Pten inhibitor bpv pic

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.

Antibodies against β‐catenin, podocalyxin (PCX) and AX2 as well as the Cdc42/RhoA/Rac1 activation assay and PTEN inhibitor BpV(pic) were purchased from Abcam (Cambridge, UK). The antibody against gp200 and the Pierce™ Co‐Immunoprecipitation Kit was obtained from Thermo Fisher Scientific (Waltham, US). The antibodies against PTEN and Alexa Fluor® 488 Phalloidin as well as cell lysis buffer were obtained from Cell Signalling Technology (Beverly, MA). Dulbecco's modified Eagle's medium (DMEM, high glucose) and Foetal bovine serum (FBS) were purchased from Gibco (Waltham, MA, US). NuPAGE Novex 4%–12% Bis–Tris Gels and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA). Transwell chambers were purchased from Corning, Inc (New York, US). The Cdc42 inhibitor, Casin and the β‐actin antibody were purchased from Sigma‐Aldrich (San Francisco, US).

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.