Journal: Journal of Biological Chemistry

Article Title: Platelet-derived Growth Factor β-Receptor, Transforming Growth Factor β Type I Receptor, and CD44 Protein Modulate Each Other's Signaling and Stability

doi: 10.1074/jbc.m114.547273

Figure Lengend Snippet: FIGURE 1. PDGFR forms a physical complex with TRI and -II. A and B, Cos1 cells were transiently transfected with PDGFR-HA (A and B), TRI-FLAG (A, left panel), TRII-FLAG (A, right panel), a truncated PDGFR (PDGFR-ECTM-HA) expressing only the extracellular and transmembrane parts of the receptor (B), and/or empty vectors. After 48 h, lysates were prepared and subjected to immunoprecipitation (IP) using FLAG or HA antibodies, and proteins were separated by SDS-PAGE. Total cell lysates were run in parallel. Immunoblotting (IB) was performed with FLAG and HA antibodies; -actin was used as a loading control. C, Cos-1 cells were transiently transfected with PDGFR-HA, TRI-FLAG, TRII-His, and/or empty vectors. After 24 h, cells were starved for another 24 h and then stimulatedwithcombinationsofPDGF-BB(7min,10ng/ml),PDGFRinhibitorimatinib(1h,5M),TGF(1h,1ng/ml),TRIinhibitorGW6604(2h,6M),DMSO control (2 h), or starvation medium alone. Cells were lysed, and the lysate was subjected to immunoprecipitation with FLAG antibodies, and proteins were separated by SDS-PAGE. Total cell lysates (TCL) were run in parallel. Proteins were detected by immunoblotting with specific antibodies against the HA and FLAG tags, phosphotyrosine, phospho-Smad2, and -actin. D, human dermal primary fibroblasts were immunoprecipitated using a TRI antibody or IgG isotype control, followed by immunoblotting with specific PDGFR and TRI antibodies. Representative data from at least three independent experiments are shown.

Article Snippet: Cell Culture—Cos1 (monkey kidney fibroblast-like cells; ATCC CRL-1650), primary human dermal fibroblasts from normal breast tissue (biopsies were taken after approval from patients undergoing breast reduction surgery at the Department of Plastic Surgery of University Hospital, Uppsala, Sweden (17)), and BJ-hTERT (telomerase immortalized human foreskin fibroblasts) cells (41) were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), supplemented with 10% fetal bovine serum (FBS; Hyclone) at 37 °C in 5% CO2.

Techniques: Transfection, Expressing, Immunoprecipitation, SDS Page, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Platelet-derived Growth Factor β-Receptor, Transforming Growth Factor β Type I Receptor, and CD44 Protein Modulate Each Other's Signaling and Stability

doi: 10.1074/jbc.m114.547273

Figure Lengend Snippet: FIGURE 2. PDGFR affects the stability and downstream signaling of TRI. A and B, human dermal primary fibroblasts were either transfected with siRNA againstPDGFRorscrambledcontrol(AandB)orleftuntransfected(C),thenstarved,andstimulated(stim)forupto1hwithTGF(1ng/ml)(A–C)andPDGF-BB (10 ng/ml) (C). A and C, cells were placed on ice to stop membrane trafficking; the cell-surface proteins were then labeled using sulfo-NHS-SS-biotin, and biotin that remained unbound was quenched. Cells were lysed, and biotinylated cell-surface proteins were precipitated using streptavidin-coupled magnetic beads. Proteins were separated by SDS-PAGE and immunoblotted for PDGFR and TRI. The amount of PDGFR and TRI left on the cell surface (relative to GAPDH) was quantified (C, lower panel indicates fold change of mean values with mean S.E. of five experiments; asterisk indicates p value 0.05 with ratio paired t test). As control, nonbiotinylated cells were treated the same way. B, cells were lysed and subjected to SDS-PAGE and immunoblotting to determine the phosphorylation status of Smad2. The blots were quantified, and phosphorylated Smad2 relative to GAPDH was determined (B, lower panel). Representative experiments of at least three independent experiments are shown. Asterisk indicates p value 0.05 using Wilcoxon matched pair signed rank test to compare siRNA control with siRNA against PDGFR. TCL, total cell lysates.

Article Snippet: Cell Culture—Cos1 (monkey kidney fibroblast-like cells; ATCC CRL-1650), primary human dermal fibroblasts from normal breast tissue (biopsies were taken after approval from patients undergoing breast reduction surgery at the Department of Plastic Surgery of University Hospital, Uppsala, Sweden (17)), and BJ-hTERT (telomerase immortalized human foreskin fibroblasts) cells (41) were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), supplemented with 10% fetal bovine serum (FBS; Hyclone) at 37 °C in 5% CO2.

Techniques: Transfection, Membrane, Labeling, Magnetic Beads, SDS Page, Control, Western Blot, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Platelet-derived Growth Factor β-Receptor, Transforming Growth Factor β Type I Receptor, and CD44 Protein Modulate Each Other's Signaling and Stability

doi: 10.1074/jbc.m114.547273

Figure Lengend Snippet: FIGURE 3. PDGF-BB induces phosphorylation of Smad2 as well as expression of PAI-1, and the PDGF-BB-induced Smad2 phosphorylation is depen- dent on the kinase activities of PDGFR and TRI, as well as on Src kinase and TGF. A, human dermal primary fibroblasts were starved and stimulated (stim) for 1 h with 1 ng/ml TGF or 2 ng/ml PDGF-BB. Cells were then lysed and subjected to SDS-PAGE and immunoblotting (IB), with P-Smad2 and Smad2 antisera, and the amount of phosphorylated Smad2 relative to total Smad2 was quantified (A, lower panel indicates fold change compared with TGF of mean values,withS.E.,ofsixexperiments;asteriskindicatespvalue0.05withratiopairedttest;threeasterisksdenotep0.01).B,humandermalprimaryfibroblasts were transfected with either siRNA against PDGFR or a scrambled control, starved, and stimulated for 30 min or 1 h with 2 ng/ml PDGF-BB. Cells were then lysed and subjected to SDS-PAGE and immunoblotting, as stated below. C–H, human dermal primary fibroblasts were starved and pretreated for 8 h with 10 M PDGFR kinase inhibitor AG1296 (C), for 2 h with 16 M TRI kinase inhibitor GW6604 (D and H), for 1 h with 20 g/ml TGF blocking antibody (E), for 1 h with1.5MSrcinhibitorSU6656(F),orfor2hwith20Mcycloheximide(G).Cellswerestarvedandstimulatedforindicatedperiodswith2ng/mlPDGF-BB(C–H) and 1 ng/ml TGF (E and H), then lysed and subjected to SDS-PAGE and immunoblotting with antibodies against phospho-Smad2, Smad2, phosphotyrosine, PDGFR (CT), phospho-STAT3, PAI-1, and GAPDH (loading control). Representative experiments from at least three independent experiments are shown. CHX, cycloheximide.

Article Snippet: Cell Culture—Cos1 (monkey kidney fibroblast-like cells; ATCC CRL-1650), primary human dermal fibroblasts from normal breast tissue (biopsies were taken after approval from patients undergoing breast reduction surgery at the Department of Plastic Surgery of University Hospital, Uppsala, Sweden (17)), and BJ-hTERT (telomerase immortalized human foreskin fibroblasts) cells (41) were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), supplemented with 10% fetal bovine serum (FBS; Hyclone) at 37 °C in 5% CO2.

Techniques: Phospho-proteomics, Expressing, SDS Page, Western Blot, Transfection, Control, Blocking Assay

Journal: Journal of Biological Chemistry

Article Title: Platelet-derived Growth Factor β-Receptor, Transforming Growth Factor β Type I Receptor, and CD44 Protein Modulate Each Other's Signaling and Stability

doi: 10.1074/jbc.m114.547273

Figure Lengend Snippet: FIGURE 5. TRI and CD44 form a ligand noninducible complex. A and B, Cos1 cells were transiently transfected with HA-tagged TRI, 6-Myc-tagged CD44, or corresponding empty tagged vectors. After 48 h, lysates were immunoprecipitated (IP) with antibodies against TRI or HA (A), CD44 or Myc (B), correspond- ing IgG isotype controls, or beads alone; proteins were separated by SDS-PAGE. Total cell lysates (TCL) were run in parallel. Immunoblotting (IB) was performed with antibodies against CD44 and TRI. C, BJ-hTERT foreskin fibroblasts were grown in 8-well chambers, starved, and stimulated (stim) for 7 min with PDGF-BB (20 ng/ml), 2 h with hyaluronan (200 g/ml), or 1 h with TGF (1 ng/ml). Cells were fixed, and PLA was performed with mouse anti-CD44 and rabbit anti-TRI antibodies, followed by anti-mouse and anti-rabbit PLA probes conjugated with priming and nonpriming oligonucleotides. F-actin was stained with FITC- conjugated phalloidin. Individual protein-protein interactions were visualized by fluorescence microscope as red dots. PLA signals per cell were quantified using Duolink Image Tool according to the manufacturer’s instructions. Average values are indicated in the graph by horizontal lines. A representative experiment out of three is shown.

Article Snippet: Cell Culture—Cos1 (monkey kidney fibroblast-like cells; ATCC CRL-1650), primary human dermal fibroblasts from normal breast tissue (biopsies were taken after approval from patients undergoing breast reduction surgery at the Department of Plastic Surgery of University Hospital, Uppsala, Sweden (17)), and BJ-hTERT (telomerase immortalized human foreskin fibroblasts) cells (41) were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), supplemented with 10% fetal bovine serum (FBS; Hyclone) at 37 °C in 5% CO2.

Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Staining, Protein-Protein interactions, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Platelet-derived Growth Factor β-Receptor, Transforming Growth Factor β Type I Receptor, and CD44 Protein Modulate Each Other's Signaling and Stability

doi: 10.1074/jbc.m114.547273

Figure Lengend Snippet: FIGURE 6. PDGFR, TRI, and CD44 all bind each other, but the complex between PDGFR and TRI is not dependent on CD44. A, Cos1 cells were transiently transfected with PDGFR-HA, TRI-FLAG, and CD44–6myc or correspondingly tagged empty vectors. After 48 h, cell lysates were immunoprecipi- tated (IP) using FLAG, HA, or Myc antibodies. Proteins were released from beads by boiling in reducing sample buffer and subjected to SDS-PAGE and immunoblotting (IB) with antibodies against CD44, HA, and FLAG or GAPDH as loading control. Total cell lysates (TCL) were analyzed in parallel. B, Cos1 cells were transiently transfected with CD44 siRNA for 24 h and then with TRI-FLAG and PDGFR-HA vectors for another 48 h. Cells were lysed and immunopre- cipitated with FLAG antibodies, separated by SDS-PAGE, and detected by immunoblotting for CD44 and the HA and FLAG tags. C, BJ-hTERT foreskin fibroblasts were grown in 8-well chambers and transiently transfected with siRNA against CD44 or a scrambled control. Following starvation, cells were stimulated for 7 min with PDGF-BB (20 ng/ml), 2 h with hyaluronan (200 g/ml), 1 h with TGF (1 ng/ml), or 1 h with 10% FBS. Cells were fixed and PLA was performed with mouse anti-PDGFR and rabbit anti-TRI antibodies, followed by anti-mouse and anti-rabbit PLA probes conjugated with priming and nonpriming oligonu- cleotides.F-actinwasstainedwithFITC-conjugatedphalloidin.Singleprotein-proteininteractionswerevisualizedbyfluorescencemicroscopeasreddots.PLA signals per cell were quantified with Duolink Image Tool according to the manufacturer’s instructions. Average is indicated in the graph by horizontal lines. D, Cos1 cells were transfected with PDGFR-HA and TRI-FLAG, in combination with varying amounts of CD44-myc (0.1, 0.5, or 2 g/sample) or empty vector. Cells were lysed, and immunoprecipitated with FLAG antibody and subjected to SDS-PAGE. The samples were then immunoblotted with specific antibodies against the FLAG and HA tags and CD44. Total cell lysates were run in parallel. Representative experiments out of three independent experiments are shown.

Article Snippet: Cell Culture—Cos1 (monkey kidney fibroblast-like cells; ATCC CRL-1650), primary human dermal fibroblasts from normal breast tissue (biopsies were taken after approval from patients undergoing breast reduction surgery at the Department of Plastic Surgery of University Hospital, Uppsala, Sweden (17)), and BJ-hTERT (telomerase immortalized human foreskin fibroblasts) cells (41) were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), supplemented with 10% fetal bovine serum (FBS; Hyclone) at 37 °C in 5% CO2.

Techniques: Transfection, SDS Page, Western Blot, Control, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Platelet-derived Growth Factor β-Receptor, Transforming Growth Factor β Type I Receptor, and CD44 Protein Modulate Each Other's Signaling and Stability

doi: 10.1074/jbc.m114.547273

Figure Lengend Snippet: FIGURE 7. Knockdown of CD44 stabilizes the levels of PDGFR and Smad2. Human dermal primary fibroblasts were transfected with siRNA against CD44 or a scrambled control, starved, and stimulated for indicated time periods with either 2 ng/ml PDGF-BB (A), 1 ng/ml TGF (B), or 10 ng/ml PDGF-BB (C). Lysates were subjected to SDS-PAGE and immunoblotting with antibodies against phospho-Smad2, Smad2, CD44, phospho-ERK1/2, ERK1/2, and GAPDH (loading control). Representative experiments of three independent experiments are shown.

Article Snippet: Cell Culture—Cos1 (monkey kidney fibroblast-like cells; ATCC CRL-1650), primary human dermal fibroblasts from normal breast tissue (biopsies were taken after approval from patients undergoing breast reduction surgery at the Department of Plastic Surgery of University Hospital, Uppsala, Sweden (17)), and BJ-hTERT (telomerase immortalized human foreskin fibroblasts) cells (41) were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), supplemented with 10% fetal bovine serum (FBS; Hyclone) at 37 °C in 5% CO2.

Techniques: Knockdown, Transfection, Control, SDS Page, Western Blot

Journal: Genes to Cells

Article Title: Derivation of human differential photoreceptor cells from adult human dermal fibroblasts by defined combinations of CRX, RAX , OTX2 and NEUROD

doi: 10.1111/gtc.12127

Figure Lengend Snippet: Induction of retina-specific genes in human dermal fibroblasts by the retroviral infection of genes for defined transcription factors. (A) RT-PCR analysis for photoreceptor-specific genes in cultured human dermal fibroblasts (NHDF) obtained from Lonza after gene transfer of several kinds of transcription factors. Recoverin, blue opsin and PDE6c genes were up-regulated by CRN transduction. ‘Negative control’: amplified water as a negative control. ‘GFP’: cultured fibroblasts after retroviral gene transfer of the GFP gene as another negative control. ‘w/o’: cultured fibroblasts without gene transfer as the other negative control. ‘SPPO’: SOX2, POU1F1, PAX6 and OTX2 . ‘SPO’: SOX2, POU1F1 and OTX2 . ‘CRN’: CRX, RAX and NEUROD . ‘Human retina’: human retinal tissue as a positive control. The amount of cDNA as a template was a half in the positive control. (B) RT-PCR analysis for photoreceptor-specific genes in cultured human dermal fibroblasts (NHDF) obtained from Promo Cell after gene transfer of several transcription factors. Recoverin, blue opsin and PDE6c genes were up-regulated by CRN or CRNO transduction. ‘w/o’: cultured fibroblasts without gene transfer as a negative control. ‘GFP’: cultured fibroblasts after retroviral gene transfer of the GFP gene as another negative control. ‘CRNO’: CRX, RAX, NEUROD and OTX2 . (C) RT-PCR analysis for photoreceptor-specific genes in cultured human dermal fibroblasts (HDF-a) obtained from ScienCell after gene transfer of several transcription factors. Recoverin, blue opsin and PDE6C genes were up-regulated by CRN or CRNO transduction. Expression levels of blue opsin were increased by additional OTX2 gene transduction. ‘w/o’: cultured fibroblasts without gene transfer as a negative control. ‘GFP’: cultured fibroblasts after retroviral gene transfer of the GFP gene as another negative control. ‘CRNO’: CRX , RAX , NEUROD and OTX2 . ‘1’, ‘2’ and ‘3’ mean independently cultured, transfected and harvested cells by the same combination of CRN genes. (D) Immunocytochemistry using antibodies to rhodopsin and blue opsin (green). Nuclei were stained with DAPI (blue). Experiments were carried out at 2 weeks after infection. The cells in the left panel and the right panel are CRN-infected Fib#2 and Fib#1, respectively. Scale bars represent 10 μm.

Article Snippet: Three strains of cultured human dermal fibroblasts were used: one was obtained from Lonza (NHDF), another was from Promo Cell (NHDF) and the other was from ScienCell (HDF-a).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Transduction, Negative Control, Amplification, Positive Control, Expressing, Transfection, Immunocytochemistry, Staining