Supplemental figures

Fig S1. Effect of Sorafenib and Brefeldin-A on MEK1b phosphorylation. HeLa cells overexpressing either MEK1b-GFP, MEK1-GFP, GFP-ERK1c or GFP-ERK1 (underlined names in each pairs of panels) were either left non-treated (NT), treated with Sorafenib (Sora, 10 mM, 24 h), with nocodazole (Noc, 100 ng/ml, 24 hr), with nocodazole plus Sorafenib (Noc+Sora, 100 ng/ml Noc +10 mM Sora, 24h), or Brefeldin-A (BFA, 2µg/ml, 2h). The cells were then harvested and subjected to a Western blot analysis with anti pMEK, pERK gMEK (C-18), and gERK(CRS) Abs as indicated. Similar results were obtained also when BFA or Sora were added for different times.

Fig S2. MEK1b is a specific ERK1c kinase, while MEK1 preferentially phosphorylates ERK1. (A) Time course of ERK1/1c phosphorylation by MEK1b-GFP. HeLa cells were transfected with MEK1b-GFP treated with nocodazole (100 ng/ml, 24 hr). (B) Time course of ERK1/1c phosphorylation by MEK1-GFP. HeLa cells were transfected with MEK1-GFP. The cells were serum starved and then treated with EGF (50ng/ml, 3 min). The proteins from either A or B separately were IPed with anti GFP Ab, and subjected each to in vitro phosphorylation using bacterial GST-ERK1/1c as substrates. In addition, the MEK inhibitor, U0126 (U), was added for 30 min together with the kinases. The amounts of ERK1/1c as well as their phosphorylation levels were determined by Western blotting using anti pERK, gMEK, and gERK(CRS) Abs. (C) Quantification of ERK1/1c phosphorylation. Known amounts of phosphorylated ERK were added to the blots (see text for details) and used as a calibration curve that enabled the comparative determination of phosphorylation levels of ERK1/1c. The results in the graphs represent means ± standard errors (SE) of 3 experiments. Squares represent ERK1 phosphorylation and triangle represents ERK1c phosphorylation.

Fig. S3. The induced expression and activity of MEK1b during G2/M phase of the cell cycle in HEK293T cells are similar to that in HeLa cells. (A) ERK1c, but not ERK1 or ERK2, undergoes MEK-dependent phosphorylation during G2/M phase of the cell cycle in HEK293T. Cells were transfected with GFP, ERK1-GFP, ERK2-GFP, or ERK1c-GFP. Each of these transfected cells was treated with nocodazole (100 ng/ml, 24 hr (+)), or left untreated (-). U0126 (5 µM, 1 hr prior to harvesting) was added to additional experiment with cell transfected with ERK1c. Then, the cells were harvested and subjected to a Western blotting with apERK, and agERK(CRS) Abs. (B) Expression and specific phosphorylation are elevated in the G2/M phase of the cell cycle in HEK293T cells. HEK293T cells were synchronized by a double-thymidine block, or left untreated (-) as a control. At the indicated times after block release, the cells were fixed and stained with DAPI. The percentage of mitotic cells out of total number of cells was counted and revealed that mitosis occurs around 10 hrs after release (data not shown). In addition, the harvested cells were subjected to a Western blot with anti gMEK1 Ab (upper panel). Equal amounts of MEK1b (middle panel; prepared by a calibrating Western blot with anti MEK (H-8) Ab) were subjected to Western blot with anti pMEK Ab (lower panel). (C) Silencing of MEK1b/ERK1c inhibits Golgi fragmentation in G2/M phase of the cell cycle in HEK293T cells. HEK293T Cells were transfected with GFP, Sh-MEK1b together with GFP (Sh-MEK1b), Sh-ERK1c together with GFP (Sh-ERK1c) or Sh-MEK1 together with GFP (Sh-MEK1). The cells were synchronized by double thymidine block, and 10 h after the release were stained with p58 Abs and visualized. (D) Quantification of Golgi fragmentation. The cells in C were counted, and percentage of cells with fragmented Golgi out of total transfected cells is presented as means ± SE of 3 experiments (n = 200). Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.01.

Fig. S4. Effect of Sorafenib and BFA on mitotic Golgi fragmentation. A) Effect of Sorafenib and BFA measured by p58 staining. GFP expressing HeLa cells were synchronized by a double-thymidine block. At point of release (time 0) the synchronized cells were treated with sorafenib (Sora; 10 mM), or left untreated, both for 10 hr. Alternatively, the GFP expressing HeLa cells were treated with BFA (2 mg/ml, 2h, or left untreated (Basal). The cells were then fixed, stained with anti p58 Ab, and visualized by a fluorescent microscope. Arrows indicate fragmented Golgi. Bar = 10 µM. (B) Quantification of Golgi fragmentation. The percentage of cells with fragmented Golgi out of all transfected cells for the treatments in A is presented as means ± SE of 3 experiments (n = 40).

Fig S5. MEK1b regulates mitotic progression. (A) Modulation of ERK1c and MEK1b expression affects cell cycle progression. HeLa cells were transfected with GFP, MEK1b-GFP, MEK1-GFP, Sh-MEK1b (+ GFP), Sh-MEK1 (+ GFP), DN-EE-MEK1b-GFP, DN-EE-MEK1-GFP, GFP-ERK1c, (ERK1c), or Sh-ERK1c. The cells were synchronized by double-thymidine block as above. At the indicated times, the cells were fixed, stained with propidium iodide and analyzed by flow cytometry for DNA content (2N or 4N). (B) Quantification of the effect of the various treatments. The ratio between G1/G2 was compared to that of the control cells in the GFP expressing cells. The results are means ± SE of 3 experiments. Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.01. (C) Correlation between the effects of MEK1b and ERK1c expression on Golgi fragmentation with that of cell cycle. HeLa cells were transfected with the plasmids like in A, the cells were synchronized by double-thymidine block and 9 h after release were stained with p58 Abs and DAPI. The cells were counted, and the percentage of cells with fragmented Golgi out of total transfected cells is presented as means ± SE of 3 experiments (n = 40). Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.01. (D) MEK1b-induced mitotic progression is mediated by ERK1c but not ERK1. HeLa cells were transfected with GFP, GFP + shERK1C, MEK1b-GFP, MEK1b-GFP + Sh-ERK1c, MEK1b-GFP + Sh-ERK1. The cells were synchronized by double-thymidine block as above, and then fixed, stained with propidium iodide and analyzed by flow cytometry for DNA content (2N or 4N). (E) Quantification of the effect of the various treatments. The ratio between G1/G2 was compared to that of the control cells in the GFP expressing cells. The results are means ± SE of 2 experiments. Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.01. ShE1c – Sh-ERK1c, ShE1 – Sh-ERK1.

- 3 -