qPCR analysis
Assuming the expression of the gene for the leptin receptor in cancerous and non-cancerous human ovarian epithelial cell line and action of leptin on ObR gene expression, cells were seeded into 96-well culture plates at a density of 1 × 104 cells/well HOSEpiC, 1 × 104 cells/well SK-OV-3, 1.5 × 104 cells/well TOV-21G, 1.5 × 104 cells/well CaOV-3 and 1 × 104 cells/well OVCAR-3. The next day the medium was changed and the cells were treated with leptin at two doses, 40 ng/mL and 100 ng/mL, for 24 h. Total RNA isolation and cDNA synthesis were performed using the TaqMan Gene Expression Cell-to-CT Kit (Applied Biosystems, Carlsbad, CA, USA) following the manufacturer’s protocol. Amplifications were performed using the StepOnePlus system (Applied Biosystems, Carlsbad, CA, USA) and the TaqMan Leptin Receptor starter (Cat. No. Hs00174497_m1) in combination with the TaqMan Gene Expression Master Mix (Applied Biosystems, Carlsbad, CA, USA), following the manufacturer’s instructions. A PCR was performed using a final volume of 20 μL, including 50 ng/reaction cDNA.
In experiments with antagonists, cells were seeded into 96-well culture plates at a density of 1.5 × 104 cells/well CaOV-3 and 1 × 104 cells/well OVCAR-3. The next day the medium was changed and the cells were treated with leptin at a dose of 40 ng/mL alone or with SHLA or Lan2 at a dose of 1000 ng/mL for 24 h. cDNA synthesis was performed using the TaqMan Gene Expression Cell-to-CT Kit (Applied Biosystems, Carlsbad, CA, USA) following the manufacturer’s protocol. Amplifications were performed using the StepOnePlus system (Applied Biosystems, Carlsbad, CA, USA) and the TaqMan Array, Human Cyclins and Cell Cycle Regulation, Fast 96-well (Cat. No. 4418768) in combination with the TaqMan Gene Expression Master Mix (Applied Biosystems, Carlsbad, CA, USA), following the manufacturer’s instructions. A PCR was performed with a final volume of 10 μL including 50 ng/reaction cDNA.
The PCR conditions were as follows: pre-incubation for 2 min at 50 °C and 10 min at 95 °C, amplification for 40 cycles (15 s at 95 °C and 1 min at 60 °C). The relative expression of genes was normalised against the endogenous reference gene GAPDH (Human GAPD Endogenous Control, number 4333764F) (ΔCq) and converted to relative expression (RQ) using the 2−ΔΔCq method. The results are expressed in the figures as relative values (RQ).
Western blot analysis
Cells were plated into 24-well plates at a density of 10 × 104 cells for OVCAR-3 cells, for CaOV-3 and 8 × 104 cells HOSEpiC cells and allowed to attach overnight. The next day the media were changed and the cells were treated with 40 μg/mL leptin alone or in combination with 1000 μg/mL SHLA or Lan-2. To examine cell cycle protein expression, cells were incubated for 72 h (OVCAR-3) or 48 h (CaOV-3, HOSEpiC). After incubation, the cells were washed with ice-cold PBS and lysed with Laemmli lysis buffer (Sigma Chemical Co., St. Louis, MO, USA). The lysed cells were then scraped, transferred to microtubes and stored at −70 °C until analysis.
Before analysis, samples were sonicated and centrifuged at 15,000 × g for 15 min at 4 °C. The quantity of proteins was determined using the Bradford method, and the clear supernatant was used for electrophoresis. Equal amounts of protein (100 µg) from each treatment group were separated by SDS-PAGE and transferred to PVDF membranes using a Bio-Rad Mini-Protean 3 apparatus (Bio-Rad Laboratories Inc., Hercules, CA, USA). The blots were blocked for 1 h in 5 % BSA with 0.1 % Tween-20 in 0.02 M TBS buffer. Blots were incubated overnight with primary antibodies specific to ObR (ab5593, abcam, Cambridge, Great Britain; dilution 1:2000). After incubation with the primary antibody, the membranes were washed three times with 0.1 % Tween-20 in 0.02 M TBS buffer and incubated for 1 h with an appropriate horseradish peroxidase-conjugated secondary antibody (#7074, Cell Signaling Technology Inc., Beverly, MA, USA; dilution 1:2000).
β-Actin was used as an internal loading control; membranes were washed for 30 min in stripping buffer (0.25 M glycine, 1 % SDS, pH 2) and reprobed by overnight incubation with primary antibodies specific to β-actin (A5316, Sigma Chemical Co., St. Louis, MO, USA; dilution 1:2000) and for 1 h with a horseradish peroxidase-conjugated secondary antibody (P0447 DAKO, Glostrup, Denmark; dilution 1:5000).
Immunopositive bands were visualised using Western Blotting Luminol Reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and ChemiDoc™ XRS+System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The relative levels of protein expression were determined using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). Individual protein levels were normalised to β-actin controls, and the ratio of protein to β-actin was normalised to 1 in the untreated control group.
To study cell cycle protein level cells were plated into 24-well plates at a density of 10 × 104 cells for OVCAR-3 cells and 9 × 104 cells for CaOV-3 cells and allowed to attach overnight. The next day the media were changed and the cells were treated with 40 μg/mL leptin alone or in combination with 1000 μg/mL SHLA or Lan-2. To examine cell cycle protein expression, cells were incubated for 72 h (OVCAR-3) or 48 h (CaOV-3). Equal amounts of protein (60 µg) from each treatment group were separated by SDS-PAGE and transferred to PVDF membranes. The blots were blocked for 1 h in 5 % BSA with 0.1 % Tween-20 in 0.02 M TBS buffer. Blots were incubated overnight with primary antibodies specific to Cyclin D1 (#2978, Cell Signaling Technology Inc., Beverly, MA, USA), cdk4 (#12790), cdk2 (#2546), cyclin A2 (#4656) at a 1:1000 dilution and E2F-2 (sc-251 Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a dilution of 1:200. After incubation with the primary antibody, the membranes were washed three times with 0.1 % Tween-20 in 0.02 M TBS buffer and incubated for 1 h with an appropriate horseradish peroxidase-conjugated secondary antibody (#7074, Cell Signaling Technology Inc., Beverly, MA, USA; dilution 1:2000 and sc-2005, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, respectively).
GAPDH was used as an internal loading control; membranes were washed for 30 min in stripping buffer (0.25 M glycine, 1 % SDS, pH 2) and reprobed by overnight incubation with primary antibodies specific to GAPDH (G-8795, Sigma Chemical Co., St. Louis, MO, USA; dilution 1:20 000) and for 1 h with a horseradish peroxidase-conjugated secondary antibody (sc-2005, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA dilution 1:2000).
Immunopositive bands were visualised using Western Blotting Luminol Reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and ChemiDoc™ XRS+System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The relative levels of protein expression were determined using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). Individual protein levels were normalised to GAPDH controls and the ratio of protein to GAPDH was normalised to 1 in the untreated control group.
To study leptin receptor signalling, the cells were treated with 40 ng/mL of leptin in combination with SHLA or Lan-2 at a concentration of 1000 μg/mL for 0, 5, 15, 30 and 60 min. Sixty micrograms of protein from each treatment group was separated by 10 % SDS-PAGE. Blots were incubated overnight at 4 °C with antibodies specific for phospho-Stat3 (Tyr705) (#9131), Stat3 (#9132), phospho-p44/42 MAPK (#9101), p44/42 MAPK (#9102), phospho-Akt (Ser473) (#9271) and Akt (#9272) at a dilution of 1:1000 (Cell Signaling Technology Inc., Beverly, MA, USA). After incubation with the primary antibody, the membranes were washed three times and incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody (#7074) at a dilution of 1:2000 (Cell Signaling Technology Inc., Beverly, MA, USA). Immunopositive bands were visualised using Western Blotting Luminol Reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and ChemiDoc™ XRS+System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The relative levels of protein expression were determined using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). Individual protein levels were normalised to the total amounts of signalling proteins and the ratio of phospho-protein to total protein was normalised to 1 in the untreated control group (time = 0 min).