Fig. 2: Functional and quiescent properties of NS-induced cells (iU87MG).

a Cell lysates were prepared from U87MG and iU87MG cells and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then analyzed by western blot analysis with the specified antibodies. Representative immunoblots show an increased level of pluripotent stem cell markers (Oct4, Sox2, and Nanog) and Nestin at the protein level in iU87MG compared to U87MG. β-Actin served as a loading control. b U87MG and iU87MG (1 × 10⁴) cells were adhered on poly-l-lysine-coated coverslip and processed for immunocytochemistry with Oct4, Sox2, and Nanog primary antibodies followed by incubation with Alexa Flour 488-conjugated secondary antibodies. Representative confocal images merged with DAPI staining represented the enhanced expression of Oct4, Sox2, and Nanog in iU87MG compared to U87MG. c Cells were trypsinized, resuspended, counted, and seeded in stem cell-specific medium in ultra-low-attachment plates. Representative phase-contrast images demonstrated higher neurospheres formation after 72 h by iU87MG compared to U87MG. Bar graph represents quantitative measurements of the number of neurospheres per field (n = 7). d Western blots analysis from cell lysates of U87MG and iU87MG showing enhanced expression of Slug, Snail, and VEGF at the protein levels in iU87MG. β-Actin served as a loading control. e Transwell invasion assay showing higher invasive property of iU87MG. Representative phase-contrast images of invaded cells to the lower surface of the insert after 36 h. Cells were stained with crystal violet and a number of cells were counted (n = 9). f U87MG and iU87MG cells were seeded in six-well plates in serum-free medium. Scratch was made using the 10-µl tip in each well. Representative phase-contrast images showed cellular migration and filling of the gap after 8 and 24 h. The average wound was calculated and represented as the percentage of wound healing (n = 5). g U87MG and iU87MG cells were trypsinized and single cells were made and processed for cell cycle analysis. Representative histogram plots demonstrating restriction at the G0/G1 phase of iU87MG as compared to U87MG. h Representative immunoblots showing higher expression of Cyclin D1, Cyclin D2, and Cyclin E at the protein level in iU87MG compared to U87MG. β-actin was used as a loading control. i Representative phase-contrast images of re-differentiated iU87MG showing differentiated morphology in the fresh medium. Images were taken after 24 h of re-seeding. j Cell cycle analysis of U87MG and re-differentiated iU87MG showing similar trends and progression into the S phase after 24 h of re-seeding in the fresh medium. k Hoechst side population (SP) assay showing enhanced Hoechst-negative side population in iU87MG denoting enhanced drug efflux potentiality of these cells. l U87MG and iU87MG cells were treated with temozolomide, paclitaxel, 5-fluorouracil, and cisplatin separately. Cell viability assay of treated cells showed enhanced drug resistance in iU87MG than U87MG in a dose-dependent manner. The significance levels were shown between two groups using asterisks in each dose. m Schematic representation of nutritional stress-mediated stochastic emergence of CSC phenotype in GBM. Results represent mean ± SD from three independent experiments