Journal: American journal of physiology. Cell physiology

Article Title: Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

doi: 10.1152/ajpcell.00467.2008

Figure Lengend Snippet: Hyperosmolarity induces ROCK-mediated LIM kinase and cof phosphorylation. A and B: cells grown in 6-well plates were preincubated for 10 min with vehicle or 10 μM Y-27632 in isotonic medium and then challenged with hyperosmolarity (300 mM sucrose) in the presence or absence of Y-27632 for the indicated times. Subsequently cells were lysed and the lysates were subjected to Western blot anaylsis using pcof and cof antibodies. Cumulated data for n = 6. B: similar experiments were performed as in A, and the lysates were probed with phospho-LIMK and LIMK2 antibodies (n = 4). Blotting with anti-LIMK1 did not provide clear, specific labeling (not shown). C: cells were preincubated with vehicle or 10 μM PAK18, a selective PAK inhibitor, for 20 min under isotonoic condition and then challenged with hyperosmolarity for 5 min in the presence or absence of the drug. Cof and pcof content were determined by Western blots. GADPH was used as a loading control. D, top: cells were pretreated in isotonic medium with or without Y-27632, treated iso-or hypertonically for 5 min (as indicated) in the absence or presence of 10 μM Y-27632, and then processed for Western blotting using an anti-phospho-p38 antibody. The blots were reprobed with anti-p38. Bottom: cells were preincubated with 10 μM SB-203580, treated in the absence or presence of the drug, and processed as stated above. Cell lysates were probed for pcof and reprobed for cof. E: similar experiments were performed as in C, and the lysates were probed with anti-p38 and anti-phospho-p38 antibodies.

Article Snippet: The ROCK inhibitor Y-27632, the p38 mitogen-activated protein kinase (designated as p38) inhibitor SB-203580 and the PAK inhibitory peptide PAK18, were from Calbiochem.

Techniques: Phospho-proteomics, Western Blot, Labeling, Control

Journal: Oncotarget

Article Title: IRE1α-XBP1 inhibitors exerted anti-tumor activities in Ewing’s sarcoma

doi: 10.18632/oncotarget.24467

Figure Lengend Snippet: (A) The chemical formulas of four types of IRE1α-XBP1 pathway inhibitors. (1) Toyocamycin, (2) 2-hydroxy-1-naphthaldehyde (HNA), (3) STF-083010 (STF) and (4) 3-ethoxy-5 6-dilbromosalicylaldehyde (3ETH). These chemical formulas are totally different from one another. (B) The IC50 values of IRE1α-XBP1 inhibitors. The IC50 values of the four inhibitors were measured in four ES cell lines. Toyocamycin had the highest inhibition of ES cell growths among the inhibitors. (C) Cell viability curve of toyocamycin in ES and Su8686 (positive control cell line). The cell viabilities of toyocamycin were investigated in four ES cell lines and Su8686 (pancreatic cancer) which have high sensitivities of toyocamycin according to published articles. Toyocamycin significantly and dose-dependently inhibited the cell viabilities in all ES cell lines. (D) Tunicamycin (TM) mediated the expression of XBP1s in an ES cell line (TC71). Western blotting showed that TM stimulation time-dependently enhanced the expression of XBP1s in the TC71 cell line. (E) Toyocamycin inhibited XBP1s expression. Under conditions of TM stimulation, toyocamycin inhibited XBP1 expression dose-dependently.

Article Snippet: After 72 h, the inhibitory effect of these inhibitors on the growth of ES cell lines was assessed using a Cell Counting Kit-8 (Dojindo JAPAN, Tokyo Japan) and a Microplate Reader (SAFIRE, TECAN, Männedorf, Switzerland).

Techniques: Inhibition, Positive Control, Expressing, Western Blot

Journal: Oncotarget

Article Title: IRE1α-XBP1 inhibitors exerted anti-tumor activities in Ewing’s sarcoma

doi: 10.18632/oncotarget.24467

Figure Lengend Snippet: To verify the anti-tumor activity of IRE1α-XBP1 inhibitors, in vivo assays were performed using mice and ES cells. (A) Regarding the associations between the inhibitors and tumor size (including volume and weight) in mice, toyocamycin dose-dependently inhibited both the tumor volume and weight compared to control animals (tumor volume: p = 0.078 and tumor weight: p = 0.025). (B) Gross features of tumors. Tumors treated with inhibitors were smaller than control ones. (C) We confirmed the tumor mass in mice by HE staining. These results demonstrated that toyocamycin exerts anti-tumor activity of ES cells in vivo . Microscopic features on HE (left and middle panels) and cleaved caspase-3 staining (right panels) of tumors treated with toyocamycin (lower panels) and control (upper panels). An examination of the HE-stained features at low power under a microscope (left panels) showed that the tumors treated with toyocamycin (left upper panel) were smaller than the control ones (left lower panel). An examination of the HE-stained features at high power indicated that the masses consisted of tumor cells in both toyocamycin-treated (middle lower panel) and control tumors (middle upper panel). The microscopic features on cleaved caspase-3 staining (right panels) indicated that the tumors treated with toyocamycin (right lower panel) had higher numbers of stained cells than control tumors (right upper panel).

Article Snippet: After 72 h, the inhibitory effect of these inhibitors on the growth of ES cell lines was assessed using a Cell Counting Kit-8 (Dojindo JAPAN, Tokyo Japan) and a Microplate Reader (SAFIRE, TECAN, Männedorf, Switzerland).

Techniques: Activity Assay, In Vivo, Control, Staining, Microscopy