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  • br Cell migration assay was performed based on the

    2020-08-30


    Cell migration assay was performed based on the modified Boyden chamber assay method using Trevigen's Cultrex® cell migration 24-well assay kit according to manufacturer's protocol, and a detailed description of the procedure is provided in the Supplementary Materials and Methods section.
    Cell viability studies of H1650 SP and MP MG 132 were performed with cisplatin treatment as previously described.21 In summary, cells (1 × 104 cells/well) were inoculated in a 96-well format for 16–18 h followed by treatment with different concentrations of cisplatin for 72 h. Cells were sequentially fixed in glutaraldehyde (0.025% w/v), stained with crystal violet solution (0.01% w/v), and resuspended in disodium hydrogen solution. Absorbance readings were taken at 540 nm to determine cell viability. Results representing 4–6 separate experiments were presented as average of percentage of cells inhibited vs. treatment with SEM.
    SOX2 protein expression in H1650 SP or H1650 MP cell lysates was performed per standard protocol as previously described.21 A detailed outline of the procedure is provided in the Supplementary Materials and Methods section.
    Preparation and characterization of cationic lipoplexes
    A detailed description of the preparation and characterization of cationic lipoplexes (CL) from combining DOTAP, DPPC and DSPE-mPEG [2000] as adapted from our previous study,21 with a detailed description of the procedure provided in the Supplementary Materials and Methods section.
    Animals study assurances
    Four- to six-week old male SCID-beige mice with mean weight of 21 gram (n = 45) (C.B-Igh-1b/GbmsTac-Prkdcscid- Lystbg N7) were procured from Taconic Biosciences (Cambridge City, IN). All animals were maintained on a standard animal chow and water ad libitum. Criteria for excluding mice from study, or early termination of treatment included significant loss (N25%) in body weight of mice from baseline and/or visual observation of general distress. Animals were randomized into 9 groups (n = 5) and allowed to acclimate for 6 days. All animal study protocols were approved by the Florida A&M University Institutional Animal Care and Use Committee (IACUC). Procedures involving animals were carried out in an AAALAC-approved facility located at Florida A&M University-College of Pharmacy and Pharmaceutical Sciences' F.H. Humphies Science Research Building.
    Tissue kinetics of fluorescent cationic lipoplexes in mice
    SCID-beige mice (n = 5) were injected (intraperitoneal, i.p.) with 100 μL of fCL. Animals were sacrificed at hourly intervals under isoflurane exposure followed by resectioning of the lungs. The kinetics of the fCL on the basis of lung tissue perfusion was investigated by digital imaging using the Carestream Molecular Imaging In-Vivo MS FX PRO (Bruker, Billerica, MA). Results representing three different readings were taken and presented as total flux (perfusion per second) vs. time (h).
    Animal model of xenograft and orthotopic tumor and treatment
    SCID-beige mouse modeling of orthotopic and xenograft lung tumors are as previously described,21–23 and treatment are detailed in the Supplementary Materials and Methods section.
    Efficacy studies
    Efficacy assessment of treatment on xenograft tumor growth progression over the treatment duration was performed on the basis of body weight variations from baseline (gram), tumor volume (mm3), tumor weight (mg), immunohistochemistry (IHC) staining and hematoxylin–eosin (H&E) staining. Xeno-graft tumor measurements were performed using an electronic  caliper on day 0, 4, 6, 8, 11 and 13, and the tumor volume estimated according to:
    IHC staining for SOX2, E-cadherin and N-cadherin, and H&E staining of resected lung tissue were performed as previously described.22 Results for body weight, tumor volume and tumor weight were presented as graphs of means with standard deviation (n = 5).
    Immunoblotting of tumor lysates
    Assessment of protein expression of xenograft tumors was performed by western blot as previously described,24 with detailed description provided in the Supplementary Materials and Methods section.
    Statistical analysis
    Where applicable, results were analyzed using the GraphPad Prism version 5.0 software (GraphPad Software, Inc.; La Jolla, CA). Statistically significant differences between groups were determined by two-tailed unpaired student t test at 95% confidence interval where P b 0.05 is considered significant.
    Results
    H1650 SP cells are upregulated for SOX2 and exhibit resistance phenotypes to chemotherapy
    Preparation and characterization of cationic lipoplexes loaded with siSOX2
    Table 1 shows parameters for characterization of cationic lipoplexes (CL) loaded with or without control siRNA (CL-siScr) and SOX2 siRNA (CL-siSOX2). Results represent means with standard deviation.