Autophagic flux response and glioblastoma sensitivity to radiation

Western blot images of the LC3A and LC3B proteins, in the pellet fraction of the T98 and U87 glioblastoma cells, 48 h and 7days after exposure to 4Gy of radiation.

In order to confirm that LC3A-LAMP2Aor LC3B-CTSD imaging indeed records “autophagic flux” changes, T98 and U87 cells were incubated with Bafilomycin A, (100 nM) for 24h. The autophagosome and lysosome fusion was disrupted as it is demonstrated in the relevant confocal microscopy images. As the drug has its major effect on slowing the degradation of LC3-II within existing autolysosomes, incubation for 24h results to accumulation of LC3A or LC3B auto-lysosomes. This, however, was more prominent in the T98 cell line.

(A) Relative quantitative analysis of mRNA levels of the LC3A, LC3B, p62, TFEB, LAMP2a and CTSD mRNA levels at 0, 2 and 7 days after exposure of T98 and U87 cells lines to 4 Gy of radiation (*P < 0.05, **P < 0.01, ***P < 0.001). (B) γH2AX nuclear foci expression and image densitometry of nuclear staining of T98 and U87 cell lines exposed to escalated doses of radiation. (C) Detection of apoptosis in T98 and U87 cell lines, assessed with confocal microscopy, following 4 Gy of irradiation, at various time points (typical confocal images, fluorescence intensity assessment and % of apoptotic cells) (40 x). (D) Caspase 9 (csp9) expression levels in the T98 and U87 cell lines, at 2 and 7 days, following exposure to 4 Gy radiation. (E) Percent of cells with SA-β-gal expression in wild type T98 and U87 cells (control), cells with silenced LC3A (shLC3A cells), and following exposure to 4 Gy of radiation (on the 4^th day).

(A) Confocal images (100 x) and Western blot analysis of T98 and U87 glioblastoma cell lines stably transfected with shLC3A, shLC3B and shTFEB, showing successful suppression of protein expression (assessed in the supernatant fraction). (B) Growth delay dose/response curves, assessed on the 7 th day after irradiation, in the T98 and U87 cell lines without and with repression of the LC3A, LC3B, LC3A/B and TFEB protein expression, using siRNA. (C) Cell viability of the T98 and U87 cell lines after 24 h exposure to temozolomide (10 μΜ) or cisplatin (10 μΜ), in wild type and transfected cells with siLC3A, siLC3B or siTFEB (*P < 0.05, ** P < 0.01, *** P < 0.001).

(A) Reduced expression of the LC3A-1 protein in the supernatant of T98 and U87 cell lines, following transfection with specific vs. random non-coding siRNAs. (B, C) Growth kinetics of the wild type and shLC3A transfected T98 (B) and U87 cell lines (C) following subcutaneous implantation in athymic mice. (D) A typical image of a U87 implanted tumor in an athymic mice showing different size of the wild type and shLC3A implanted tumors, 12 days after irradiation.

In vitro growth curves of T98 and U87 control cells and cells transfected with LC3A shRNA.