Fig. 4: TMZ induces apoptosis and autophagy in C2C12 and RH30 3D culture.

a, b Bright field image of C2C12 a and RH30 b 3D culture which shows the morphology of untreated and TMZ treated cells (100, 250 µM, 4 h) in 3D culture. c–f Viability assay was done by adding the live/dead solution to cells 48 and 96 h after treatment with TMZ (0–500 µM). Cells were incubated for 2 h in the dark at room temperature, rinsed twice with DPBS, and confocal microscopy was used to capture live/dead cell images in C2C12 c and RH30 d cells. e, f Quantification of live/dead assay was measured by calculating the ratio of live: total cells which showed a significant decrease in viability of C2C12 e and RH30 f cells treated with different concentrations of TMZ. The data showed TMZ significantly induces cell death in both C2C12 and RH30 cells (P < 0.001) while TMZ induces more cell death in RH30 compared to C2C12 cells. g, h IF labeling of C2C12 cells g and RH30 cells h by cleaved PARP following treatment with TMZ (100 µM, 72 h) increased number of cells with cleaved PARP in TMZ treated cells in comparison to control cells which is the hallmark of increase of apoptosis in these cells. i, j After treatment of C2C12 i and RH30 j cells with TMZ (100 µM, 72 h), cells were IF labeled with autophagosome markers, LC3 and P62. Data showed that TMZ increases LC3 puncta (green) which is localized with p62 compared to corresponding time match control, a hallmark of autophagy induction in C2C12 and RH30 3D culture