Journal: bioRxiv

Article Title: PDGFRα/β heterodimer activation negatively affects downstream ERK1/2 signaling and cellular proliferation

doi: 10.1101/2023.12.27.573428

Figure Lengend Snippet: ( A,B ) Venus expression (white or green) as assessed by fluorescence analysis of HCC15 cells transduced with PDGFRα-V1 and PDGFRβ-V2 in the absence (A) or presence (B) of PDGF-BB ligand for 5 min. Nuclei were stained with DAPI (blue; A,B). Scale bars: 20 µm. ( C ) Scatter dot plot depicting fluorescence intensity for the PDGFRα/β heterodimer cell line in the absence or presence of PDGF-BB ligand for 5 min. Data are mean±s.e.m. * P <0.05 (two-tailed, paired t -test). Colored circles correspond to independent experiments. Summary statistics from biological replicates consisting of independent experiments (large circles) are superimposed on top of data from all cells; n ≥38 technical replicates across each of three biological replicates. ( D ) Scatter dot plots depicting Pearson’s correlation coefficient of the PDGFRα/β heterodimer cell line with an anti-PDGFRα antibody (left) and an anti-PDGFRβ antibody (right) following PDGF-BB ligand stimulation for 5 min. Data are mean±s.e.m. Colored circles correspond to independent experiments. Summary statistics from biological replicates consisting of independent experiments (large circles) are superimposed on top of data from all cells; n =25 technical replicates across each of three biological replicates. ( E,F ) PDGFRα (E) and PDGFRβ (F) antibody signal (white or magenta; E,F) and/or Venus expression (white or green; E,F) as assessed by (immuno)fluorescence analysis of the PDGFRα/β heterodimer cell line. Insets in E and F are regions where white arrows are pointing. Nuclei were stained with DAPI (blue; E,F). White arrows denote colocalization; white outlined arrows denote lack of colocalization. Scale bars: 20 µm (main images), 3 µm (insets).

Article Snippet: The following antibodies were used for western blotting: PDGFRα (1:1000; D13C6; 5241; Cell Signaling Technology, Inc., Danvers, MA, USA); PDGFRβ (1:1000; 28E1; 3169; Cell Signaling Technology, Inc.); β-tubulin (1:1000; E7; E7; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); Lamin B1 (1:1000; D4Q4Z, 12586; Cell Signaling Technology Inc.); phospho-PDGFRα (Tyr 849)/PDGFRβ (Tyr857) (1:1000; C43E9; 3170; Cell Signaling Technology, Inc.); phospho-ERK1/2 (Thr202/Tyr204) (1:1000; 9101; Cell Signaling Technology, Inc.); ERK1/2 (1:1000; 9102; Cell Signaling Technology, Inc.); phospho-AKT (Ser473) (1:1000; 9271; Cell Signaling Technology Inc.); AKT (1:1000; 9272; Cell Signaling Technology Inc.); Myosin ID (1:500; H-1; sc-515292; Santa Cruz Biotechnology, Inc); GAPDH (1:50000; 1E6D9; 60004-1-Ig; Proteintech Group Inc, Rosemont, IL, USA); horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20,000; 111035003; Jackson ImmunoResearch Inc., West Grove, PA, USA); and horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000; 115035003; Jackson ImmunoResearch Inc.).

Techniques: Expressing, Fluorescence, Transduction, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: PDGFRα/β heterodimer activation negatively affects downstream ERK1/2 signaling and cellular proliferation

doi: 10.1101/2023.12.27.573428

Figure Lengend Snippet: ( A ) Immunoprecipitation (IP) of dimerized PDGFRα/β receptors with GFP-Trap nanobody from cells that were unstimulated or treated with PDGF-BB ligand for 2–5 min followed by western blotting (WB) with an anti-PDGFRα (left) or an anti-PDGFRβ antibody (right). WCL, whole-cell lysates. ( B ) Line graph depicting quantification of band intensities from n =3 biological replicates as in A. Data are mean±s.e.m. * P <0.05 (two-tailed, ratio paired t -test). ( C ) Immunoprecipitation of dimerized PDGFRα/β receptors with GFP-Trap nanobody from cells that were unstimulated or treated with PDGF-BB ligand for 2 min to 4 h followed by western blotting with an anti-phospho (p)-PDGFR antibody. ( D ) Line graph depicting quantification of band intensities from n =3 biological replicates as in C. Data are mean±s.e.m. * P <0.05; (two-tailed, ratio paired t -test).

Article Snippet: The following antibodies were used for western blotting: PDGFRα (1:1000; D13C6; 5241; Cell Signaling Technology, Inc., Danvers, MA, USA); PDGFRβ (1:1000; 28E1; 3169; Cell Signaling Technology, Inc.); β-tubulin (1:1000; E7; E7; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); Lamin B1 (1:1000; D4Q4Z, 12586; Cell Signaling Technology Inc.); phospho-PDGFRα (Tyr 849)/PDGFRβ (Tyr857) (1:1000; C43E9; 3170; Cell Signaling Technology, Inc.); phospho-ERK1/2 (Thr202/Tyr204) (1:1000; 9101; Cell Signaling Technology, Inc.); ERK1/2 (1:1000; 9102; Cell Signaling Technology, Inc.); phospho-AKT (Ser473) (1:1000; 9271; Cell Signaling Technology Inc.); AKT (1:1000; 9272; Cell Signaling Technology Inc.); Myosin ID (1:500; H-1; sc-515292; Santa Cruz Biotechnology, Inc); GAPDH (1:50000; 1E6D9; 60004-1-Ig; Proteintech Group Inc, Rosemont, IL, USA); horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20,000; 111035003; Jackson ImmunoResearch Inc., West Grove, PA, USA); and horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000; 115035003; Jackson ImmunoResearch Inc.).

Techniques: Immunoprecipitation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: PDGFRα/β heterodimer activation negatively affects downstream ERK1/2 signaling and cellular proliferation

doi: 10.1101/2023.12.27.573428

Figure Lengend Snippet: ( A,C ) Western blot (WB) analysis of whole-cell lysates (WCL) from the PDGFRα/β heterodimer cell line following a time course of PDGF ligand stimulation from 2 min to 4 h with anti-phospho (p)-ERK1/2 (A) or anti-phospho (p)-AKT (C) antibodies. ( B,D ) Line graphs depicting quantification of band intensities from n =3 biological replicates as in A and C. Data are mean±s.e.m. ** P <0.01 (two-tailed, ratio paired t -test). ( E,F ) Colony growth in soft agar anchorage-independent growth assays for the HCC15 cell line (E) or the PDGFRα/β heterodimer cell line (F) after 10 days in RPMI growth medium in the absence or presence of PDGF-BB ligand in six-well plate wells. ( G,H ) Scatter dot plots depicting quantification of colony count (G) or colony area (H) from n =3 biological replicates as in E and F. Data are mean±s.e.m. * P <0.05; ** P <0.01 (two-tailed, paired t- test within each cell line and a two-tailed, unpaired t -test with Welch’s correction between each cell line). Colored symbols correspond to independent experiments.

Article Snippet: The following antibodies were used for western blotting: PDGFRα (1:1000; D13C6; 5241; Cell Signaling Technology, Inc., Danvers, MA, USA); PDGFRβ (1:1000; 28E1; 3169; Cell Signaling Technology, Inc.); β-tubulin (1:1000; E7; E7; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); Lamin B1 (1:1000; D4Q4Z, 12586; Cell Signaling Technology Inc.); phospho-PDGFRα (Tyr 849)/PDGFRβ (Tyr857) (1:1000; C43E9; 3170; Cell Signaling Technology, Inc.); phospho-ERK1/2 (Thr202/Tyr204) (1:1000; 9101; Cell Signaling Technology, Inc.); ERK1/2 (1:1000; 9102; Cell Signaling Technology, Inc.); phospho-AKT (Ser473) (1:1000; 9271; Cell Signaling Technology Inc.); AKT (1:1000; 9272; Cell Signaling Technology Inc.); Myosin ID (1:500; H-1; sc-515292; Santa Cruz Biotechnology, Inc); GAPDH (1:50000; 1E6D9; 60004-1-Ig; Proteintech Group Inc, Rosemont, IL, USA); horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20,000; 111035003; Jackson ImmunoResearch Inc., West Grove, PA, USA); and horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000; 115035003; Jackson ImmunoResearch Inc.).

Techniques: Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: PDGFRα/β heterodimer activation negatively affects downstream ERK1/2 signaling and cellular proliferation

doi: 10.1101/2023.12.27.573428

Figure Lengend Snippet: ( A ) Schematic depicting experimental workflow for identification of PDGFR dimer-specific interacting proteins. ( B ) Filters applied to the mass spectrometry data set and Venn diagram displaying shared protein identifications between samples within the filtered data. ( C ) Heat map of heavy (H)/light (L) SILAC ratios for identified proteins involved in trafficking. ( D ) Immunoprecipitation (IP) of myristoylated Venus and dimerized PDGFR receptors with GFP-Trap nanobody from cells that were unstimulated or treated with PDGF ligand for 5 min followed by western blotting (WB) with an anti-MYO1D antibody. WCL, whole-cell lysates. ( E ) MYO1D antibody signal (white or magenta) and/or Venus expression (white or green) as assessed by (immuno)fluorescence analysis of the PDGFRα/β heterodimer cell line following PDGF-BB ligand stimulation for 5 min. Inset in E is a region where white arrows are pointing. Nuclei were stained with DAPI (blue). White arrows denote colocalization; white outlined arrows denote lack of colocalization. Scale bars: 20 µm (main images), 3 µm (insets). ( F ) Scatter dot plot depicting Pearson’s correlation coefficient of the PDGFRα homodimer, PDGFRα/β heterodimer or PDGFRβ homodimer cell line Venus signal with an anti-MYO1D antibody signal following PDGF ligand stimulation for 5 min as in E. Data are mean±s.e.m. *** P <0.001 (two-tailed, unpaired t -test with Welch’s correction). Colored circles correspond to independent experiments. Summary statistics from biological replicates consisting of independent experiments (large circles) are superimposed on top of data from all cells; n =10 technical replicates across each of three biological replicates.

Article Snippet: The following antibodies were used for western blotting: PDGFRα (1:1000; D13C6; 5241; Cell Signaling Technology, Inc., Danvers, MA, USA); PDGFRβ (1:1000; 28E1; 3169; Cell Signaling Technology, Inc.); β-tubulin (1:1000; E7; E7; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); Lamin B1 (1:1000; D4Q4Z, 12586; Cell Signaling Technology Inc.); phospho-PDGFRα (Tyr 849)/PDGFRβ (Tyr857) (1:1000; C43E9; 3170; Cell Signaling Technology, Inc.); phospho-ERK1/2 (Thr202/Tyr204) (1:1000; 9101; Cell Signaling Technology, Inc.); ERK1/2 (1:1000; 9102; Cell Signaling Technology, Inc.); phospho-AKT (Ser473) (1:1000; 9271; Cell Signaling Technology Inc.); AKT (1:1000; 9272; Cell Signaling Technology Inc.); Myosin ID (1:500; H-1; sc-515292; Santa Cruz Biotechnology, Inc); GAPDH (1:50000; 1E6D9; 60004-1-Ig; Proteintech Group Inc, Rosemont, IL, USA); horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20,000; 111035003; Jackson ImmunoResearch Inc., West Grove, PA, USA); and horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000; 115035003; Jackson ImmunoResearch Inc.).

Techniques: Mass Spectrometry, Multiplex sample analysis, Immunoprecipitation, Western Blot, Expressing, Fluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: PDGFRα/β heterodimer activation negatively affects downstream ERK1/2 signaling and cellular proliferation

doi: 10.1101/2023.12.27.573428

Figure Lengend Snippet: ( A,B,D,E ) Western blot (WB) analysis of whole-cell lysates (WCL) from the PDGFRα/β heterodimer cell line treated with Silencer Select negative control (A,D) or Silencer Select siRNA against MYO1D (B,E) following a time course of PDGF-BB ligand stimulation from 2 min to 4 h with anti-phospho (p)-ERK1/2 (A,B) or anti-phospho (p)-AKT (D,E) antibodies. ( C,F ) Line graphs depicting quantification of band intensities from n =3 biological replicates as in A, B, D and E. Data are mean±s.e.m. * P <0.05 (two-tailed, ratio paired t -test). ( G,H ) Colony growth in soft agar anchorage-independent growth assays for the PDGFRα/β heterodimer cell line treated with Silencer Select negative control (G) or Silencer Select siRNA against MYO1D (H) after 10 days in RPMI growth medium in the absence or presence of PDGF-BB ligand in six-well plate wells. ( I,J ) Scatter dot plots depicting quantification of colony count (I) or colony area (J) from n =3 biological replicates as in G and H. Data are mean±s.e.m. * P <0.05; *** P <0.001 (two-tailed, paired t- test within each siRNA treatment and a two-tailed, unpaired t -test with Welch’s correction between each siRNA treatment). Colored symbols correspond to independent experiments.

Article Snippet: The following antibodies were used for western blotting: PDGFRα (1:1000; D13C6; 5241; Cell Signaling Technology, Inc., Danvers, MA, USA); PDGFRβ (1:1000; 28E1; 3169; Cell Signaling Technology, Inc.); β-tubulin (1:1000; E7; E7; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); Lamin B1 (1:1000; D4Q4Z, 12586; Cell Signaling Technology Inc.); phospho-PDGFRα (Tyr 849)/PDGFRβ (Tyr857) (1:1000; C43E9; 3170; Cell Signaling Technology, Inc.); phospho-ERK1/2 (Thr202/Tyr204) (1:1000; 9101; Cell Signaling Technology, Inc.); ERK1/2 (1:1000; 9102; Cell Signaling Technology, Inc.); phospho-AKT (Ser473) (1:1000; 9271; Cell Signaling Technology Inc.); AKT (1:1000; 9272; Cell Signaling Technology Inc.); Myosin ID (1:500; H-1; sc-515292; Santa Cruz Biotechnology, Inc); GAPDH (1:50000; 1E6D9; 60004-1-Ig; Proteintech Group Inc, Rosemont, IL, USA); horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20,000; 111035003; Jackson ImmunoResearch Inc., West Grove, PA, USA); and horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000; 115035003; Jackson ImmunoResearch Inc.).

Techniques: Western Blot, Negative Control, Two Tailed Test