Apc mouse anti human cd44

1 × 10⁵ MCF7 cells were treated with atovaquone (5 μM and 10 μM) for 48 hours in 6-well plates, grown as a monolayer. Then, cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 12 hours, MCF7 cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen cat.559942) antibodies for 15 minutes on ice. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Invitrogen) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live population, as identified by the LIVE/DEAD dye staining, was analyzed for CD24/CD44 expression. Data were analyzed using FlowJo software.

The growing cells cultured in 100 mm dishes were washed with 1× PBS, trypsinized, centrifuged, and resuspended in 2% FBS in Hank’s balanced salt solution (HBSS). The viable cell number was counted using an automatic cell counter (trypan blue exclusion), and the cell density was adjusted to 1 × 10⁶ cells/100 μL. For CD24 and CD44 staining, 20 μL of antibodies (anti-CD24, anti-CD44 and PE-mouse IgG isotype control and APC-mouse IgG isotype control) were added to 100 μL of cell suspension and incubated at 4 °C for 30 min per the manufacturer’s protocol. However, for ABCG2 staining, 5 μL of APC Mouse Anti-Human CD338 and APC-mouse-IgG isotype control were added to 100 μL cell suspension and incubated at 4 °C for 30 min, according to the manufacturer’s instructions. After incubation, the cells were washed three times with HBSS and centrifuged at 2000 rpm for 3 min. Then, the cells were resuspended in 300 μL 1× PBS containing 1 μg/mL DAPI and were passed through tube filters to avoid cell aggregation. Finally, the cells were analyzed using flow cytometry. PE mouse anti-human CD24, APC mouse anti-human CD44, PE mouse IgG2α, κ isotype control (555574), APC mouse IgG2α, and κ isotype control antibodies were purchased from BD Bioscience (San Jose, CA, USA), and anti-mouse IgG₁ isotype control (MAB002) was purchased from R & D Systems, Inc. (Minneapolis, MN, USA).

1 × 10⁵ MCF7 cells were plated in 6-well plates in complete media supplemented with 10% heat-inactivated FBS. Next day, cells were treated with DPI (5, 10, 50 nM) for 5 days. Vehicle alone (DMSO) control cells were processed in parallel. Briefly, 30,000-50,000 live cells, as identified by 7-AAD dye staining, were analyzed for CD24/CD44 expression. We employed CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen) antibodies for FACS-analysis, using the BD LSR Fortessa (BD Bioscience). Results are the average of three biological replicates (repeats) and are expressed as percentages of mean fluorescence intensity, normalized to the control. ^* p<0.05, ^** p<0.01, ^*** p<0.001. One-way ANOVA was used with Bonferroni's multiple comparisons test.

After a 72 h treatment, SUM159 and MCF10A cultures were trypsinized, counted, washed with phosphate-buffered saline (PBS) and stained with cell surface markers: FITC anti-human CD326 (EpCAM) (BioLegend, Inc., San Diego, CA), PE-Cy7 Mouse Anti-Human CD24 (BD Biosciences, San Jose, CA), and APC Mouse Anti-Human CD44 (BD Biosciences, San Jose, CA). One million cells were incubated with the antibodies for 15 minutes in dark on ice with manufacturer recommended concentrations. After washing with PBS, cells were analyzed using the LSRFortessa cell analyzer (BD Biosciences, San Jose, CA). Side and forward scatter were used to eliminate debris and cell doublets. Unstained and IgG2a, κ isotypes with PE-Cy7, FITC, and PE (BD Biosciences, San Jose, CA) stained samples were used as controls.

Trypsinized cells were incubated with anti-human CD44 antibodies (allophycocyanin) (BD Pharmingen, APC Mouse Anti-Human CD44, 559942, USA), anti-human CD24 antibodies (phycoerythrin) (BD Pharmingen, and PE Mouse anti-human CD24, 555428, USA) on days 1, 3, 5 and 7. Next, cells were resuspended in FACS buffer (1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in PBS) and then aquired using a FACSAria flow cytometer (Becton Dickinson, San Jose, California, USA). Results were analyzed with FlowJo Data Analysis Software (FlowJo LLC, OR, USA).

Rabbit anti-human TMEM98 (Catalog #NBP1-84154), FAT4 (Catalog #NBP1-78381) and GPR64 (Catalog #NBP1-84906) were purchased from Novus (Littleton, CO), RAP1A, APC- mouse anti-human CD44 from BD Pharmingen (San Jose, CA), PE-mouse anti-human CD24 and PE-murine IgG isotype from Biolegend (San Diego, CA), PerCP-Cy5.5- anti-rabbit IgG from Santa Cruz Biotechnology (Dallas, Tx), PE-anti- human pan-cytokeratin from Abcam (Cambridge, MA) and APC-mouse IgG isotype control from (BD Biosciences). Mouse anti-fibronectin (FN1) was prepared as ascites from a hybridoma cell line that was purchased from ATCC.