*4.2. Full-length cDNA Cloning of the MARK4 Gene*

Total RNA was isolated from the placenta of Landrace sows using RNAiso Plus (TaKaRa, Tokyo, Japan) and then was treated with DNase I using Recombinant RNase-free DNase I kit (TaKaRa, Tokyo, Japan) to degrade genomic DNA. 1% agarose gels electrophoresis and spectrophotometric analysis (260/280 ratio) were used to assess the quantity and quality of isolated RNA.

The cDNA was synthesized with PrimeScript 1 st strand cDNA Synthesis kit (TaKaRa, Tokyo, Japan) using total RNA (1 μg) from the placenta as template and Oligo dT18 as primer according to the manufacturer's instructions. Degenerated primer pairs of MARK4F/MARK4R (Table 1) were designed based on highly conserved regions from the available sequences of various vertebrate species. PCR amplification was performed with 1 μL of reverse-transcribed (RT) reactions in a total volume of 50 μL and 1 μL Tks Gflex DNA Polymerase (1.25 U/μL; TaKaRa, Tokyo, Japan). The PCR cycling conditions were one cycle of 94 ◦C for 1 min, 35 cycles of 98 ◦C for 10sec, 55 ◦C for 15sec, and 68 ◦C for 1 min, followed by one cycle of 72 ◦C for 5 min. The PCR products were purified with MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Tokyo, Japan) and sequenced by Takara Biotechnology (Dalian) Co.Ltd (Dalian, China). Sequencing was performed in both forward and reverse directions by using an ABI PRISMTM3730XL DNA Sequencer (Applied Biosystems, Waltham, MA, USA). The forward and reverse sequences were assembled using SeqMan NGen15 software in DNASTAR Lasergene 15.2 (DNASTAR, Madison, WI, USA), through which the core fragment of MARK4 gene was obtained. According to the sequence information of this fragment, gene-specific primers were designed for the 3 RACE and 5 RACE.



Rapid amplification of the 3 end was performed using the 3 -full RACE Core Set with PrimeScriptTM RTase (TaKaRa, Tokyo, Japan) following the manufacturer's instructions. The primers used for 3 RACE are shown in Table 1. Firstly, total RNA (1 μg) from placenta was reverse-transcribed using 3 RACE Adaptor (Table 1) as the primer for the synthesis of first strand cDNA. Then, the cDNA was amplified by a specific forward primer MARK4,3-F1 and 3 RACE Outer Primer containing the anchor sequence. After the first PCR, 1 μL of Outer PCR reactions was re-amplified using 3 RACE

Inner Primer and a specific forward primer MARK4,3-F2. The nested PCR product was separated by electrophoresis using 1% agarose gels and sequenced by the methods aforementioned.

Rapid amplification of the 5 end was conducted with SMARTerTM RACE 5 /3 cDNA Amplification Kit (TaKaRa, Tokyo, Japan) according to the manufacturer's instructions. Briefly, total RNA (1 μg) was reverse-transcribed with a specific reverse primer MARK4,5-R1(Table 1). After the synthesis of first strand cDNA, 2 μL of RT reactions was amplified by prime pairs MARK4,5-R2 and UPM Primer. Then, 1 μL of the first PCR product was used as a template for the nested PCR, which was performed with UPS Primer and MARK4,5-R3. The nested PCR product was separated by 1% agarose gel test and sequenced following the methods aforementioned.

#### *4.3. Bioinformatics Analysis*

The full-length cDNA of MARK4 gene was obtained using SeqMan NGen15 software in DNASTAR Lasergene (version 15.2) to assemble the core fragment, 3 end and 5 end sequences. The resulting nucleotide sequence was edited and analyzed by Open Reading Frame (ORF) Finder on NCBI (https://www.ncb-i.nlm.nih.gov/orffinder), and then translated into amino acids (AA) using standard genetic codes. The molecular weight (MW) and isolectric point (PI) of the Mark4 protein were predicted using the compute PI/MW software at https://web.expasy.org/compute\_ pi. Multiple alignments were generated by the MegAlign 15 program in DNASTAR Lasergene (version 15.2). The secondary and three-dimensional (3D) structures of Mark4 protein were predicted by the SABLE program (http://sable.cchmc.org) and the SWISS-MODEL program (https: //swissmodel.expasy.org) as previously described [34], respectively. Illustration of the MARK4 model was performed in PyMOL 2.2 program (https://pymol.org). Phylogeny tree was inferred by the MEGA7 program, and distance analysis was conducted using the Neighbour-Joining (NJ) algorithm. 1000 bootstrap-replications were generated to evaluate the reliability for each code.

#### *4.4. Porcine Placental Trophobalst Cell Isolation and Culture*

The isolation and culture of porcine placental trophobalst cells were performed as previously described with some modifications [35]. Briefly, placental villous tissue, obtained from vaginal delivery, were dissected from fetal amnion and rinsed thoroughly in cold PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin, and then cut into 1–3 mm 3 pieces. The tissue fragments were digested with 0.125% (*w/v*) Type I collagenase (trypsin) at 37 ◦C for 30 min with continuous shaking, followed by filtration through a 70 μm cell strainer. The filtrate was further purified by Percoll gradient centrifugation. Placental trophoblast cells were collected from the appropriate layers between 35% and 45% Percoll density gradient separated layers, and cultured in DMEM/F12 supplemented with 10% FBS, 1% (*v/v*) ITS, 10 ng/mL of EGF, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 ◦C under 5% CO2 as previously described [35]. The purity of trophoblast cells isolated from full-term placentas was determined by flow cytometry as previously described [36], using FITC fluorescein-labeled antibody against cytokeratin-7 (Santa Cruz Tech, Dallas, CA, USA) as a specific marker of trophoblast cells.

#### *4.5. Cell Transfection and Drug Treatment*

DNA constructs including Myc-MARK4 and Flag-DKK1 were made by Generay Biotech Company (Shanghai, China) using pEGFP-N1 expression vector. shRNA sequences against MARK4 or DKK1 were contrived and synthesized by Genepharma Company (Shanghai, China) using pGpU6/GFP/Neo shRNA expression vector. After transfection efficiency detection, the optimal shRNA of MARK4 or DKK1 was chosen and named sh-MARK4 and sh-DKK1. Cells were plated at a concentration of <sup>6</sup> × 105–2 × <sup>10</sup>6/dish in 60-mm dishes. 2 <sup>μ</sup>g interference or expression plasmids DNA were mixed with X-treme GENE HP Reagent (Roche, Basel, Switzerland) and Opti-MEMI media (Invitrogen, Carlsbad, CA, USA) following the instruction. The transfection mixture was then added into each dish for 48 h to allow the expression of DNA or shRNA constructs described above.

In order to induce lipid accumulation in trophoblast cells in vitro, cells were treated with 400 μM Fatty Acid (FA) Supplement containing 2 mol of linoleic acid and 2 mol of oleic acid per mole of albumin (L9655; Sigma-Aldrich, Saint Louis, MO, USA) in triplicate as previously described [10,37]. The optimal treatment concentration of 400 μM was chosen based on results of concentration gradient studies (Figure S5) indicating that fat accumulation was significantly increased by 50, 100 and 200 μM fatty acids when compared to 0μM, with the most significant increase following the 400 μM treatment. Treatment media without fatty acids was added with bovine serum albumin (FA free) to maintain the same osmolarity. In some experiment, cells were treated with one of the following specific agonists or inhibitors: 2 μM GW1929 (PPARγ-specific agonist; MCE, Shanghai, China), 20 μM LiCL (GSK3β inhibitor; Millipore, Billerica, MA, USA), or 10 μM JW74 (WNT signaling pathway specific inhibitor; MCE, Shanghai, China) for the amount of time specified in the individual figures.

#### *4.6. Oil Red O Staining*

After 24 h of FA treatment, cells were fixed in 4% paraformaldehyde for 30 min at room temperature for Oil Red O staining. Each well was then briefly washed in PBS and 60% isopropanol and then stained for 10 min in a 60% working Oil Red O solution (Sigma-Aldrich). For quantification of Oil Red O staining, cells were extracted by 100% isopropanol for colorimetric analysis at an optical density of 490 nm as previously described [10].

#### *4.7. Cell Viability and Reactive Oxygen Species (ROS) Assay*

Cell viability was detected using cell counting kit-8 (CKK-8; KeyGen BioTECH, Nanjing, China). The isolated cells were seeded into 96-well plates at a density of 5 × 103 and cultured with 0, 400 and 500 μM fatty acids for the amount of time specified in Figure S5, respectively. 10 μL CKK-8 solution was then added into each well and incubated for 2 h at 37 ◦C. Absorbance was measured at 450 nm using a Multiskan Go Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

The intracellular level of ROS test was performed using Oxygen Species Assay Kit (KeyGen BioTECH, Nanjing, China) according to the manufacturer's instructions. Briefly, the dye loading was performed by incubating the cells with 10 μM 2 , 7 -dichlorofluorescin diacetate (DCFH-DA) at 37 ◦C for 1 h. The production of ROS was examined using a Luminescence Spectrophotometer (Promega Corporation, Madison, WI, USA) by measuring the fluorescence intensity of DCF at emission wavelength of 525 nm.

#### *4.8. Lipid Accumulation Assay*

The Bodipy 493/503 lipid probes (D-3922; Thermo Scientific) was used to visualize fatty acid accumulation in cultured trophoblast cells as previously described [38]. Briefly, cells were washed in PBS and 4% paraformaldehyde in PBS was added to fix the cells for 30 min at room temperature. After fixation, cells were washed in PBS containing 0.1% Triton X-100 for 5 min. Bodipy dye was diluted in PBS at a concentration of 10 μg/mL and applied to cells for 15 min. For nuclei staining, 10 μg/mL of 4 , 6-diamidino-2-phenylindole (DAPI) solution was incubated with each sample for 30 min, and then the samples were examined on confocal laser scanning microscope (Zeiss LSM 700 META, Jena, Germany). For quantification of lipid accumulation, triglyceride (TG) content in cultured trophoblast cells was evaluated with a spectrophotometer (Thermo Scientific) at 510 nm using Tissue Triglyceride Assay Kit (APPLYGEN, Beijing, China) as previously described [17]. Because phloretin blocks receptor (transport proteins)-mediated fatty acid transport and accumulation [39,40] it was used to determine receptor-mediated fatty acid accumulation by subtracting the TG content in the presence of phloretin (500 μM) from those in the absence of phloretin as previously described [21].

#### *4.9. Immunofluorescence Assay*

β-catenin immunofluorescence analysis was performed as previously described [10]. Briefly, cells were grown on coverslips, fixed with 4% paraformaldehyde for 30 min and permeabilized

with 0.25% Triton X-100 for 10 min. After blocked with 5% BSA-supplemented PBS for 1 h, cells were incubated overnight at 4 ◦C with rabbit anti-β-Catenin primary antibody (8480, dilution 1:300, Cell Signaling Technology, Danvers, MA, USA), followed by incubation of Goat anti-rabbit Cy3 fluorescein-labeled secondary antibody (BA1032, dilution 1:500, Boster, China). Meanwhile, the cell nuclei were counterstained with 4 , 6-diamidino-2-phenylindole for 10 min, and then the samples were mounted on glass slides and examined on confocal laser scanning microscope (Zeiss LSM 700 META, Jena, Germany). Quantification of the fluorescence intensity from the red channel (β-Catenin) was performed using the Image J software (NIH Image).

#### *4.10. Measurement of LPL Activity*

For LPL activity detection, cells were harvested after the medium removed, washed with ice-cold PBS and lysed with cell lysis buffer (20 mM Tris, 150mM NaCl, 1% Triton X-100). The lysate was centrifuged at 10,000× *g* for 5 min at 4 ◦C. Then LPL enzyme activity was measured in the supernatant by the enzyme fluorescence method using Biovision LPL Activity assay kit (Biovision Incorporated, Milpitas, CA, USA) according to the manufacturer's instructions as previously described [9]. Results were normalized to the amount of protein (mU per mg of bulk cellular protein). Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

#### *4.11. Real-time Quantitative PCR Analysis*

Total RNA was extracted from cultured cells with the High Pure RNA tissue kit (Omega Bio-Tek, Norcross, GA, USA) and 500 ng of total RNA was reverse transcribed using PrimeScript RT Master Mix Kit (TaKaRa, Tokyo, Japan). Real-time RT-PCR was conducted on the Step One Plus Real-Time PCR System (ABI, Waltham, MA, USA) with the following program: 95 ◦C for 30 sec, 95 ◦C for 5 sec, 60 ◦C for 30 sec, 95 ◦C for 15 sec, 60 ◦C for 1 min, and 95 ◦C for 15 sec, with 40 cycles of steps 2 and 3. Primers were synthesized by Invitrogen (Shanghai, China). Amplication was performed in 25 μL reaction system containing specific primers (Table S1) and SYBR Premix Ex Taq II (TaKaRa, Tokyo, Japan). Relative gene expression was calculated using the comparative Ct method with the formula 2−ΔΔCt [41]. The two reference genes GAPDH and HPRT1 were used. The geometric mean of relative gene expression was calculated and used for further analysis as previously reported [42].

#### *4.12. Protein Extraction and Western Blotting Analysis*

Total protein from cultured trophobalst cells was extracted using cell lysis buffer (Beyotime Co, China) by procedures as previously described [38]. Nuclear protein isolation was performed using Nuclear and Cytoplasmic Protein Extraction Kit (KenGEN BioTECH, Nanjing, China) according to the manufacturer's instructions as previously reported [11]. The concentration of protein was quantified using BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA). Proteins (50 μg) were separated by SDS-PAGE and transferred to PVDF nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). After blocking in 5% fat-free milk for 1 h at room temperature, the membranes were incubated with rabbit anti-Mark4(4834, 1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA), β-Catenin (8480, dilution 1:1000, Cell Signaling Technology), GAPDH (2118, dilution 1:1000, Cell Signaling Technology), and Phospho-Mark4 (SAB4504258, 1:500 dilution, Sigma, Saint Louis, MO, USA) antibody, Goat anti-DKK1 (LS-B194, dilution 1:1000, LifeSpan BioSciences, Seattle, WA, USA) antibody, or Mouse anti-LaminA (sc-376248, dilution 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) antibody overnight at 4 ◦C, followed by incubation with Donkey anti-goat, Goat anti- mouse or rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies (HAF109, HAF007 and HAF008, dilution 1:2000, RD SYSTEMS, Minneapolis, USA) for 1 h at room temperature. Proteins were visualized using the LumiGLO Reagent and Peroxide system (Cell Signaling Technology, Danvers, USA), and then the blots were quantified using Bio-Rad ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, USA). Band density was normalized according to the GAPDH content.

#### *4.13. Statistical Analysis*

All the data were obtained from at least three independent experiments. Statistical analyses were conducted using SPSS Statistics 20.0 software (IBM SPSS, Armonk, NY, USA). Data were analyzed using One-way ANOVA for comparisons among groups, followed by Duncan test. Results were expressed as means ± SEM. A *p*-value < 0.05 was considered statistically significant, and very significant was indicated when *p* < 0.01.

#### **5. Conclusions**

In summary, our present study demonstrates that MARK4 stimulates fatty acid accumulation in porcine trophoblast cells, which could contribute to a lipotoxic placental milieu in conditions associated with elevated maternal fatty acids such as excessive back-fat during pregnancy of sows. Moreover, WNT/β-catenin signal is essential for MARK4 promoting lipogenesis in pig placental trophoblasts (Figure 8). Thus, our results indicate that MARK4 has potential as a regulator of lipotoxicity associated with maternal obesity in the pig placenta.

**Figure 8.** A proposed model for role of MARK4 in regulating lipogenesis in pig placental trophoblast cells. MARK4 promotes lipogenesis by activating WNT/β-catenin signaling pathway. Arrows indicates a positive regulation and bar-headed lines show negative regulation. Interactions depicted are based

on studies performed in various tissues (in some cases placenta) and have been previously published.

**Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/20/5/ 1206/s1, Figure S1: The full-length cDNA of MARK4 gene in porcine and the deduced amino acid sequence, Figure S2: Clustal W alignment of the MARK4 protein from pig and other organisms, Figure S3: The secondary structure of the MARK4 protein in porcine constructed by the SABLE program, Figure S4: Phylogenetic tree based on MARK4 sequences made by MEGA 7.0 software using the neighbor-joining method, Figure S5: Identification of optimal fatty acid (FA) concentration for induction of FA accumulation in pig primary cytotrophoblasts, Table S1: Primer sets used for real-time PCR.

**Author Contributions:** All the authors contributed to this manuscript. Planed experiments: L.T. and P.S.; Performed experiments: L.T. and D.S.; Analyzed data: L.T. and A.W.; Contributed reagents/materials/analysis tools: L.T., A.W. and D.S.; Wrote the paper: L.T.

**Funding:** This research was surported by National Nature Science Foundation of China (No. 31702120), Fundamental Research Funds for the Central Universities of China (No. KJQN201831) and grants from the Nature Science Foundation of Jiangsu Province of China (No. BK20150672).

**Acknowledgments:** We sincerely acknowledge staffs in Research Farm and National Experimental Teaching Demonstration Center of Animal Science of Nan Jing Agricultural University for their helpful assistance in samples collection and offering technical platform.

**Conflicts of Interest:** The authors declare no conflict of interest.
