Figure 2. SGK1 knockdown reduces CoCSC growth, invasive ability and chemoresistance.

Tu12, Tu21 and Tu22 were infected with copGFP control, control shRNA or SGK1 shRNA lentiviral particles (MOI=6.5). 72h post-transfection, cells were subjected to (A) light microscopy, and (B, left) RT-PCR analysis for SGK1 or 18S housekeeping gene as internal control. Scale bars, 100μm. After additional 5 days of puromycin selection (1.5-2μg/mL), cells were analyzed by immunofluorescence for SGK1 Ser422 (B, right). Scale bar, 100μm. (C) Decreased CoCSC clonogenicity following SGK1 knockdown. One hundred thousand cells infected with control or SGK1 shRNA lentiviral particles were plated in each well of a 6-well plate. 2 weeks later, colonies were fixed and stained with 0.2% crystal violet (CV) in 10% ethanol. (D) Bar graph (left) and optical imaging (right) of CV-stained transwells showing reduced invasive potential of SGK1-silenced Tu12 cells. Scale bar, 100μm. Fifteen thousand cells infected as in (C) were seeded on growth factor reduced Matrigel (2mg/ml)-coated transwells and allowed to invade for 48h. Data are presented as relative ratio of invasion from one experiment representative of two independent experiments carried out in triplicate (***p<0.001) (the regions representing invaded cells were selected using the “Magic Wand” tool, and the highlighted pixels counted using the histogram command in Adobe Photoshop). (E) Merged fluorescent TUNEL assay and optical imaging (left; scale bars, 100 or 50μm), and cleaved caspase-3 (CC3) Asp175 immunofluorescence (right, scale bar, 100μm) of Tu12, Tu21 and Tu22 cells infected as in (C) following 1μM Oxaliplatin treatment for 48h. Enlarged images (I, II, III) show CC3 reactivity in apoptotic bodies. (F) Bar graph showing mean percentage (±SD) of apoptotic cells calculated considering three different microscopic fields of TUNEL assay and CC3 staining (**p<0.01, ***p<0.001).