Fig. 2

Knockdown of USP5 expression suppresses stemness properties in lung cancer. A CL1-5-shLacZ, shUSP5#1, and shUSP5#2 cells were cultured at 1,000 cells per well in low-attachment plates to assess sphere formation. Left panel, representative stitched brightfield images were produced using NIS-Elements software (Nikon). Scale bar: 200 μm. Right panel, quantification of spheres generated by CL1-5-shLacZ, shUSP5#1 or shUSP5#2 cells after 14 days. B shLacZ-, shUSP5#1-, and shUSP5#2-transduced cells were dissociated into single cells, and viable cells were plated in low-attachment 96-well plates for a limiting-dilution assay. The percentage of wells with spheres was measured and used to compute the limiting dilution to determine the frequency of sphere-initiating cells. The frequency and P-value were calculated using ELDA software. C and D Representative flow cytometric analysis (C) and quantification (D) of CD44⁺ subpopulations of shLacZ, shUSP5#1 and shUSP5#2 cells. E The mRNA levels of stemness-related factors in CL1-5-shLacZ, shUSP5#1 and shUSP5#2 cells. F CL1-5-shLacZ and shUSP5#2 cells (25,000, 2,500 or 250 cells) were resuspended in a Matrigel solution (1:1 Matrigel:PBS) and subcutaneously injected into NOD/SCID mice. Tumor incidence was assessed for each dilution of cells for 8 weeks, and the CSC frequency was calculated using ELDA software. In all bar plots, *P < 0.05 and ***P < 0.001 by a two-tailed Student’s t test