Journal: Human Mutation

Article Title: Temperature as a Key Modulator: Investigating Phosphorylation Patterns of p.Asn666 PDGFRB Variants and Their Role in Downstream Signaling

doi: 10.1155/humu/6664372

Figure Lengend Snippet: (a) All PDGFR β substitutions (p.Asn666Lys, p.Asn666Ser, p.Asn666His, and p.Asn666Tyr) increase basal phosphorylation at 37°C. (b) Exposure to 32°C for 6 h further increases phosphorylation in the p.Asn666Tyr, p.Asn666Lys, and p.Asn666His substitutions. Total phosphorylated PDGFR β levels were measured by ELISA. Nontransduced immortalized fibroblasts were referred to as “nt,” while fibroblasts transduced with PDGFRB wild-type vector were denoted as “WT PDGFRB .” (a) One-way ANOVA with Dunnett's test compared wild-type PDGFRB and variants at 37°C (⁣ ∗∗∗∗ p < 0.0001). (b) Cells were incubated at 32°C or 37°C, and two-way ANOVA with Šídák's test compared phosphorylation between temperatures for each variant (⁣ ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: In brief, ELISA analysis was conducted using a DuoSet IC PDGFR β kit (# DYC1767-2, R&D Systems, Minneapolis, MN) following the guidelines provided by the manufacturer.

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation, Incubation, Variant Assay

Journal: Human Mutation

Article Title: Temperature as a Key Modulator: Investigating Phosphorylation Patterns of p.Asn666 PDGFRB Variants and Their Role in Downstream Signaling

doi: 10.1155/humu/6664372

Figure Lengend Snippet: Effect of reduced temperature (32°C) and ligand stimulation on PDGFR β -specific tyrosine residue phosphorylation. Representative immunoblots showing phosphorylation of PDGFR β at p-Y-740, p-Y-751, p-Y-771, p-Y-1009, and p-Y-1021. At 37°C, p.Asn666Lys and p.Asn666Ser variants exhibited nearly identical signal intensity, whereas p.Asn666His and p.Asn666Tyr showed a distinct pattern. At 32°C, all variants exhibited a variant-specific increase in phosphorylation. PDGF stimulation (10 ng/mL) altered phosphorylation patterns, increasing the upper PDGFR β band, likely representing the fully mature, membrane-associated receptor. Full immunoblots, also overexposed, are shown in Figures and .

Article Snippet: In brief, ELISA analysis was conducted using a DuoSet IC PDGFR β kit (# DYC1767-2, R&D Systems, Minneapolis, MN) following the guidelines provided by the manufacturer.

Techniques: Residue, Phospho-proteomics, Western Blot, Variant Assay, Membrane

Journal: Human Mutation

Article Title: Temperature as a Key Modulator: Investigating Phosphorylation Patterns of p.Asn666 PDGFRB Variants and Their Role in Downstream Signaling

doi: 10.1155/humu/6664372

Figure Lengend Snippet: Effects of lower physiological temperature (32°C) on PDGFR β downstream signaling. (a) Immunoblots showing temperature-dependent changes in p-AKT-Ser473, p-AKT-Thr308, p-STAT1, p-STAT3-Tyr705, p-STAT6, and p-PLC γ 1, in immortalized fibroblasts expressing the four p.Asn666 variants. GAPDH served as a loading control. Full immunoblots are shown in Figure . (b,c) Quantification of downstream signaling is presented as the ratio of phosphorylated protein to GAPDH. (b) Normalized values for each variant compared to the wild type at 37°C. (c) Rations for each variant at 32°C versus 37°C. Symbols represent fold changes: “-” (≥ 30% decrease), “≈” (−30% to +50%), “(+)” (150–200%), “+” (2- to 4-fold), “++” (4- to 10-fold), “+++” (10- to 50-fold), “++++” (> 50-fold). Bar charts summarizing these data are provided in Figures and .

Article Snippet: In brief, ELISA analysis was conducted using a DuoSet IC PDGFR β kit (# DYC1767-2, R&D Systems, Minneapolis, MN) following the guidelines provided by the manufacturer.

Techniques: Western Blot, Expressing, Control, Variant Assay

Journal: BMC Cancer

Article Title: Therapeutic potential of TAS-115 via c-MET and PDGFRα signal inhibition for synovial sarcoma

doi: 10.1186/s12885-017-3324-3

Figure Lengend Snippet: c-MET and PDGFRα signals are crucial for the proliferation of SS cells. a Phosphorylation status of RTKs in 3 SS cell lines. b Yamato-SS (3 × 10 3 ), SYO-1 (5 × 10 3 ) and HS-SY-II (1 × 10 4 ) cells were transfected with siRNAs against c-MET. Transfected cells were cultured for 96 h and relative cell proliferation rates were measured using a WST-1 assay. Bars represent the SD. * p < 0.05. c Growth of Yamato-SS (3 × 10 3 ), SYO-1 (5 × 10 3 ) and HS-SY-II (1 × 10 4 ) cells transfected with siRNAs against PDGFRα. Relative cell proliferation rates were determined using a WST-1 assay after 96 h. Bars represent the SD. * p < 0.05

Article Snippet: An antibody against p-PDGFRα (Tyr 762 ; AF21141) was purchased from R&D systems (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Transfection, Cell Culture, WST-1 Assay

Journal: BMC Cancer

Article Title: Therapeutic potential of TAS-115 via c-MET and PDGFRα signal inhibition for synovial sarcoma

doi: 10.1186/s12885-017-3324-3

Figure Lengend Snippet: TAS-115 suppresses phosphorylation of c-MET and PDGFRα, as well as their downstream effectors. a Yamato-SS cells (c-MET-dependent SS cells) were treated with 0.001–10 μM of TAS-115 or control (0.1% DMSO) for 3 h. b SYO-1 and HS-SY-II cells (PDGFRα-dependent SS cells) were treated with 0.001–10 μM of TAS-115 or control (0.1% DMSO) for 3 h

Article Snippet: An antibody against p-PDGFRα (Tyr 762 ; AF21141) was purchased from R&D systems (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Control

Journal: BMC Cancer

Article Title: Therapeutic potential of TAS-115 via c-MET and PDGFRα signal inhibition for synovial sarcoma

doi: 10.1186/s12885-017-3324-3

Figure Lengend Snippet: Inhibitory activities of TAS-115 and pazopanib on c-MET, PDGFRα and their downstream effectors in vitro. a Yamato-SS cells (c-MET-dependent SS cells) were treated with 0.1 μM TAS-115 or pazopanib or control (0.1% DMSO) for 3 h. b Yamato-SS cells were treated with 0.001–20 μM of pazopanib or control (0.1% DMSO) for 3 h. c SYO-1 and HS-SY-II (PDGFRα-dependent) SS cells were treated with 10-μM TAS-115 or pazopanib or control (0.1% DMSO) for 3 h, followed by an additional treatment with rhPDGF-BB at a concentration of 10 ng/ml for the last 15 min

Article Snippet: An antibody against p-PDGFRα (Tyr 762 ; AF21141) was purchased from R&D systems (Minneapolis, MN, USA).

Techniques: In Vitro, Control, Concentration Assay

Journal: BMC Cancer

Article Title: Therapeutic potential of TAS-115 via c-MET and PDGFRα signal inhibition for synovial sarcoma

doi: 10.1186/s12885-017-3324-3

Figure Lengend Snippet: Inhibitory effect of TAS-115 and pazopanib on c-MET, PDGFRα and their downstream effectors in vivo. a , b Mice bearing Yamato-SS (c-MET-dependent) cells and mice bearing SYO-1 (PDGFRα-dependent) SS cells were treated with orally administered TAS-115 (200 mg/kg) or pazopanib (100 mg/kg) or control for consecutive 3 days and euthanized 3 h after the final administration

Article Snippet: An antibody against p-PDGFRα (Tyr 762 ; AF21141) was purchased from R&D systems (Minneapolis, MN, USA).

Techniques: In Vivo, Control

Journal: Cell reports

Article Title: Kaposi’s sarcoma herpesvirus activates the hypoxia response to usurp HIF2α-dependent translation initiation for replication and oncogenesis

doi: 10.1016/j.celrep.2021.110144

Figure Lengend Snippet: (A) Number of infected hMSCs cultured in KS-like media. Infected human MSCs were incubated in an IncuCyte Zoom acquiring green fluorescence images. The number of infected cells was plotted over time, and bar graphs show 24 and 72 hpi (n = 3; mean ± SD; *p < 0.05 and **p < 0.01, unpaired t test). (B) Number of lytically infected hMSCs cultured in KS-like media. The number of RFP expressing hMSCs was acquired as in (A) (n = 3; mean ± SD; *p < 0.05 and **p < 0.01, unpaired t test). (C) Immunoblot of cyclinD1, HIF2α, and PDGFRA wild-type (WT) and phosphorylated form and its cognate ligands (PDGFA and PDGFB) in silenced KSHV-negative and KSHV-positive hMSCs (72 hpi) cultured in KS media. Bar graph represents the fold change in the western blot band intensity of each protein relative to the siControl calculated using ImageJ (n = 3; mean ± SD; *p < 0.05, **p < 0.001, and ***p < 0.0001, two-way ANOVA with Tukey’s post-test). (D) KSHV proteins levels in silenced KSHV-positive hMSCs cultured in KS-like media 72 hpi. (E) VEGF secreted levels in cell-free supernatants of cells from (C). siHIF1β was used as a positive control for VEGF reduction (mean ± SD; *p < 0.0001, two-way ANOVA with Tukey’s post-test). (F) Representative image of immunohistochemical (IHC) staining for HIF2α, KSHV LANA, and K8.1 in a case of K8.1-positive nodular KS. Abundant cytoplasmic expression of HIF2α (brown) was seen, as well as many cells with positive nuclei, in areas with numerous LANA + cells (brown). Only rare K8.1 positive cells (red) were seen. Magnification, 10× (top) and 60× (bottom). Scale bar, 50 μm. (G) Representative HIF2α and KSHV LANA IHC of a K8.1-negative case of nodular KS. Abundant cytoplasmic expression of HIF2α was seen, as well as many cells with positive nuclei, in areas with numerous LANA + cells, but no K8.1 expression was seen. Magnification, 10× (top) and 60× (bottom). Scale bar, 50 μm. (H) Representative image of IHC staining for HIF2α, KSHV LANA, and K8.1 in a case of K8.1-positive plaque-stage KS. Cytoplasmic and nuclear expression of HIF2α was seen in areas with numerous LANA + cells. K8.1-positive cells (red) were seen in these areas, indicative of lytic replication. Magnification, 10× (top) and 60× (bottom). Scale bar, 50 μm. (I) Table showing ten selected cases for HIF2α IHC, including seven cases with some K8.1-positive cells and three cases that were K8.1 negative, consistent with a tighter viral latency in these lesions. All cases had significant HIF2α in the nuclei as well as cytoplasm of many cells independently of histopathological stage or evidence of lytic replication in the lesions.

Article Snippet: Polyclonal antibodies were used for KSHV gB (ThermoFisher Scientific), KSHV RTA (ABBIOTEC), human PDGFRA (R&D Systems), human phospho-PDGFRA (R&D Systems), eIF4E2 (GeneTex), eIF4E1 (Santa Cruz Biotechnology), eIF4G1 (Novus Biologicals) and eIF4G3 (GeneTex).

Techniques: Infection, Cell Culture, Incubation, Fluorescence, Expressing, Western Blot, Positive Control, Immunohistochemical staining, Immunohistochemistry

Journal: Cell reports

Article Title: Kaposi’s sarcoma herpesvirus activates the hypoxia response to usurp HIF2α-dependent translation initiation for replication and oncogenesis

doi: 10.1016/j.celrep.2021.110144

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Polyclonal antibodies were used for KSHV gB (ThermoFisher Scientific), KSHV RTA (ABBIOTEC), human PDGFRA (R&D Systems), human phospho-PDGFRA (R&D Systems), eIF4E2 (GeneTex), eIF4E1 (Santa Cruz Biotechnology), eIF4G1 (Novus Biologicals) and eIF4G3 (GeneTex).

Techniques: Immunohistochemistry, Virus, Recombinant, Lysis, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Amplification, Reverse Transcription, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Control, Software, Real-time Polymerase Chain Reaction, Western Blot

Journal: Pharmaceutical Biology

Article Title: Effects and mechanisms of pirfenidone, prednisone and acetylcysteine on pulmonary fibrosis in rat idiopathic pulmonary fibrosis models

doi: 10.1080/13880209.2016.1247879

Figure Lengend Snippet: The expression of cytokines in rat abdominal aorta.

Article Snippet: TGF-β1, TNF-α and PDGF in the serum of rat abdominal aorta were determined using TGF-β1 ELISA kit (catalog number MB100B, R&D Systems, Minneapolis, MN), TNF-α ELISA kit (catalog number DY2279, R&D Systems, USA) and PDGF ELISA kit (catalog number DYC1767-1, R&D Systems, USA), according to the manufacturer’s protocols.

Techniques: Expressing

Journal: Pharmaceutical Biology

Article Title: Effects and mechanisms of pirfenidone, prednisone and acetylcysteine on pulmonary fibrosis in rat idiopathic pulmonary fibrosis models

doi: 10.1080/13880209.2016.1247879

Figure Lengend Snippet: Correlations of caveolin-1 (Cav-1) expression with cytokines in rat. (a) Correlation of Cav-1 with transforming growth factor-β1 (TGF-β1) expression. (b) Correlation of Cav-1 with tumor necrosis factor-α (TNF-α) expression. (c) Correlation of Cav-1 with platelet derived growth factor (PDGF) expression.

Article Snippet: TGF-β1, TNF-α and PDGF in the serum of rat abdominal aorta were determined using TGF-β1 ELISA kit (catalog number MB100B, R&D Systems, Minneapolis, MN), TNF-α ELISA kit (catalog number DY2279, R&D Systems, USA) and PDGF ELISA kit (catalog number DYC1767-1, R&D Systems, USA), according to the manufacturer’s protocols.

Techniques: Expressing, Derivative Assay