Fig. 2

Irradiation of glioblastoma cells increases the levels of CD147 protein in their EVs. a Immunoblots for CD147 on the EVs of T98G and U-87 MG cells are shown for the 0 or 8 Gy dose at 24, 48, and 72 h for three experiments. The ‘C’ label represents the 0 Gy control samples, and the ‘IR’ label is for samples receiving 8 Gy of ionizing radiation. Immunoblot for CD147 in lysates of T98G cells, harvested after the 72 h collection of EVs, was also performed. CD147 levels were normalized relative to the Ponceau S stain. Graphical representations of these results are also displayed. b Immunoblot for CD147 on EVs collected at 24 h from T98G cells receiving increasing doses of γ-rays. The level of CD147 in the cells at 24 h was also measured. c Immunoblot for CD147 in EVs from T98G cells with CD147 knockdown, showing that CD147 still increases in response to γ-irradiation, even in partial knockdowns. Plasmid 1 clone 1 was the knockdown used in experiments. The scramble control (Scr) has saturated signal due to the high levels of CD147 in its EVs relative to the knockdowns. Clones 2 and 3 of plasmid 1 and clones 1, 3, and 4 of plasmid 2 demonstrate that even following knockdown, CD147 is increased in EVs when cells are irradiated. Immunoblots for CD147 on the cell lysates of the selected clones is also shown relative to a control c to provide a relative knockdown efficiency for cellular CD147. d Immunoblots on EVs of 0 or 8 Gy γ-irradiated U-118 MG cells, with the EVs from γ-irradiated U-87 MG cells used as a positive control. Despite EVs from U-118 MG cells not having CD147, they contained MCT1 and MCT4, which are found in EVs and co-localize with CD147. Ponceau S Red Stain was used as loading control. e Sucrose gradients determined that CD147 and CD63 sediment together at the correct density for EVs