Journal: iScience

Article Title: MASTL is enriched in cancerous and pluripotent stem cells and influences OCT1/OCT4 levels

doi: 10.1016/j.isci.2022.104459

Figure Lengend Snippet: High MASTL levels correlate with OCT1 and mammosphere formation (A) Western blotting of β3-integrin (ITGB3), OCT1, MASTL, and GAPDH in MDA-MB-231 cells grown as a monolayer (2D) or as mammospheres. (B) Relative protein expression of MASTL to GAPDH, experimental setup shown in (A). (n = 3 biologically independent experiments, unpaired t-test, mean ± SD). (C) Western blotting of OCT1, MASTL, and GAPDH in MDA-MB-231 cells silenced with siControl or siOCT1 for 48 h. (D) Relative protein expression of MASTL to GAPDH, experimental setup shown in (C), two datapoints collected after 96 h and three after 48 h (n = 5 biologically independent experiments, one sample t-test, mean ± SD). (E) Western blotting of OCT1, MASTL, and tubulin in MDA-MB-231 cells overexpressing EGFP-control (enhanced green fluorescent protein) or hemagglutinin (HA)-tagged OCT1. (F) Relative protein expression of MASTL to tubulin, experimental setup shown in (E). (n = 3 biologically independent experiments, one sample t-test, mean ± SD). (G) Representative flow cytometry histograms of MDA-MB-231 cells treated with DMSO control or DCF-DA (2ʹ,7ʹ-Dichlorofluorescin Diacetate) to measure ROS activity after silencing with siControl (gray), siMASTL#6 (red), or siMASTL#7 (orange) for 48 h. (H) Geometric Mean of the DCF-DA signal (ROS activity) in the experimental setup described in (G). (n = 3 biologically independent experiments, unpaired t-test, mean ± SD). (I) Representative images of tetracycline-induced shControl and shMASTL#3 MDA-MB-231 cells grown in mammosphere culture conditions (7 days) and stained with Calcein (Nikon Eclipse Ti-E widefield microscope, Hamamatsu Orca C13440 Flash 4.0 ERG [b/w] sCMOS camera and Plan Apo lambda 20×/0.80, WD 1,000-μm objective). (J) Average mammosphere size (average ferret diameter of spheres in μm) and spheres/1,000 cells plated, experimental setup shown in (I). (n = 3 biologically independent experiments, eight replicate wells/experiment, unpaired t-test, mean ± SEM). (K) Western blotting of OCT1, MASTL, and GAPDH in tetracycline-induced (4 days) shControl and shMASTL#3 MDA-MB-231 cells. (L) Relative protein expression of MASTL to GAPDH, experimental setup shown in (K). (n = 3 biologically independent experiments, one sample t-test, mean ± SD). (M) Representative flow cytometry histograms of CD44 expression in tetracycline-induced (4 days) shControl and shMASTL#3 MDA-MB-231 cells. (N) Geometric mean of CD44 expression in the experimental setup described in (M). (n = 5 biologically independent experiments, unpaired t-test, mean ± SD). See also Figure S2 .

Article Snippet: Images were captured with (Nikon Eclipse Ti-E widefield microscope, Hamamatsu Orca C13440 Flash 4.0 ERG [b/w] sCMOS camera and Plan Apo lambda 20×/0.80, WD 1,000-μm objective).

Techniques: Western Blot, Expressing, Flow Cytometry, Activity Assay, Staining, Microscopy