Fluorescein isothiocyanate labeled phalloidin

Plasmids, specifically pReceiver-M61C-LAMC2, were constructed by GeneCopoeia (USA) and amplified at the School of Basic Medical Science, Zhejiang University. Chitosan (CS, molecular weight, 100,000) was purchased from Qingdao Yunzhou Biochemistry (China). Fluorescein-isothiocyanate-labeled phalloidin, Triton X-100, CelLytic buffer, and hyaluronic acid (HA) were purchased from Sigma Chemical Co. (MO, USA). A Label IT^® TM-Rhodamine labeling kit was purchased from Mirus Bio, LLC. (USA). Alamarblue and Lipofectamine LTX Plus reagent were purchased from Invitrogen (USA). Bovine serum albumin was purchased from Shanghai Sangon Biological Engineering Technology and Services (China). Hoechst 33258 DNA dye was purchased from Beyotime (China). Anti-laminin γ2 mouse monoclonal antibody, anti-integrin β4 mouse monoclonal antibody and anti-plectin rabbit monoclonal antibody were purchased from Abcam (USA). Goat anti-mouse IgG conjugated with Alexa 488 and goat anti-mouse IgG conjugated with Alexa 594 were purchased from Invitrogen (USA). Goat anti-rabbit IgG conjugated with rhodamine was purchased from Jackson ImmunoResearch Laboratories (USA). A human laminin-5 ELISA kit was purchased from R&D Technologies. Flat pure titanium plates 10 × 10 × 1 mm in size were purchased from Zhejiang Guangci Medical Appliance Company (China).

Fluorescent actin staining assays were performed as previously described with slight modifications [54 (link)]. Briefly, before cell suspension was added to a 6-well plate, sterile tweezers were used to place sterile cell coverslips into a 6-well plate. Cell culture conditions and co-bacteria culture times were same as those described in the bacterial adhesion assay. After incubation for 3 h, the coverslips were washed with PBS six times, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X-100. The cells were stained with fluorescein isothiocyanate-labeled phalloidin (Sigma-Aldrich, Shanghai, China) to visualize actin filaments and stained with propidium iodide (PI) (Beyotime, Shanghai, China) for nucleus visualization. At least 50 HeLa cells were calculated for A/E lesions forming number counting for each bacteria strain.

The full-length WT cDNA and mutant human ENPP1 cDNA entailing each of the three variants, c.656G>A, c.1530G>C, and c.876_880delTAAAG, were cloned into a pcDNA3.1(+) vector (GenScript). HEK293 and Cercopithecus aethiops kidney (COS7) cells were transfected using jetPEI (Illkirch, France). We measured the activity of nucleotide phosphodiesterase (NPP) 24 hours after transfection of HEK293 cells using pNP-TMP as substrate [7 (link),20 (link)]. Expression of human ENPP1 protein was detected by Western blot using a rabbit anti-human ENPP1 antibody, #5342, 1:1,000 (Cell Signaling, Boston, MA). An anti-human β-actin antibody, #4970, 1:1,000, was used to normalize protein loading (Cell Signaling). The cellular localization of the ENPP1 protein was analyzed in transfected COS7 cells using a monoclonal anti-human ENPP1 antibody, 1:100 (3E8, kind gift from Dr. Fabio Malavasi, Torino, Italy). Cells were stained with fluorescein isothiocyanate labeled phalloidin, #P5282, 1:40 (Sigma-Aldrich, Taufkirchen, Germany), to visualize plasma membrane localization. The PPi concentration in the medium of transfected HEK293 cells was measured 20 min after incubation with 20 μM GTP. PPi was quantified as previously described [10 (link),12 (link)].

hESCs grown in 24-well plates were treated with siRNA or Adenovirus for 2 days and then exposed to a decidualization stimulus of 8-Br-cAMP plus MPA for 3 days and then fixed with 4% paraformaldehyde (w/v) for 30 min at room temperature. Next, the cells were washed with PBS and permeabilized with 0.5% Triton X-100 in PBS at room temperature. Subsequently, the cells were blocked with 3% BSA in PBS and incubated with primary antibodies against ATF3 (1:50, 18655s, CST) at 4 °C overnight. Next, the cells were washed with PBST and then incubated with Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:100, A-21203, ThermoFisher Scientific), and fluorescein isothiocyanate-labeled phalloidin (1:300; P5282, Sigma, St. Louis, MO, USA) at room temperature. Cell nuclei were stained with DAPI.

Sunitinib malate (Sutent, Pfizer) was suspended in solvent 1, which contained carboxymethylcellulose sodium (0.5% w/v), NaCl (1.8% w/v), Tween 80 (0.4%, w/v), benzyl alcohol (0.9 w/v), and deionized water (added to final volume) adjusted to pH 6.0. Peptide HM-3 (Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp) was synthesized by GL Biochem Ltd. (Shanghai, China) and had a purity in excess of 99%. Other reagents included Matrigel (BD Biosciences), Fluorescein Isothiocyanate labeled Phalloidin (Sigma-Aldrich), Rhotekin RBD and PAK-1 PBD agarose conjugates (Millipore). The antibodies used for immunohistochemistry included: anti-CD31 polyclonal antibodies (sc-28188, Santa Cruz Biotech), anti-CD34 polyclonal antibodies (ZA-0550). Docetaxel was from Jiangsu Hengrui Pharmaceutical Co. LTD in China.

The HTR-8/SVneo trophoblast cell line was kindly provided by Dr. C.H. Graham, Queen's University, Kingston, Ontario, Canada [27] (link). The HTR-8/SVneo cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS),2 mM L-glutamine, penicillin(50 units/mL, and streptomycin(50 μg/ml) at 37°C in 5% CO₂[25] (link). Primary antibodies FAK, Phospho-FAK(Tyr397), MMP-2, MMP-9,TIMP-1, TIMP-2, integrins α1, α5, αV and β1 were purchased from Cell Signal technology Inc., BOSTER BIO-Engeneering, Ltd., or GeneTex, Inc. The secondary antibody horseradish peroxidase-conjugated IgG was the product of LKP, Inc. FAK inhibitor PF-573,228 and fluorescein isothiocyanate labeled Phalloidin were purchased from SIGMA. The thermo Scientific Pierce BCA Protein Assay Kit was used for determination of protein concentrations in cell lysates.