Figure 7. Time-course analysis of puncta formation and ATG gene expression changes in C2-ceramide-treated HeLa cells. (A) HeLa/GFP-LC3B cells were treated with DMSO (white bar) or 100 µM of C2-ceramide (black bar) for the indicated time period (h) and the number of puncta per cell was counted. The bars indicate the mean ± SD of 129, 159, 127, 135, 128, 145, 148, 152, 167, and 109 cells, respectively (bars from left to right). HeLa cells were treated with DMSO (white bar) or 100 µM of C2-ceramide (black bar) for the indicated time period (h), and quantitative RT-PCR was performed to determine the WIPI1 (B), MAP1LC3B (C), ULK1 (D), GABARAPL1 (E), and ATG13 (F) mRNA levels. The means ± SDs are shown as the relative fold-induction when the values obtained in DMSO-treated cells were set as 1. The GAPDH level was used as an internal standard. *P < 0.05 (5 areas for puncta formation assay and n = 2 for quantitative RT-PCR).