Materials and antibodies
Fetal bovine serum (FBS), Ham's F12 medium (F12), Dulbecco’s modified Eagle’s high glucose and pyruvate medium (DMEM), Lipofectamine 3000, Halt protease and phosphatase inhibitor cocktail and Opti-MEM® I Reduced-Serum Medium (Opti-MEM; #31985070) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Penicillin-streptomycin, MK2206 and LY294002 were obtained from Beyotime (Shanghai, China). L-Met and L-Leu (≥98%) were from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal anti-human β-casein antibody, rabbit polyclonal anti-human MRCKα antibody, rabbit monoclonal anti-mouse Cyclin D1 antibody, rabbit polyclonal anti-mouse sterol regulatory element-binding protein 1 (SREBP1) antibody, and mouse monoclonal anti-β-actin antibody were purchased from ABCAM (Cambridge, UK). Rabbit polyclonal anti-human STAT5 antibody, rabbit polyclonal anti-human phosphorylated STAT5 (Tyr694) antibody, rabbit polyclonal anti-human PI3K antibody and rabbit polyclonal anti-human phosphorylated PI3K (Tyr317) antibody were from Bioss antibodies (Beijing, China). Rabbit polyclonal anti-human mTOR antibody and rabbit polyclonal anti-human phosphorylated mTOR (Ser2448) antibody were obtained from ImmunoWay Biotechnology Company (Plano, TX, USA). Mouse monoclonal anti-human protein kinase B (AKT) antibody, mouse monoclonal anti-human phosphorylated AKT (Thr308) antibody, rabbit polyclonal anti-mouse JAK2 antibody and rabbit polyclonal anti-mouse phosphorylated JAK2 (Tyr1007/1008) antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat anti-rabbit HRP-conjugated IgG and goat anti-mouse HRP-conjugated IgG were obtained from ZSGB-BIO company (Beijing, China).
Cell culture and treatments
BMEC were obtained from mammary gland tissues of euthanized Holstein cows in mid-lactation at a commercial abattoir (Harbin, China), and were cultured as reported previously [21]. Briefly, BMEC were routinely cultured in DMEM-F12 with penicillin-streptomycin (100 units/mL) and 10% FBS (complete medium) at 37° C in a humidified atmosphere containing 5% CO2. The BMEC from 4-10 passages were preserved in liquid nitrogen until experimental analysis.
To study the effects of AA stimulation on relative protein levels of specific genes, BMEC were thawed, acclimated for one passage and seeded in uncoated six-well cell culture plates (Corning, NY, USA) at densities of 4 × 105/well. Cells grew to approximately 70% - 80% confluence at 37° C, i.e., still in their log phase of growth, and then were serum-starved for 8 h. Next, complete medium was replaced with Opti-MEM (containing basal concentrations of 0.09 mM Met and 0.3 mM Leu; without serum and antibiotics) to which we applied various doses of additional Met (0, 0.2, 0.4, 0.6 and 0.8 mM) or Leu (0, 0.2, 0.4, 0.6 and 0.8 mM). For this purpose, 0.22 μm-filtered stock solutions (1000-fold) of Met and Leu in Opti-MEM were freshly made. After incubation for 24 h at 37° C, cells were harvested for Western blot analyses (see below).
MRCKα knockdown
To study the effects of MRCKα knockdown on Met- or Leu-stimulated β-casein synthesis, treatments were arranged in a 2 × 3 factorial design with introduction of scrambled small interfering RNA (siRNA) (SC) or MRCKα siRNA (SI), and either no Met or Leu added (B, basal medium, basal levels of Met and Leu in the control culture medium were 0.09 and 0.3 mM, respectively), Met added (M, 0.6 mM) or Leu added (L, 0.6 mM), simultaneously. The MRCKα siRNA and the scrambled siRNA used in the present study were synthesized by GenePharma (Suzhou, China); MRCKα siRNA was: 5’-GCAACUGAAGCUUAUGAAATT-3’ (sense); 5’-UUUCAUAAGCUUCAGUUGCTT-3’ (antisense). Cells were seeded at 4 × 105 cells/well in a six-well cell culture plate (Corning) and next day attached cells were transfected with MRCKα siRNA (67 nM) using Lipofectamine 3000 according to the manufacturer’s instructions (Thermo Fisher Scientific). As a control, cells were transfected with scrambled siRNA (67 nM; GenePharma). Cells were transfected in antibiotics- and serum-free Opti-MEM at 37° C and simultaneously stimulated with Met or Lys. After 24 h, cells were harvested for Western blot and real time-qPCR analysis (see below).
MRCKα overexpression
The MRCKα encoding plasmid was obtained as described in Wang et al. [17]. Cells were transfected with 2.5 µg MRCKα encoding plasmid or the empty vector pcDNA3.1(+) (used as control) in Lipofectamine 3000 according to the manufacturer’s instruction (Thermo Fisher Scientific). Cells were transfected in antibiotics-free and serum-free Opti-MEM at 37° C and simultaneously the exposure to MK2206 (see below).
PI3K and AKT inhibition
To study the effects of PI3K inhibition on Met/Leu-stimulated β-casein synthesis, treatments were arranged in a 2 × 3 factorial design with blank (untreated control) or PI3K inhibition (LY294002, 43 µM), and either no Met and Leu added (B, basal medium), Met added (M, 0.6 mM) or Leu added (L, 0.6 mM), simultaneously. Following serum starvation in Opti-MEM containing neither antibiotics nor serum for 24 h, cells were treated in Opti-MEM for 24 h (37° C) and then harvested for Western blot analysis. To evaluate the effect of inhibition of AKT activity under condition of MRCKα overexpression, a 2 × 2 factorial experiment was conducted with simultaneously transfecting of empty vector (EV) or MRCKα overexpression (OE), and with blank (untreated control) or AKT inhibition (MK2206, 1 µM). Transfection and MK2206 treatment (1 µM) occurred simultaneously in antibiotics- and serum-free Opti-MEM media for 24 h (37° C) after which cells were harvested for Western blot analysis (see below).
Quantitative real-time PCR (RT-PCR)
Total RNA was extracted from BMEC with a RNeasy Mini Kit (Qiagen, Dusseldorf, Germany). RNA concentrations were measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), ensuring that each value of OD260nm/280nm was between 1.8 and 2.0. Then, total RNA (500 ng) was reverse transcribed into cDNA using a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Quantitative PCR was performed with the SYBR® Premix™ EX Taq II (TaKaRa) on an Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific). The primer pairs used for qPCR are presented in Table 1. The running program was one cycle of 30 s at 95° C followed by 40 cycles of amplification at 95° C for 5 s, 60° C for 34 s and 72° C for 1 min, with reaction specificity confirmed by subsequent melting curve analysis. Data analysis was performed with the 2−ΔΔCT method [22], where ribosomal protein S9 (RPS9) was used as the internal reference, because geNorm algorithm [23] identified this gene as one of the most stable genes [9].
Western blot analysis
Cells were lysed in Western and IP Cell Lysate solution containing Halt protease and phosphatase inhibitor cocktail (all from Beyotime) for 10 min on ice and then heated at 95° C for 10 min. Protein concentrations were determined with a BCA protein assay kit (Beyotime). The lysate was subsequently electrophoresed (25 µg total protein per lane) on ExpressPlus precast PAGE gels (4-20%; GenScript, Piscataway, NJ, USA), and then blotted to nitrocellulose membranes (0.45 µm; PALL, Port Washington, NY, USA). The membranes were probed with primary antibodies overnight and then with goat anti-mouse or goat anti-rabbit HRP-conjugated secondary antibodies for 1 h and eventually visualized with ECL Western Blot Detection Reagents (Thermo Fisher Scientific) using the Bio-Rad Gel Doc™ XR system. The relative intensities of the positive immunobands were calculated with ImageJ Gel Analysis software tool (version 1.52a; https://imagej.nih.gov/ij/). β-actin was used as internal reference protein.
Statistical analyses
Data are reported as means ± SEM of three independent experiments. Comparison of protein and mRNA levels under various Met or Leu concentration treatments with their unstimulated condition was attained by analysis of variance (ANOVA) followed by a Dunnett post-hoc test. For the other treatments, comparison was attained by analysis of ANOVA followed by a Tukey’s post-hoc test. Factors in the linear model included AA addition (AA), silencing of MRCKα (SI), and their interaction (AA × SI); AA, inhibition of PI3K (LY), and their interaction (AA × LY); or MRCKα overexpression (OE), inhibition of AKT (MK), and their interaction (OE × MK). Analyses were performed with SPSS Statistics software (version 17; IBM, NY, USA). Differences were considered statistically significant at P ≤ 0.05 and as tendencies for 0.05 < P < 0.10.