Journal: Cell Death & Disease

Article Title: Intrinsic regulation of hemangioma involution by platelet-derived growth factor

doi: 10.1038/cddis.2012.58

Figure Lengend Snippet: Expression of PDGF receptors in proliferating and involuting hemangiomas. ( a ) Real-time RT-PCR analysis of PDGFR- α , - β , and PPAR γ in proliferating and involuting hemangiomas (data normalized to 18S rRNA and presented as relative to normal skin; * P <0.05 compared with normal skin, † P <0.05 compared with proliferating hemangioma; n =3). ( b ) Immunostaining of proliferating hemangiomas for PDGFR- α and PDGFR- β (images taken at × 20, inserts show higher magnification; brown=DAB staining, blue=hematoxylin). ( c ) Immunofluorescence double labeling for mesenchymal cell marker α -SMA and PDGFR- β (images taken at × 20, green=PDGFR- β , red= α -SMA)

Article Snippet: The lysates were also used to measure tyrosine phosphorylated PDGF receptors α (PathScan Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit; Cell Signaling Technology, Pickering, Ontario, Canada) and β (PathScan Phospho-PDGF Receptor β (Tyr751) Sandwich ELISA Kit; Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Immunostaining, Staining, Immunofluorescence, Labeling, Marker

Journal: Cell Death & Disease

Article Title: Intrinsic regulation of hemangioma involution by platelet-derived growth factor

doi: 10.1038/cddis.2012.58

Figure Lengend Snippet: Phosphorylated PDGF receptors in proliferating hemangiomas. Immunostaining of proliferating hemangioma specimens for phospho-PDGFR- β ( a ) and phospho-PDGFR- α ( b ) (images taken at × 20, green=PDGFR, blue=DAPI)

Article Snippet: The lysates were also used to measure tyrosine phosphorylated PDGF receptors α (PathScan Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit; Cell Signaling Technology, Pickering, Ontario, Canada) and β (PathScan Phospho-PDGF Receptor β (Tyr751) Sandwich ELISA Kit; Cell Signaling Technology).

Techniques: Immunostaining

Journal: Cell Death & Disease

Article Title: Intrinsic regulation of hemangioma involution by platelet-derived growth factor

doi: 10.1038/cddis.2012.58

Figure Lengend Snippet: Expression of PDGF signaling axis in hemangioma-derived CD133+ cells. ( a ) Expression of PDGFRs in hemSCs and bm-MPCs (data normalized to 18 S rRNA and presented as relative to bm-MPCs; two different hemSC preparations run in triplicates were averaged). ( b ) Expression of PDGF transcripts in hemSCs (data presented as in ( a ); * P <0.05 compared with bm-MPCs; right panel shows the expression of PDGF-A, PDGF-C, and PDGF-D at a lower scale). ( c ) PDGF-BB levels in media from cells cultured for 48 h in EBM2/20%FBS (with growth factors) (* P <0.05 compared with bm-MPCs). ( d ) PDGF-BB levels in cell lysates determined by ELISA (* P <0.05 compared with bm-MPCs). ( e ) Levels of phosphorylated PDGFR- α and - β in hemSCs with or without exogenous PDGF-BB stimulation (cells were stimulated with 10 ng/ml PDGF-BB; * P <0.05 compared with unstimulated cells). ( f ) Schematic illustration of the hypothesized PDGF polypeptides and the corresponding receptors on hemangioma stem cell surface

Article Snippet: The lysates were also used to measure tyrosine phosphorylated PDGF receptors α (PathScan Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit; Cell Signaling Technology, Pickering, Ontario, Canada) and β (PathScan Phospho-PDGF Receptor β (Tyr751) Sandwich ELISA Kit; Cell Signaling Technology).

Techniques: Expressing, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Intrinsic regulation of hemangioma involution by platelet-derived growth factor

doi: 10.1038/cddis.2012.58

Figure Lengend Snippet: Effect of PDGF-BB on hemSC phenotype and cellular proliferation. ( a ) Fluorescence immunostaining for mesenchymal markers α -SMA, calponin and desmin in hemSCs. Umbilical artery SMCs were used as control for the mural markers. Effect of exogenous PDGF-BB on hemSC migration ( b ) and proliferation ( c ) (data presented as relative to control media treated cells; n =3)

Article Snippet: The lysates were also used to measure tyrosine phosphorylated PDGF receptors α (PathScan Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit; Cell Signaling Technology, Pickering, Ontario, Canada) and β (PathScan Phospho-PDGF Receptor β (Tyr751) Sandwich ELISA Kit; Cell Signaling Technology).

Techniques: Fluorescence, Immunostaining, Control, Migration

Journal: Cell Death & Disease

Article Title: Intrinsic regulation of hemangioma involution by platelet-derived growth factor

doi: 10.1038/cddis.2012.58

Figure Lengend Snippet: PDGF-BB inhibits adipogenic differentiation in hemSCs. ( a ) Representative oil red O staining of hemSCs cultured in adipogenic differentiation media supplemented with 10 ng/ml of PDGF-AA, -AB, or -BB. ( b ) Quantitative analysis of adipogenic differentiation in the presence of PDGF polypeptides was assessed by real-time RT-PCR for adipogenesis-specific transcription factors, C/EBP α and PPAR γ (data normalized to 18 S rRNA and presented as relative to adipogenic media; * P <0.05 compared with the adipogenic media; n =3). ( c ) Effect of adipogenic differentiation on PDGFR expression in hemSCs at day 7. ( d ) Oil red O staining of hemSCs cultured with different growth factors (each at 10 ng/ml) illustrating specific inhibition of hemSC adipogenesis by PDGF signaling

Article Snippet: The lysates were also used to measure tyrosine phosphorylated PDGF receptors α (PathScan Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit; Cell Signaling Technology, Pickering, Ontario, Canada) and β (PathScan Phospho-PDGF Receptor β (Tyr751) Sandwich ELISA Kit; Cell Signaling Technology).

Techniques: Staining, Cell Culture, Quantitative RT-PCR, Expressing, Inhibition

Journal: Cell Death & Disease

Article Title: Intrinsic regulation of hemangioma involution by platelet-derived growth factor

doi: 10.1038/cddis.2012.58

Figure Lengend Snippet: PDGF-BB, but no other PDGF polypeptides, prevent full functional differentiation of hemSCs. ( a ) Oil red O staining of hemSCs cultured in low (10 ng/ml) and high (50 ng/ml) PDGF polypeptide levels. PDGF-AA and –AB were ineffective against preventing adipogenic differentiation at 10 and 50 ng/ml. ( b ) Quantitative analysis of early adipogenesis-specific transcription factors (C/EBP α and PPAR γ ) and late marker of adipocytes, FABP-4 (* P <0.05 compared to adipogenic media; n =3). ( c ) Expression of FABP-4 in hemSCs treated with 50 ng/ml PDGF-AA. ( d ) Inhibition of adipogenesis by PDGF-BB in bm-MPCs (data presented relative to adipogenic media; * P <0.05 compared with adipogenic media; n =3)

Article Snippet: The lysates were also used to measure tyrosine phosphorylated PDGF receptors α (PathScan Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit; Cell Signaling Technology, Pickering, Ontario, Canada) and β (PathScan Phospho-PDGF Receptor β (Tyr751) Sandwich ELISA Kit; Cell Signaling Technology).

Techniques: Functional Assay, Staining, Cell Culture, Marker, Expressing, Inhibition

Journal: Cell Death & Disease

Article Title: Intrinsic regulation of hemangioma involution by platelet-derived growth factor

doi: 10.1038/cddis.2012.58

Figure Lengend Snippet: Inhibiting cell-autogenous PDGF signaling enhances adipogenesis. ( a ) Oil red O staining of hemSCs exposed to adipogenic media with or without PDGFR inhibitors, AG-370 and AG-1296, and PDGFR neutralizing antibodies. ( b ) Quantitative analysis of C/EBP α and PPAR γ in cells exposed to PDGF inhibitors as in ( a ) (data presented relative to control media; * P <0.05 compared with control media; † P <0.05 compared with adipogenic media; n =3). ( c ) Effect of PDGFR- α and PDGFR- β- neutralizing antibodies and chemical inhibitors on reversing the inhibition by exogenous PDGF-BB (* P <0.05 compared with PDGF-BB treatment)

Article Snippet: The lysates were also used to measure tyrosine phosphorylated PDGF receptors α (PathScan Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit; Cell Signaling Technology, Pickering, Ontario, Canada) and β (PathScan Phospho-PDGF Receptor β (Tyr751) Sandwich ELISA Kit; Cell Signaling Technology).

Techniques: Staining, Control, Inhibition

Journal: Cell Death & Disease

Article Title: Intrinsic regulation of hemangioma involution by platelet-derived growth factor

doi: 10.1038/cddis.2012.58

Figure Lengend Snippet: PDGF-BB employs PDGFR- β in hemSCs. shRNA-mediated knockdown of PDGFR- α ( a ) and PDGFR- β ( b ) in hemSCs at sub-passage 1 (subP1; following 2 weeks of puromycin selection) and sub-passage 6 (six serial passages after puromycin selection) (* P <0.05 compared with control shRNA). ( c ) shRNA-knockdown of PDGFR- α and PDGFR- β in hemSCs reduced the inhibitory effect of PDGF-BB on hemSC adipogenesis (* P <0.05 compared with control shRNA). ( d ) Schematic illustrating the findings of the study and our working hypothesis. High levels of PDGF-BB from hemSCs during proliferation mediate intracellular signaling through PDGFR- β (predominantly PDGFR- ββ homodimer with contribution from PDGFR- αβ heterodimer) to inhibit C/EBP α and PPAR γ expression and adipogenesis (i.e., involution). Upon regression of the vessels and removal of the PDGF-BB source, adipogenesis is triggered

Article Snippet: The lysates were also used to measure tyrosine phosphorylated PDGF receptors α (PathScan Phospho-PDGF Receptor α (Tyr849) Sandwich ELISA Kit; Cell Signaling Technology, Pickering, Ontario, Canada) and β (PathScan Phospho-PDGF Receptor β (Tyr751) Sandwich ELISA Kit; Cell Signaling Technology).

Techniques: shRNA, Knockdown, Selection, Control, Expressing