Reagents

An antibody against PDGFRα (AF 1602) was purchased from R&D Systems (Minneapolis, MN, USA). The antibodies recognizing phospho-PDGFRα (pY849, #3170), phospho-PLCγ1 (pTyr783, #2821), PLCγ1 (#2822), phospho-STAT3 (pTyr705, D3A7, #9145), STAT3 (79D7, #4904), phospho-Akt (pSer473, D9E, #4060), Akt (#9272S), phospho-ERK1/2 (pThr202/pThr204, #9101) and ERK1/2 (137F5, #4695) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SUMO1 (sc-5308) and ubiquitin (sc-8017) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The α-tubulin antibody (T6074) was from Sigma-Aldrich (St Louis, MO, USA). Primary antibodies against HA (NB600-363) and the transferrin receptor (TfR) (NB100-92243) were from Novus Biologicals (Centennial, CO, USA). Antisera recognizing Alix (HP95) [42] and PDGFRβ (CTβ) [43] were prepared in house. The secondary antibodies used for immunoblotting included goat anti-mouse IgG (62-6520), goat anti-rabbit IgG (65-6120) and rabbit anti-goat IgG (81-1620), were from Invitrogen (Waltham, MA, USA). The inhibitors MG132 (C2211) and chloroquine (C6628) were from Sigma-Aldrich (St Louis, MO, USA). The inhibitor ginkgolic acid (345,887) was purchased from Merck KGaA and bortezomib (#2244) was from Cell Signaling Technology (Danvers, MA, USA).

Mutagenesis

QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, 210,518) was used to mutate lysine residue 917 of PDGFRα to an arginine residue. Thermal cycling for PCR was performed using plasmid pcDNA3-PDGFRα as the template and primers containing the mutation were designed by the online tool (https://www.chem.agilent.com/store/primerDesignProgram.jsp). The amplification products were then digested by Dpn I enzyme and transformed into XL10-Gold Ultracompetent Cells provided in the kit following the manual. The plasmids were isolated and sequenced to confirm the presence of mutation. Wild-type (WT) and K917R mutant PDGFRα constructs were further used for transient transfection and to create stable cell lines.

Cell culture and plasmid transfection

African green monkey kidney fibroblast-like cells COS-7 (purchased from ATCC, CRL-1651), human embryonic kidney (HEK) 293 cells (purchased from ATCC, CRL-1573), and human retinal pigment epithelial-1 (RPE-1) cells (kindly provided by Soren Christensen, University of Copenhagen, Denmark; ATCC, CRL-4000) were cultured in Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS) (Biowest). Porcine aortic endothelial (PAE) cells were cultured in Ham’s F-12 Nutrient Mix, GlutaMAX™ Supplement (Thermo Fisher Scientific) containing 10% FBS. WT and K917R mutant PDGFRα stable inducible cell lines were cultured in DMEM media, supplemented with 0.8 µg/ml puromycin and 10% FBS. For starvation, cells were incubated in DMEM medium supplemented with 0.1% FBS for 24 h. Cells were transiently transfected with plasmids using Lipofectamine 3000 reagent (Invitrogen), following the protocol provided by the vendor.

Cell lysis, immunoprecipitation and immunoblotting

After various treatments, cells were washed once with phosphate-buffered saline (PBS), pre-chilled at 4℃ and then lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 10% glycerol, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl), supplemented with 1 mM Pefa Block, 1 mM sodium orthovanadate (Na3VO4) and 20 mM N-ethylmaleimide (NEM), on ice for 5 min. The lysates were centrifuged at 13,000 rpm for 15 min at 4℃ and the supernatants were collected. To prepare cell lysates for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), 20 µl of 6× SDS sample buffer (500 mM Tris-HCl, pH 6.8, 30% glycerol, 10% SDS, 600 mM dithiothreitol, 0.01% bromophenol blue) was added to 100 µl of the supernatants. For immunoprecipitation, antibodies were added to the supernatants at a final concentration of 0.2 µg/ml and samples were incubated overnight at 4℃ with end-over-end rotation; Dynabeads Protein A/Protein G (Invitrogen) was then added and incubation was prolonged for one more hour. The beads were washed with lysis buffer three times and heated in SDS sample buffer at 95℃ for 3 min, followed by SDS-PAGE and immunoblotting. After electrophoresis, proteins were electro-transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P). The membranes were blocked in PBS containing 0.1% Tween 20 (PBST) and 5% bovine serum albumin (BSA) for 1 h, incubated with primary antibodies diluted in PBST containing 1% BSA overnight at 4℃, washed three times in PBST for 5 min each time, incubated in peroxidase-conjugated secondary antibodies for 1 h, and then washed three times in PBST. Proteins were visualized by SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and a charge-coupled device (CCD) camera (Bio-Rad). Image Lab (Bio-Rad) was used to quantify the bands.

Biotinylation of cell surface PDGFRα

After starvation and stimulation, cells were washed once with pre-chilled PBS, incubated in 0.3 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Thermo Scientific) in PBS for 1 h on ice, blocked with 50 mM Tris-HCl, pH 7.4 for 5 min, washed once with PBS, and lysed in RIPA buffer for 5 min on ice. After centrifugation at 13,000 rpm for 15 min at 4℃, prewashed Pierce Streptavidin Agarose (Thermo Scientific) was added to the supernatant and incubated end-over-end for 1 h at 4℃, followed by three times washes in lysis buffer and heating at 95oC in SDS sample buffer.

Establishment of tet-inducible cell lines

Lenti-X Tet-One Inducible Expression System (Takara Bio USA) was used to establish tet-inducible cell lines. In-Fusion HD Cloning Kit (Takara Bio USA) was used to clone WT or K917R mutant PDGFRα into the pLVX-TetOne vectors and lentiviral particles were produced using Lenti-X Packaging Single Shots (VSV-G), containing transfection reagent and lentiviral packaging plasmids. 293T cells were transfected with indicated plasmids and lentiviral particles were harvested 24- and 48-hours post-transfection. The collected medium was diluted twice with F-12 medium containing 10% FBS and added to a 6-well plate with PAE cells supplemented with 8 µg/ml polybrene. After 16 h, the medium was replaced with DMEM medium supplemented with 10% FBS and 0.8 µg/ml puromycin. The surviving PAE cells were cultured in medium with puromycin and induced with 100 ng/ml doxycycline to express WT or K917R mutant PDGFRα.

Cell proliferation assay

Single clones selected from tet-inducible PAE cells were seeded into 96 well plates at a density of 500 cells per well and cultured for 24 h. The media was replaced with 100 µl of F-12 media supplemented with 1% FBS and 1–20 ng/ml PDGF-AA or PDGF-BB in the absence or presence of 100 ng/ml doxycycline, while 10% FBS was used as a positive control. After incubation for 3 days, 10 µl of WST-1 reagent (Roche, 11,644,807,001) was added into the well, incubated at 37℃ in 5% CO2 for 4 h. The absorbance was then measured using an ELISA reader at 440 nm, while 650 nm was used as the reference wavelength.

Statistical analysis

Data were analyzed with Microsoft Excel or GraphPad Prism. The values are shown as mean ± standard deviation for experiments with more than three repeats. The statistical significance was determined by unpaired, two-tail student t-test. *p < 0.05.