Casin

PMVECs were isolated from normal SD rat lungs as previously described.20 Experimental data were obtained from cells between passages two to six. PMVECs were divided into two groups as follows: the sham group consisted of PMVECs that were cultured in DMEM supplemented with normal rat serum (5%); and the CBDL group consisted of PMVECs that were incubated in DMEM containing 5% CBDL rat serum (1, 2, 3 and 4 weeks) for 24 hours.
For drug treatment groups, PMVECs were added with 0.05 μM PTEN inhibitor BpV(pic) (Abcam，Cambridge, UK) or 5 μM Casin (Sigma‐Aldrich) for 24 hours in different groups. PMVECs were transfected at 70% confluence for 36 hours with 50 nM short interfering (si)RNAs targeting rat AX2 or a negative control siRNA (NC siRNA) using HiPerfect reagent (Qiagen). Briefly, the siRNA was complexed with 20 μL of transfection reagent and diluted with M199 to 120 μL. Complete fresh medium was added at 3 hours after transfection, and the cells were further incubated for 24‐36 hours before administration of subsequent treatments.

Cell migration was examined using a Transwell chamber assay with 24‐well chambers (pore size 8 μm; Corning). For drug treatment groups, PMVECs were added with 5 μM Casin (Sigma‐Aldrich) for 24 hours in different groups. Approximately 500 μL of DMEM medium containing 5% serum was added to the lower wells, and PMVECs were seeded at 10 × 10⁴ cells onto the upper wells containing 300 μL of DMEM medium without serum. The chambers were subsequently incubated for 12 hours and 24 hours at 37°C. The cells were fixed in 4% polyoxymethylene solution for 5 minutes and stained with 1% crystal violet. The migrated cells were manually calculated in three random microscopic fields using a fluorescence microscope.

Murine macrophage RAW264.7 was brought from FuHeng BioLogy (Shanghai, China). RAW264.7 cells were cultured in Dulbecco’s modified Eagle medium (Basal Media, Shanghai, China) with 10% fetal bovine serum (Sigma, St. Louis, MO, USA) at 37 °C with 95% air, 5% CO₂. To build cell model of inflammation, cells were exposed to 100 ng/mL LPS (Sigma, St. Louis, MO, USA) for 24 h. Cells were pre-exposed to 1 μM CASIN (Sigma, St. Louis, MO, USA) for 6 h to selectively inhibit CDC42 activity, and pre-treated with 1 μM and 5 μM selective JNK inhibitor SP600125 (Sigma, St. Louis, MO, USA) for 6 h to selectively inhibit JNK pathway; DMSO (dimethyl sulfoxide) was treated as controls.

The preparation of GST fusion proteins and the pull down assay for active Rac1, Cdc42 and RhoA was performed according to a previously reported procedure.23 Briefly, PMVECs were grown in six wells culture‐plates for 2 days and stimulated with 5% normal or CBDL rat serum (4 weeks). For treatment groups, PMVECs were added with 0.05 μM PTEN inhibitor BpV(pic) (Abcam) or 5 μM Casin (Sigma‐Aldrich) for 24 hours in different groups. After treatment, the cells were washed with ice‐cold PBS and scraped into 0.3 mL of lysis buffer. The lysates were cleared by centrifugation. A sample from the supernatant was set aside for determination of total level of GTPases, and equal volumes of lysates were then incubated with Rhotekin‐RBD –agarose (for RhoA‐GTP) or PAK‐1 PBD–agarose (for Cdc42 or Rac1‐GTP) for 1 hour at 4°C followed by three washes with lysis buffer. The beads were boiled in sodium dodecyl sulphate (SDS) sample buffer. Total amounts of Rho proteins from cell lysates were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and measured by Western blotting using specific antibodies. The amount of the GTP‐bound form was normalized to the total amount of Rho GTPase in cell lysates. All experiments were performed three times independently.

Cell proliferation was measured and quantified by Ki‐67 staining following each treatment. PMVECs were added with 5 μM Casin (Sigma‐Aldrich) for 24 hours in different groups.Six‐μm‐thick paraffin sections of fixed PMVECs were blocked by 10% bovine serum albumin for 2 hours. Next, the sections were incubated overnight at 4°C with primary antibody, Ki‐67 (1:1000，abcam, Cambridge, MA, ab15580).For immunofluorescence staining of Ki‐67, fluorescence‐tagged secondary antibody and 4′, 6‐diamidino‐2‐phenylindole were used. The section was investigated using a confocal microscope. Each assay was performed three times in triplicate.

HeLa229 cells were prepared in 24-well plates with 12 mm coverslips, and invasion assays were performed essentially as described (9 (link)). Density gradient purified EBs were used at a multiplicity of infection (MOI) of 20. Where appropriate, host cells were treated with medium containing 100 µM EIPA, 20 µM casin, 25 µM wiskostatin, 25 µM Ehop-016, or 200 µM CK666 (all purchased from Sigma-Aldrich) for 15 min prior to infection. Cultures were either mock treated or maintained with inhibitors during infection and subsequent incubation. Infections were done on ice with rocking for 1 h and then shifted to 37°C for 45 min. The cultures were fixed with 4% paraformaldehyde, and extracellular or intracellular EBs were differentially labeled with murine LPS-specific or rabbit MOMP antibodies, respectively. Detection was accomplished with secondary antibody conjugated to Alexa-594 (anti-mouse) or Alexa-488 (anti-rabbit). Percentages of invaded chlamydiae were computed by enumeration of internal and external chlamydiae in 10 fields of view. The percentage EB internalization was calculated via the formula ([total EBs – external EBs]/total red EBs) × 100 = percent (%) invasion.

In vitro migration in response to SDF-1α was analyzed as previously described^{47 (link)}. Lin⁻ cells (1 × 10⁵) from FL of E14.5 Hem-1^+/+ and Hem-1^−/− embryos were pre-incubated with vehicle or 10 µM CASIN (Sigma) for 2 h to inhibit the activity of CDC42. They were plated in the upper well of a 24-well Transwell chamber separated with a filter containing 5.0 μm pore size (Corning, Corning, NY, USA) in IMDM medium with 2% bovine serum albumin (BSA). After a 4-h incubation against an SDF-1 gradient (100 ng/ml) in the lower chamber, all cells that migrated through the filter were collected. These cells were then stained with c-Kit-APC and Sca-1-PE and analyzed using an LSRII flow cytometer (BD Bioscience) for the percentages of LSK cells within PI-negative cells. The numbers of migrating LSK cells were calculated by multiplying the total number of migrating Lin⁻ cells by the percentage of migrating LSK cells.