fig2

Figure 2. Effects of TMZ treatment on GBM cells and GBM-derived EVs. Cell viability was assessed by MTT assay in (A) T98G, (B) U87MG, (C) U251MG, and (D) U373MG cells treated with 100, 200, 400, or 800 µM TMZ for 24, 48, or 72 h. Data are reported as a percentage compared to the control (untreated cells; CTRL), presented as mean ± SD of three independent biological replicates (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. control (Student’s t-test); (E) Representative AFM images of CTRL (untreated) or TMZ-treated T98G, U87MG, U251MG, and U373MG cells. The Nu and PNu/CE areas are highlighted by green- and blue-dotted lines, respectively; Quantitative value of Rq measured in the Nu and PNu/CE area of CTRL or TMZ treated (F) T98G, (G) U87MG, (H) U251MG, and (I) U373MG cells; (J) Representative 3D reconstruction from AFM images of isolated EVs and (K) relative diameter measurements (mean diameter is shown as a black dotted line) of CTRL or TMZ-treated T98G, U87MG, U251MG, and U373MG; (L) Particle concentration measured by NTA. Data are presented as mean ± SD of three independent biological replicates (n = 3); ^*P < 0.05, ^***P < 0.001 vs. control (Student’s t-test); (M) Cryo-EM images of EVs isolated from T98G, U87MG, U251MG, and U373MG cells treated with TMZ. AFM: Atomic force microscopy; cryo-EM: cryo-electron microscopy; CTRL: control; EVs: extracellular vesicles; GBM: glioblastoma; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NTA: nanoparticle tracking analysis; Nu: nuclear region; PNu/CE: perinuclear/cellular edge; Rq: root mean square roughness; SD: standard deviation; TMZ: temozolomide.