Figure 1.

Cell cycle arrest in HeLa S3 cells by v-Src-induced tyrosine phosphorylation. (a) Schematic depiction of our synchronization method. Cells were synchronized by a double thymidine block and cultured for the indicated times in thymidine-free medium with or without 1 µg/ml Dox. (b) HeLa S3/TR/v-Src-wt cells were stained with propidium iodide (PI) for analyzing cell cycle progression by flow cytometry. The histogram [Dox(+), 0 h] is presented with the control histogram of untreated cells synchronized at the G₁/S boundary [Dox(−), 0 h]. (c) HeLa S3 cells expressing inducible v-Src(K295M) [HeLa S3/TR/v-Src(K295M)] were cultured with or without 1 µg/ml Dox for 24 h, and whole cell lysates were analyzed by Western blotting (WB) using anti-Src (N-16), anti-actin, and anti-pTyr antibodies. (d) HeLa S3/TR/v-Src(K295M) cells were synchronized as depicted in Fig. 1a. Synchronized cells at the G₁/S boundary were released for 24 h or 30 h with or without 1 µg/ml Dox and stained with PI for analyzing cell cycle progression by flow cytometry. (e) HeLa S3/TR/v-Src-wt cells were synchronized as depicted in Fig. 1a. Whole cell lysates were analyzed by Western blotting using anti-phospho-ERK1/2 (pERK1/2), anti-ERK2, anti-Src (N-16) and anti-α-tubulin antibodies. Asy stands for asynchronous HeLa S3/TR/v-Src-wt cells.