Cell culture, vector transfection, RNAi gene silencing, gene expression analysis, and Western blot detection
ACC cell culture and vector transfection were performed as previously described . Briefly, the human ACC cell lines SW-13 and NCI-H295R (authenticated and supplied by American Type Cell Collection) were maintained under sterile conditions in a humidified incubator at 37.0 C with 5% CO2. SW-13 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% certified fetal bovine serum (FBS) and 10,000 U/mL penicillin/streptomycin; designated as complete medium (CM). NCI-H295R cells were grown in DMEM/F12 supplemented with 5% NuSerum, 10,000 U/mL penicillin/streptomycin, 5 μg/ml of insulin, 5 μγ/ml of transferrin, and 5 ng/ml of selenium (all reagents from Applied Biosystems); designated complete medium as well (CM). In general, cell strains underwent no more than 10 passages before experiments were performed.
Myc-DDK tagged pCMV6-Entry and pCMV6-Entry/SLC12A7-ORF plasmid expression vectors (Origene) were transfected into SW-13 cells using Lipofectamine 3000 (ThermoFisher) according to the manufacturer’s recommendations in 6-well plates with cells grown to 70–80% confluence. Stable clones of pCMV6-Entry and pCMV6-Entry/SLC12A7 vectors were selected in CM containing 800 μg/ml G-418 (Life Technologies). Multiple clones were then pooled into populations to avoid clonal variability. Selected SW-13 cell populations were designated SW-13/V (pCMV6 vector-transfected) and SW-13/S (pCMV6/SLC12A7-transfected) and were utilized to determine the effects of constitutive overexpression of SLC12A7 on the malignant behavior of SW-13 cells. Parental, un-transfected SW-13 cells were used as an additional reference control.
RNAi gene silencing of NCI-H295R cells were carried out with 3 unique 27-mer siRNA duplexes (designated siA, siB, and siC) targeting SLC12A7 (Human) using the standard protocol as previously described . Universal scrambled negative control siRNA was used as non-specific control (all from Origene). Lipofectamine 3000-mediated transfection was carried out in Opti-MEM medium according to the manufacturer’s recommendations (ThermoFisher) in 6-well plates with starting densities of 100,000 cells/well. Transfection medium was replaced with CM after 6 h of transfection. Cells were lysed for RNA extraction and gene expression analysis at 24 h post-transfection.
De novo and altered expression levels of SLC12A7, CEBPG, ID1, NFAT5, and SMAD5 mRNA were determined by gene expression analysis using a TaqMan assay (Applied Biosystems). Briefly, RNA was isolated from cells using the RNeasy Plus Mini Kit (Qiagen). Quantity and quality of isolated RNA was assessed by spectrophotometry (NanoDrop Technologies). Two hundred ng of RNA was used for cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad). Real-time quantitative PCR was performed on a CFX96 Real-Time System thermal cycler (Bio-Rad) using TaqMan PCR master mix (Applied Biosystems) with primers and probes (Applied Biosystems) specific to target genes. Relative expression was quantified using expression levels of housekeeping gene ribosomal protein large P0 (RPLP0) as a reference control (Hs99999902_m1; Applied Biosystems). Relative gene expressions were calculated using the Livak method . Gene expression analysis was performed in triplicate.
Enforced overexpression of SLC12A7 protein in SW-13 cells was confirmed via Western blot technique using anti-SLC12A7 rabbit polyclonal antibody (Novus), anti-rabbit-HRP goat antibody (Santa Cruz Biotech), mini-PROTEAN TGX gel (BioRad), PVDF blotting membrane (BioRad), and enhanced chemiluminescence detection reagents (ThermoFisher Scientific) according to the manufacturer’s protocols. Equivalent protein loading was ensured via Western blot detection of beta-actin expression using anti-β−actin mouse monoclonal antibody followed by anti-mouse-HRP goat secondary antibody (Santa Cruz Biotech). Western blot analysis was performed in duplicate.
Cell growth and clonogenic assays
Cell growth and clonogenic analysis was performed as previously described . Briefly, for growth assays, 50,000 cells per well of SW-13, SW-13/V, and SW-13/S cells were plated in 6-well plates and grown in CM at 37 °C. Total cell numbers and viability were assessed by staining cells with 0.4% Trypan Blue (Life Technology) and counting cells using a hemocytometer (Hausser Scientific) each day for 6 days. For clonogenic assays, 5000 cells per well of SW-13, SW-13/V, and SW-13/S were plated in 6-well plates and allowed to grow for 5 days in CM at 37 °C. On day 6, cells were washed with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde for 10 min, stained with 0.05% crystal violet for 30 min, washed in deionized water, and tabulated under microscopy. Independent colonies with 8 or more cells were counted and averaged from 6 wells. Cell and colony growth analysis was performed in 2 independent experiments with 6 wells for each cell type analyzed.
Migration and invasion assay
Migration and invasion analyses were performed as previously described . For migration, 100,000 cells of SW-13, SW-13/V, SW-13/S, and NCI-H295R were allowed to migrate through 8 μM pores in modified Boyden chambers (BD Biosciences) from upper chambers containing serum-free medium to lower chambers containing CM at 37 °C. After designated time points, cells that migrated to the lower side of the membrane towards a higher FBS concentration were fixed in 3.7% formaldehyde for 10 min, stained with 0.05% crystal violet for 30 min, washed using deionized water, and tabulated under microscopy. For invasion, 100,000 cells of SW-13, SW-13/V, SW-13/S, and NCI-H295R were allowed to invade in modified Boyden chambers from upper chambers containing serum-free medium through a layer of reconstituted Matrigel (BD Biosciences), which functions as a surrogate for the extracellular matrix, to lower chambers containing CM at 37 °C. After 24 h, the Matrigel was removed and cells that invaded through the Matrigel and migrated to the lower side of the membrane were fixed, stained, washed, and tabulated as above. Migration and invasion assays were performed in 2 independent experiments using 3 wells per each cell type analyzed.
Adhesion and detachment assay
For cell adhesion assays, 10,000 cells per well of SW-13, SW-13/V, SW-13/S or 250,000 cells per well of NCI-H295R were seeded in 24-well plates. The cell numbers were titrated to avoid overcrowding by trial experiments. Cells were incubated in CM at 37 °C for designated time points to allow for cell adherence to well surfaces. After each time point, cells were washed twice with PBS to remove non-adhered cells. The remaining, firmly attached cells were then fixed with 3.7% formaldehyde for 10 min, stained with 0.05% crystal violet for 30 min, washed using deionized water, and tabulated under microscopy. For cell detachment assays, 100,000 cells per well of SW-13, SW-13/V, and SW-13/S cells were seeded in 24-well plates. Cells were incubated in CM at 37 °C overnight. The following day cells were detached with a cell dissociation solution containing ethylenediaminetetraacetic acid tetrasodium salt dehydrate (EDTA; Sigma Aldrich) at multiple time points up to 15 min. The remaining attached cells were then fixed, stained, washed, and tabulated as above. Adhesion and detachment assays for both cells lines were performed in 2 independent experiments using 3–4 wells per each cell type/experimental condition analyzed.
Analysis of cell membrane organization by light and immunofluorescence microscopy
The effect of SLC12A7 overexpression on SW-13 cell extension repertoire was performed as previously described . Briefly, cells were grown on glass coverslips in CM overnight, fixed with 3.7% formaldehyde, stained with 0.05% crystal violet, washed using deionized water, and photographed under 400X magnification. Cell extensions on isolated, non-overlapping cells from 20 randomly selected photomicrographs for each cell type (SW-13, SW-13/V, and SW-13/S) were classified as three major types (lamellipodia, lobopodia, and filopodia) of cell surface extensions, averaged, and tabulated. For immunofluorescence analysis, cells were grown on sterile glass cover slips in CM and after 24 h cells were fixed in cold acetone-methanol (1:1) for 10 min. Immunostaining was then performed for SLC12A7 and EZR proteins using mouse anti-SLC12A7 (Santa Cruz Biotech) and rabbit anti-EZRIN (Abcam) primary antibodies and anti-mouse FITC or anti-rabbit TR secondary antibodies (Santa Cruz Biotech), followed by UltraCruz mounting agent containing 4′,6-diamidino-2-phenylindole – DAPI (Santa Cruz Biotech). Dilutions and incubations were carried out per the manufacturer’s recommendations. A Zeiss AX10 confocal microscope with AxioVision 4.8 program was used for immunofluorescence analysis and photomicrographs were taken at a total magnification of 1000X. Analysis of cell membrane organization by light and immunofluorescence was performed in duplicate.
Transcription factor expression array analysis
To assess possible altered transcription factor repertoire downstream to SLC12A7 enforced overexpression, transcription factor gene expression analysis was performed using the RT2 Profiler PCR Array Human Transcription Factors (PAHS-075Z; Qiagen) per the manufacturer’s instructions. Briefly, 500 ng of total RNA from SW-13, SW-13/V, and SW-13/S cells were purified using the DNA Elimination Mix, subjected to cDNA synthesis using the RT2 First Strand Kit, and then amplified with real-time quantitative PCR analysis using a CFX96 Real-Time System thermal cycler (Bio-Rad) using RT2 SYBR Green qPCR Mastermix (Qiagen). Using parental SW-13 cells as a reference control, relative expression levels of 84 transcription factors were analyzed with SABioscience PCR Array Data Analysis web-based software (Qiagen). Transcription factor expression array analysis was performed in duplicate. To confirm the significance of altered transcription factor signaling identified in SW-13/S cells, candidate transcription factor expression was measured using quantitative PCR techniques (refer above) in NCI-H295R cells treated for SLC12A7 siRNA knock down.
Continuous variables were analyzed using a two-tailed t test for normally distributed variables, or the Mann-Whitney U test for non-normally distributed variables. For variables with greater than two dependent values, a one-way analysis of variance (ANOVA) or Kruskal-Wallis tests were utilized for normally or non-normally distributed populations, respectively, with multiple comparisons made with a Tukey’s test. To assess the influence of two or more independent variables on one continuous dependent variable, a two-way ANOVA was utilized and multiple comparisons were made with a Tukey’s test. A p-value ≤ 0.05 was considered significant. Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software).