Fig. 2: TMZ induces autophagy in C2C12 and RH30 cell lines.
From: Autophagy modulates temozolomide-induced cell death in alveolar Rhabdomyosarcoma cells
a C2C12 and RH30 cells were treated with TMZ (100 μM, 36, 72, 96 h) and autophagy hallmark was detected in both C2C12 and RH30 cell lines using immunoblotting. TMZ-induced LC3β lipidation, Atg5-12 conjugation, and Beclin-1 expression in both cell lines. Beta-actin was used as loading control. Data are representative of three independent experiments using different cultures. b, c RH30 and C2C12 cells were treated with TMZ (100 μM, 72 h) and using immunocytochemistry LC3 puncta and changes in lysosomal activity (LysoTracker red staining) has been investigated. The results showed that TMZ increased LC3 puncta and LysoTracker red fluorescence intensity and co-localization of LC3 puncta and LysoTracker in both RH30 and C2C12 cells. d Transmission electron microscopy showed that in treated RH30 and C2C12 cells there are accumulated autophagosome-like structures compared to control and normal cells after 72 h treatment. Arrows show the autophagolysosomes containing the cargo (magnification ×11,600). e C2C12 cells were treated with TMZ (100 µM, 72 h) and then treated with Baf-A1 (100 nM, 1, 2, 3, 4 h) to evaluate for autophagy flux. TMZ + Baf-A1 treatment induces more lipidated LC3β and reduces the degradation of p62 in RH30 cells. Beta-actin was used as the loading control. Data are representative of three independent experiments. f C2C12 cells were treated with TMZ (100 μM, 72 h) and Baf-A1 (100 nM, +4 h) followed by immunocytochemistry to evaluate LC3 puncta and changes in lysosomal activity (LysoTracker red staining). The results showed that TMZ increased LC3 puncta and LysoTracker red fluorescence intensity in C2C12 cells. On the other hand, Baf-A1 and TMZ + Baf-A1 significantly decreased the LysoTracker red fluorescence in the presence of an accumulation of LC3 puncta showing the inhibition of autophagy flux by Baf-A1. g, h Representative figures of LC3 puncta and fluorescence intensity for LysoTracker in C2C12 cells in the presence of TMZ, Baf-A1, and TMZ/Baf-A1 treatment. These results showed that the number of LC3 puncta is significantly higher in cells which are treated with Baf-A1 and TMZ + Baf-A1. However, the fluorescence intensity of LysoTracker was lower in Baf-A1, and TMZ + Baf-A1 treated cells. i RH30 cells were treated with TMZ (100 µM, 72 h) and then treated with Baf-A1 (100 nM, 1, 2, 3, 4 h) to evaluate for autophagy flux. TMZ/Baf-A1 treatment induces more LC3β lipidation and reduces the degradation of p62 in RH30 cells compared to TMZ and Baf-A1 single treatment. Beta-actin was used as the loading control. Data are representative of three independent experiments. j RH30 cells were treated with TMZ (100 μM, 72 h) and Baf-A1 (100 nM, +4 h) followed by immunocytochemistry to evaluate LC3 puncta and changes in lysosomal activity (LysoTracker red staining). The results showed that TMZ increased LC3 puncta and LysoTracker fluorescence intensity in RH30 cells. On the other hand, Baf-A1 and TMZ + Baf-A1 significantly decreased the LysoTracker red fluorescence showing the inhibition of autophagy by Baf-A1. k, l Representative figures of LC3 puncta and fluorescence intensity for LysoTracker in RH30 cells in the presence of TMZ, Baf-A1, and TMZ/Baf-A1 treatment. These results showed that many LC3 puncta are significantly higher in cells which are treated with Baf-A1 and TMZ + Baf-A1. However, the fluorescence intensity of LysoTracker was lower in Baf-A1, and TMZ + Baf-A1 treated cells
