (b) Semi-quantitative analysis of the proportions of SA-β-Gal positive cells in exposure to indicated drug treatments for 6 days. (c) The protein expression of p16 and p21 at 48 h post drug treatment were detected by Western blot analysis. Bar: 200 μm. Data were representatives of at least 3 biological replicates, shown as mean ± SD. Significance threshold was determined by One-way ANOVA: ****: P < 0.0001.

Article Snippet: After 24 h, culture media were refreshed containing various concentrations (1, 10, 50, 100 and 200 ng/mL) of recombinant rat leptin (R&D Systems, USA) and the cells were incubated for 24 and 48 h. The cells were subjected to CCK-8 (Bioss, CHN) analysis according to the product guide.

Techniques: Staining, Expressing, Western Blot

Journal: Experimental cell research

Article Title: Leptin promotes tendon stem/progenitor cell senescence through the AKT-mTOR signaling pathway.

doi: 10.1016/j.yexcr.2024.114274

Figure Lengend Snippet: Fig. 6. Leptin impedes Achilles tendon wound repair. The repair of tendon tissues 14 days after a puncture injury was examined in rats receiving weekly intra- peritoneal injection of PBS, leptin or combination of leptin and rapamycin (n = 3 in each group). (a) HE staining of the Achilles tendon tissues. (b) Masson staining of the Achilles tendon tissues. Representative images of immunofluorescence staining of (c) CD68, (d) p-mTOR, (e) p-p70S6K, and (f) Collagen I in Achilles tendon tissues in three groups of animals were shown.

Article Snippet: After 24 h, culture media were refreshed containing various concentrations (1, 10, 50, 100 and 200 ng/mL) of recombinant rat leptin (R&D Systems, USA) and the cells were incubated for 24 and 48 h. The cells were subjected to CCK-8 (Bioss, CHN) analysis according to the product guide.

Techniques: Injection, Staining, Immunofluorescence

Journal: Physiology & behavior

Article Title: Sucrose solution, but not liquid sucrose diet, leads to leptin resistance irrespective of the time of day that sucrose is available

doi: 10.1016/j.physbeh.2022.114002

Figure Lengend Snippet: Leptin response in Experiment 1. Rats were tested on Days 26 and 30 of the experiment with each rat acting as its own control. Panel A shows cumulative energy intake 12 and 36 h after injection and Panel B shows weight gain of the rats following injection. An asterisk indicates a significant difference between PBS and leptin treatment within a dietary group. Differences were determined by analysis of variance and post-hoc paired t-test. Data are means + sem for groups of 10 rats.

Article Snippet: Starting at 5 p.m. each rat received an i.p. injection of 2 mg/kg leptin (Recombinant rat leptin, R&D Systems, Minneapolis, MN) or PBS in a volume of 2 ml/kg.

Techniques: Control, Injection

Journal: Physiology & behavior

Article Title: Sucrose solution, but not liquid sucrose diet, leads to leptin resistance irrespective of the time of day that sucrose is available

doi: 10.1016/j.physbeh.2022.114002

Figure Lengend Snippet: The top panel shows the leptin response of rats in Experiment 2 tested on Days 36, before the feeding schedule was changed, and again on Days 44 and 53 when rats were offered sucrose at different times of day. The response was measured as a change in 12-h energy intake. An asterisk indicates a significant difference between PBS- and leptin-injected rats within a dietary group, determined by unpaired t-test. The bottom panels show blood glucose measured on Day 42 when the sucrose schedule had been changed for 3 days. Glucose was measured at noon and again at 4 p.m. by tail bleeding. Values within a panel that do not share a common superscript are significantly different at P < 0.05, determined by one-way analysis of variance and post-hoc Tukey’s Honest Significant Difference Test. Values are means + sem for groups of 12 rats.

Article Snippet: Starting at 5 p.m. each rat received an i.p. injection of 2 mg/kg leptin (Recombinant rat leptin, R&D Systems, Minneapolis, MN) or PBS in a volume of 2 ml/kg.

Techniques: Injection

Journal: Cell metabolism

Article Title: Age-related ciliopathy: Obesogenic shortening of melanocortin-4 receptor-bearing neuronal primary cilia.

doi: 10.1016/j.cmet.2024.02.010

Figure Lengend Snippet: Figure 6. Chronic leptin-melanocortin signaling shortens MC4R+ cilia, and shortening MC4R+ cilia causes leptin resistance (A) Ift88 KD induces leptin resistance. Top: scramble or Ift88-KD AAV was injected into both DMH and PVH or only the PVH of Mc4r-Cre rats (n = 4–5) at 7 weeks old. Saline and leptin were injected into the lateral ventricle (i.c.v.) 2 and 3 weeks later, respectively. Bottom: food intake after i.c.v. injection is expressed as a percentage of preinjection food intake. Paired t test; *p < 0.05; **p < 0.01; ns, not significant. See also Figures S7A and S7B. (B) Ablation of leptin signaling inhibits the age-related shortening of MC4R+ cilia. Top: MC4R-immunoreactive cilia in the DMH of lean and obese Zucker rats. Scale bars, 10 mm. Bottom: MC4R-immunoreactive cilia were longer in obese Zucker rats (n = 3). One-way ANOVA followed by Bonferroni’s post hoc test, *p < 0.05 and ***p < 0.001. (C) Chronic leptin signaling shortens MC4R+ cilia. Wild-type rats (12 weeks old) were injected with saline (n = 4) or leptin (5 mg, n = 5) into the lateral ventricle every other day for 5 weeks under DR (fed 60% of NC consumed by ad-libitum-fed, age-matched rats). Leptin injection shortened MC4R-immunoreactive cilia in both DMH (left images) and PVH. Scale bars, 10 mm. Unpaired t test; *p < 0.05. (D) Serum leptin concentration negatively correlates with MC4R+ ciliary length. Linear regression analysis (Pearson’s correlation test) between MC4R+ ciliary length in the DMH (data from Figure 3A) and serum leptin concentrations measured in wild-type rats of the same diet and age (n = 4 rats except 3-week-old NC-fed rats: ciliary length, n = 7; and leptin n = 12). (E) Hypothetical mechanism by which chronic leptin-melanocortin signaling shortens MC4R+ primary cilia. LepR, leptin receptor.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER SulfoLink Coupling Resin Thermo Fisher Scientific Cat # 20401 Lipofectamine 2000 transfection reagent Thermo Fisher Scientific Cat # 11668030 Avidin-biotinylated peroxidase complex Vector Laboratories Cat # PK-6100 3,3’-diaminobenzidine tetrahydrochloride Merck Cat # 281751 Avidin-biotin blocking kit Vector Laboratories Cat # SP-2001 Alexa594-conjugated CTb Thermo Fisher Scientific Cat # C34777 FITC-conjugated tyramide (Tyramide Signal Amplification FITC Systems) Parkin Elmer Cat # SAT701001KT Alexa488-conjugated streptavidin Thermo Fisher Scientific Cat # S11223 Alexa594-conjugated streptavidin Thermo Fisher Scientific Cat # S11227 RNAscope negative control probe Advanced Cell Diagnostics Cat # 310043 RNAscope probe for Cilk1 Advanced Cell Diagnostics Cat # 1177181-C1 Cy3-conjugated tyramide (TSA Plus Cyanine 3) Akoya Biosciences Cat # NEL744001KT Food pellets for NC, HFD and DR rats Research Diets Cat # D12450H (NC) Cat # D12451 (HFD) Pyrogen-free 0.9% saline Otsuka N/A Recombinant rat leptin R&D systems Cat # 598-LP RNAlater Merck N/A MTII Sigma-Aldrich Cat # M8693 Fluorescent microbeads Thermo Fisher Scientific Cat # F8801, F8803 Critical commercial assays MEGAshortscript T7 Transcription Kit Life Technologies Cat # AM1354 mMESSAGE mMACHINE T7 Ultra Kit Life Technologies Cat # AM1345 MEGAClear kit Life Technologies Cat # AM1908 NucleoSpin Gel and PCR Clean-up Takara Bio Cat # 740609 KAPA Express Extract Kit Kapa Biosystems Cat # KK7100 RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat # 323100 Mouse/Rat Leptin Quantikine ELISA Kit R&D Systems Cat # MOB00B RNeasy Lipid Tissue Mini Kit Qiagen Cat # 74804 iTaq Universal SYBR Green One-Step Kit Bio-Rad Cat # 1725150 Experimental models: Cell lines

Techniques: Injection, Saline, Concentration Assay

Journal: Cell metabolism

Article Title: Age-related ciliopathy: Obesogenic shortening of melanocortin-4 receptor-bearing neuronal primary cilia.

doi: 10.1016/j.cmet.2024.02.010

Figure Lengend Snippet: Figure 7. Interventions to prevent age-related shortening of MC4R+ cilia (A) DR regenerates MC4R+ cilia in old rats. Left: MC4R-immunoreactive cilia in the DMH of 20-month-old wild-type rats fed NC ad libitum or after 8 weeks of DR. Scale bars, 10 mm. Right: length of MC4R-immunoreactive cilia is compared between NC (n = 5) and DR (n = 3) rats (unpaired t test, *p < 0.05 and **p < 0.01). (B and C) Molecular intervention to prevent the age-related shortening of MC4R+ cilia. Scramble AAV or AAV-dsRed-pSico-Cilk1 shRNA (Cilk1 KD) was bilaterally injected into the DMH and PVH of 9-week-old Mc4r-Cre rats, which were then ad libitum fed HFD for 7 weeks. Pseudocolored confocal images in the DMH 7 weeks after AAV injection (B) show that Mc4r-Cre-positive neurons, labeled with dsRed only (single arrowheads), lost MC4R-immunoreactive cilia due to aging in scramble rats, but retained them in Cilk1-KD rats. Double arrowheads indicate an infected Mc4r-Cre-negative cell, which expressed both dsRed and EGFP. Scale bars, 10 mm. Cilk1-KD rats had longer MC4R-immunoreactive cilia in the DMH, but not in the PVH, than scramble rats (B, scramble, n = 8; Cilk1 KD, n = 6, unpaired t test, **p < 0.01; ns, not significant). Cilk1-KD rats exhibited reduced body weight gain (C, two-way ANOVA followed by Bonferroni’s post hoc test, *p < 0.05). (D) A model for the mechanism of age-related obesity. MC4R+ primary cilia of hypothalamic neurons progressively shorten with age. This ‘‘age-related ciliopathy’’ is promoted by overnutrition, which chronically sensitizes MC4Rs to melanocortins, whereas it is inhibited by dietary restriction. The shortening of MC4R+ cilia impairs neuronal sensitivity to leptin-melanocortin satiety signals, thereby decreasing metabolism and BAT thermogenesis, increasing food intake, and ultimately developing obesity and leptin resistance.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER SulfoLink Coupling Resin Thermo Fisher Scientific Cat # 20401 Lipofectamine 2000 transfection reagent Thermo Fisher Scientific Cat # 11668030 Avidin-biotinylated peroxidase complex Vector Laboratories Cat # PK-6100 3,3’-diaminobenzidine tetrahydrochloride Merck Cat # 281751 Avidin-biotin blocking kit Vector Laboratories Cat # SP-2001 Alexa594-conjugated CTb Thermo Fisher Scientific Cat # C34777 FITC-conjugated tyramide (Tyramide Signal Amplification FITC Systems) Parkin Elmer Cat # SAT701001KT Alexa488-conjugated streptavidin Thermo Fisher Scientific Cat # S11223 Alexa594-conjugated streptavidin Thermo Fisher Scientific Cat # S11227 RNAscope negative control probe Advanced Cell Diagnostics Cat # 310043 RNAscope probe for Cilk1 Advanced Cell Diagnostics Cat # 1177181-C1 Cy3-conjugated tyramide (TSA Plus Cyanine 3) Akoya Biosciences Cat # NEL744001KT Food pellets for NC, HFD and DR rats Research Diets Cat # D12450H (NC) Cat # D12451 (HFD) Pyrogen-free 0.9% saline Otsuka N/A Recombinant rat leptin R&D systems Cat # 598-LP RNAlater Merck N/A MTII Sigma-Aldrich Cat # M8693 Fluorescent microbeads Thermo Fisher Scientific Cat # F8801, F8803 Critical commercial assays MEGAshortscript T7 Transcription Kit Life Technologies Cat # AM1354 mMESSAGE mMACHINE T7 Ultra Kit Life Technologies Cat # AM1345 MEGAClear kit Life Technologies Cat # AM1908 NucleoSpin Gel and PCR Clean-up Takara Bio Cat # 740609 KAPA Express Extract Kit Kapa Biosystems Cat # KK7100 RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat # 323100 Mouse/Rat Leptin Quantikine ELISA Kit R&D Systems Cat # MOB00B RNeasy Lipid Tissue Mini Kit Qiagen Cat # 74804 iTaq Universal SYBR Green One-Step Kit Bio-Rad Cat # 1725150 Experimental models: Cell lines

Techniques: shRNA, Injection, Labeling, Infection