Journal: STAR Protocols

Article Title: Simultaneous isolation of CD45 tumor-infiltrating lymphocytes, tumor cells, and associated fibroblasts from murine breast tumor model by MACS

doi: 10.1016/j.xpro.2022.101951

Figure Lengend Snippet:

Article Snippet: CD140b Antibody, anti-mouse, REAfinity- APC-770 (1:50 dilution) , Miltenyi Biotec , Cat# 130-118-469, RRID: AB_2733095.

Techniques: Recombinant, Flow Cytometry, Staining, Isolation, Cell Isolation, Software, Real-time Polymerase Chain Reaction, Blocking Assay, Gentle

Journal: Communications Biology

Article Title: Activation of TMEM16E scramblase induces ligand independent growth factor receptor signaling and macropinocytosis for membrane repair

doi: 10.1038/s42003-025-07465-6

Figure Lengend Snippet: a Schematic of phospholipid rearrangement by TMEM16E-dependent scramblase and conformational changes of GFRs (EGFR and PDGFR). GFRs consist of an extracellular domain (gray), transmembrane domain (blue), juxtamembrane domain (magenta), and tyrosine kinase domain (purple). Positively charged amino acids of the juxtamembrane domain interact with negatively charged phosphatidylserine (PS) in inactive GFRs. Treatment with A23187 induces TMEM16E-dependent scramblase. The interaction between the juxtamembrane domain and PS is broken, and the juxtamembrane region dimerizes and is converted to an active form by clustering with negatively charged PI(4,5)P 2 . Activation of GFRs induces macropinocytosis. The images and every material element by the authors using Microsoft Office PowerPoint 365 (Microsoft). b Representative images of 1 μM A23187-induced scrambling in cells expressing RF-Dead, RF-Sac1, or RF-PJ with TMEM16E. Cells were treated with 100 nM rapamycin for 10 min in DMEM before imaging. The white dashed square box is magnified in the images in the right panel. Scale bars, 10 μm, and magnified images scale bar, 2 μm. c Analysis of AV fluorescence intensity at 15 min (RF-Dead, n = 11; RF-Sac1, n = 13; RF-PJ, n = 12). ROIs selected plasma membrane (PM), and cytoplasmic regions (Cyto). The ratio of the cytoplasmic region to the plasma membrane (F Cyto /F PM ratio) was calculated. Analysis was performed by one-way ANOVA followed by Dunnett’s post-hoc test (Compared to Dead, PM Sac1, ns = 0.9218; PM PJ, ns = 0.3227; Cyto Sac1, ns = 0.844; Cyto PJ, * P = 0.0345; F Cyto /F PM ratio Sac1, ns = 0.9936; F Cyto /F PM ratio PJ, **** P < 0.0001). Data are mean ± SEM.

Article Snippet: Human cDNAs of EGFR (Plasmid #133749) and PDGFR (Plasmid #136455) were purchased from Addgene, and coding regions were subcloned into a pcDNA 3.1(+) vector.

Techniques: Activation Assay, Expressing, Imaging, Fluorescence, Clinical Proteomics, Membrane

Journal: Communications Biology

Article Title: Activation of TMEM16E scramblase induces ligand independent growth factor receptor signaling and macropinocytosis for membrane repair

doi: 10.1038/s42003-025-07465-6

Figure Lengend Snippet: a Schematic diagram of the action of inhibitors on EGFR and PDGFR. Afatinib inhibits phosphorylation by covalent binding to C797 located in the tyrosine kinase (TK) domain of EGFR. Imatinib binds to the ATP allosteric pocket and blocks the binding of ATP. The images and every material element by the authors using Microsoft Office PowerPoint 365 (Microsoft). b Representative images of TMEM16E-dependent scramblase calculated as AV fluorescence in the plasma membrane (PM) over that in cytoplasm (Cyto) for control, after application of 1 μM afatinib for 1 h, and after 20 μM imatinib for 1 h in DMEM. The yellow dashed line indicates the plasma membrane region and the white dashed square box is magnified in the images in the right panel. Scale bars, 10 μm, and magnified images scale bar, 2 μm. c AV fluorescence intensity 15 min after induction of scramblase in cells treated with GFR inhibitors (Control, n = 16; Afatinib (afat), n = 7; Imatinib (Imat), n = 17). The ratio of the cytoplasmic region (Cyto) to the plasma membrane (PM) was calculated. Analysis was performed by one-way ANOVA followed by Dunnett’s post-hoc test (Compared to Control, PM Afat, ns = 0.9189; PM Imat, ns = 0.5111; Cyto Afat, *** P = 0.0002; Cyto Imat, ** P = 0.0063; F Cyto /F PM ratio Afat, **** P < 0.0001; F Cyto /F PM ratio Imat, * P = 0.0392). d Protein gels of co-immunoprecipitates performed with anti-HA or IgG in cells expressing TMEM16E-GFP, PDGFR-Hax3, and EGFR-Hax3. The input represents 5% of the lysate used for immunoprecipitation (IP) ( n = 3). The result of co-immunoprecipitation with anti-HA, TMEM16E- GFP was detected with PDGFR-HAx3 and EGFR-HAx3. The summary histogram indicates an interaction of PDGFRxHA3 and EGFR-HAx3 with TMEM16E-GFP. Analysis was performed by one-way ANOVA followed by Dunnett’s post-hoc test (Compared to only cells expressing TMEM16E, with PDGFR, ** P = 0.0026; with EGFR, ** P = 0.0021). e RT-PCR determined the endogenous expression levels of EGFR and PDGFR in HEK293T cells ( n = 6). *** P < 0.001, compared with control, analyzed by Student’s t-test. f Protein gels of co-immunoprecipitates performed with anti-HA or IgG in cells expressing EGFR-HAx3, TMEM16E-GFP, E584Q-GFP, and TMEM16F-GFP. g Analysis of co-immunoprecipitation with anti-HA and TMEM16E-GFP, E584Q-GFP, and TMEM16F-GFP. The summary shows that EGFR-HAx3 interacts with TMEM16E and E584Q, but not TMEM16F ( n = 4). Analysis was performed by one-way ANOVA followed by Dunnett’s post-hoc test (Compared to cells expressing EGFR-HAx3 with TMEM16E WT, with E584Q, ns = 0.8639; with TMEM16F, **** P < 0.0001). h Protein gels of co- immunoprecipitates performed with anti-HA or IgG in cells expressing TMEM16E-GFP and HAx3-M 1 R. Co-immunoprecipitation with anti-HA was detected only with HAx3-M 1 R, but not with TMEM16E-GFP ( n = 3). Analysis of the interaction between TMEM16E-GFP with HAx3-M 1 R. ns = 0.2040, when compared with IgG, analyzed by Student’s t-test. Data are mean ± SEM.

Article Snippet: Human cDNAs of EGFR (Plasmid #133749) and PDGFR (Plasmid #136455) were purchased from Addgene, and coding regions were subcloned into a pcDNA 3.1(+) vector.

Techniques: Phospho-proteomics, Binding Assay, Fluorescence, Clinical Proteomics, Membrane, Control, Expressing, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Protective effects of glutamine in a rat model of endotoxemia.

doi: 10.3892/mmr.2012.1007

Figure Lengend Snippet: Figure 4. Immunolocalization of (A-C) platelet-derived growth factor-B (PDGF-B) and (D-F) PDGF receptor-β (PDGFR-β) in brain tissue of (A,D) controls, (B,E) lipopolysaccharide (LPS) group at 72 h, and the (C,F) glutamine (Gln) treatment group at 72 h. PDGF-B immunoreactivity was observed in the cyto plasm, PDGFR-β immunoreactivity was observed in the membrane of cortical neurons.

Article Snippet: Rabbit anti-rat NF-κB, PDGF-B, and PDGFR-β antibodies and ABC kits were purchased from Boster (Wuhan, China).

Techniques: Derivative Assay, Membrane

Journal: Molecular medicine reports

Article Title: Protective effects of glutamine in a rat model of endotoxemia.

doi: 10.3892/mmr.2012.1007

Figure Lengend Snippet: Figure 6. Western blot analysis of PDGF receptor-β (PDGFR-β) protein at various time points following injection of lipopolysaccharide (LPS). Upper row depicts the control group from left to right as 1-5 (2, 6, 12, 24 and 72 h) and the treatment group as 6-10 (2, 6, 12, 24 and 72 h). Lower row depicts the control group from left to right as 1-5 (2, 6, 12, 24 and 72 h) and the LPS group as 6-10 (2, 6, 12, 24 and 72 h).

Article Snippet: Rabbit anti-rat NF-κB, PDGF-B, and PDGFR-β antibodies and ABC kits were purchased from Boster (Wuhan, China).

Techniques: Western Blot, Injection, Control

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 cells were transfected with control shRNA (sc-108060; Santa Cruz Biotechnology), PDGFR- α shRNA (sc-29443-SH; Santa Cruz Biotechnology) plasmid DNA, or PDGFR- β shRNA (sc-29442-SH; Santa Cruz Biotechnology) at a concentration of 0.25 μ g/cm 2 surface area.

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 cells were transfected with control shRNA (sc-108060; Santa Cruz Biotechnology), PDGFR- α shRNA (sc-29443-SH; Santa Cruz Biotechnology) plasmid DNA, or PDGFR- β shRNA (sc-29442-SH; Santa Cruz Biotechnology) at a concentration of 0.25 μ g/cm 2 surface area.

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 cells were transfected with control shRNA (sc-108060; Santa Cruz Biotechnology), PDGFR- α shRNA (sc-29443-SH; Santa Cruz Biotechnology) plasmid DNA, or PDGFR- β shRNA (sc-29442-SH; Santa Cruz Biotechnology) at a concentration of 0.25 μ g/cm 2 surface area.

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: 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 cells were transfected with control shRNA (sc-108060; Santa Cruz Biotechnology), PDGFR- α shRNA (sc-29443-SH; Santa Cruz Biotechnology) plasmid DNA, or PDGFR- β shRNA (sc-29442-SH; Santa Cruz Biotechnology) at a concentration of 0.25 μ g/cm 2 surface area.

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: 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 cells were transfected with control shRNA (sc-108060; Santa Cruz Biotechnology), PDGFR- α shRNA (sc-29443-SH; Santa Cruz Biotechnology) plasmid DNA, or PDGFR- β shRNA (sc-29442-SH; Santa Cruz Biotechnology) at a concentration of 0.25 μ g/cm 2 surface area.

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 cells were transfected with control shRNA (sc-108060; Santa Cruz Biotechnology), PDGFR- α shRNA (sc-29443-SH; Santa Cruz Biotechnology) plasmid DNA, or PDGFR- β shRNA (sc-29442-SH; Santa Cruz Biotechnology) at a concentration of 0.25 μ g/cm 2 surface area.

Techniques: shRNA, Knockdown, Selection, Control, Expressing