Journal: Journal of Biomedical Science

Article Title: Many but not all pathogen-associated molecular patterns aggravate neurogenic heterotopic ossification after spinal cord injury

doi: 10.1186/s12929-026-01237-y

Figure Lengend Snippet: Expression of PRRs by mouse and human skeletal muscle cells. Expression of PRR mRNA transcripts in mouse satellite cells (SC), FAPs (FP), endothelial cells (EC) and monocyte/macrophages (MΦ) sorted from skeletal muscles. Whole mouse bone marrow (BM) cells and splenocytes (SP) were used as positive controls for these transcripts. Human PDGFRα + FAPs were sorted from muscles surrounding NHO biopsies, and CD14 + monocytes were isolated from peripheral blood from healthy donors. A TLR1, B TLR2, C TLR3, D TLR4, E TLR5, F TLR6, G TLR7, H TLR8, I TLR9, J STING1, K RIGI, L MDA5 (IFIH1), M PRK, N NOD1, O NOD2, P Dectin-1 (CLEC7A), Q Dectin-2 (Clec4n/CLEC6A), R Mincle (CLEC4E). For each gene, left hand histogram represents expression of the mouse gene in mouse cells and the right-hand histogram represents expression of the human gene in human cells. For mouse cells, relative mRNA expression was quantified relative to house-keeping gene Hprt . For human cells, values were normalized using three references genes HPRT , RPLP0 and PPIA for FAPs and ACTB , GAPDH and PPIA for CD14 + cells. Each dot represents a mouse or a human donor, bars represent mean ± SD

Article Snippet: Human MPCs were trypsinized and incubated for 30 min with biotinylated anti-human PDGFRα (Cat# BAF322, R&D Systems) goat polyclonal antibody and CD56-PE (clone B159, BD Pharmigen) monoclonal antibody in PBS 2% FCS, 2 mM EDTA or with control isotypes IgG1 PE (Cat# A07796, Beckman Coulter) and biotinylated goat IgG (Cat# BAF108, R&D Systems).

Techniques: Expressing, Muscles, Isolation

Journal: Journal of Biomedical Science

Article Title: Many but not all pathogen-associated molecular patterns aggravate neurogenic heterotopic ossification after spinal cord injury

doi: 10.1186/s12929-026-01237-y

Figure Lengend Snippet: OSM and IL-1 neutralization in monocyte-conditioned media strongly inhibit hFAPs mineralization. A OSM, IL-1α and IL-1β concentrations quantified in conditioned media from human CD14 + macrophages stimulated with 200 ng/ml Pam2CSK4 (CM Pam2CSK4 ), 200 ng/ml Pam3CSK4 (CM Pam3CSK4 ) or non-stimulated (CM ∅ ). B OSM, IL-1α and IL-1β concentrations in CM Pam2CSK4 , CM Pam3CSK4 and CM ∅ were correlated with hFAP calcium mineralization measured by Alizarin Red staining after 2 weeks of culture in osteogenic conditions in the presence of the conditioned media. Each dot represents a different conditioned medium sample. C–F Mouse anti-human OSM antibody, isotype control antibody and IL-1RA were used to neutralize OSM and IL-1 in human CD14 + monocyte conditioned media as indicated below each chart. Calcium mineralization of hFAPs cultured for 2 weeks in osteogenic conditions with C CM Pam2CSK4 or D CM Pam3CSK4 was measured using Alizarin Red staining and quantified by spectrophotometry. E, F RUNX2 protein quantification by Western blot using Stain-Free normalization for hFAPs cultured for 2 weeks in osteogenic conditions with E CM Pam2CSK4 or F CM Pam3CSK4 . In C-F, each dot represents a hFAP individual donor. Bars represent mean ± SD, One-way ANOVA Dunnett’s multiple comparison test versus CM ∅ in ( A ) and ( B ), versus CM Pam2CSK4 in ( C ) and ( E ), or versus CM Pam3CSK4 in ( D ) and ( F ), * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Human MPCs were trypsinized and incubated for 30 min with biotinylated anti-human PDGFRα (Cat# BAF322, R&D Systems) goat polyclonal antibody and CD56-PE (clone B159, BD Pharmigen) monoclonal antibody in PBS 2% FCS, 2 mM EDTA or with control isotypes IgG1 PE (Cat# A07796, Beckman Coulter) and biotinylated goat IgG (Cat# BAF108, R&D Systems).

Techniques: Neutralization, Staining, Control, Cell Culture, Spectrophotometry, Western Blot, Comparison

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: A novel population of subepithelial platelet-derived growth factor receptor ?-positive cells in the mouse and human colon

doi: 10.1152/ajpgi.00001.2013

Figure Lengend Snippet: The information of primary and secondary antibodies

Article Snippet: PDGFRα , Goat , AF-307-NA , 1:100 , R & D Systems.

Techniques:

Journal: Journal of Neuroscience

Article Title: Bone Morphogenetic Protein Inhibition Promotes Neurological Recovery after Intraventricular Hemorrhage

doi: 10.1523/jneurosci.0013-11.2011

Figure Lengend Snippet: Figure2. IVHsuppressesproliferationofoligodendrocyteprogenitors.A,Cryosectionsfromtheforebrainofrabbitpupswerelabeled withOlig2andKi67antibody.CellsdoublelabeledforthetwoantibodiesindicateproliferatingOLs(arrowhead).B,Thebargraphdepicts meanSEM(n5–6each).ThedensityofproliferatingOlig2cellsinVZandSVZwasreducedinpupswithIVHthanwithoutIVHatday 3, not day 7. C, Representative immunofluorescence of cryosections from 3-d-old E29 pups with and without IVH double labeled for PDGFR-andKi67-specificantibody.CellscolabeledwiththetwoantibodiesindicateproliferatingPDGFR-positiveOLs(arrowheads). NoteproliferatingPDGFR-positiveOLinpupswithoutIVH,butnotinpupswithIVH.D,ThebargraphdepictsmeanSEM(n5–6 each).ThedensityofmitoticallyactivePDGFRcellsinVZandSVZwasreducedinpupswithIVHthanwithoutIVHatday3,butnotatday 7. E, Cryosections from 3-d-old E29 pups were labeled with O4- and Ki67-specific antibodies. Cells colabeled with the two antibodies indicateproliferatingO4-positiveOLs(arrowheads).F,ThebargraphdepictsmeanSEM(n5–6each).ThedensityofproliferatingO4 ()cellswascomparableinpupswithandwithoutIVH.Insetsshowimmunolabelingunderhighmagnification.Scalebar,50m.*p 0.05***p0.001forthecomparisonbetweenpupswithandwithoutIVH.

Article Snippet: The primary antibodies used in experiments included goat polyclonal BMP2 (catalog #sc-6895, Santa Cruz Biotechnology), goat polyclonal BMP4 (catalog #sc-6896, Santa Cruz Biotechnology), rabbit monoclonal Ki67 (catalog #275R-14, Cell Marque), goat polyclonal Olig2 (catalog #AF-2418, R&D Systems), rabbit polyclonal Olig1 (catalog #sc-48787, Santa Cruz Biotechnology), mouse monoclonal GFAP (catalog #G6171; Sigma-Aldrich), rat monoclonal myelin basic protein (catalog #AB7439, Abcam), mouse monoclonal caspase-3 (clone 3CSP01, catalog #MS1121-P; Thermo Scientific), goat polyclonal PDGFR (catalog #AF307 NA, R&D Systems), mouse monoclonal NG2 (catalog #5384, Millipore), mouse monoclonal Adenomatous Polyposis Coli, CC1 clone (APC) antibody (catalog #OP80, EMD Chemicals).

Techniques: Immunofluorescence, Labeling

Journal: Journal of Neuroscience

Article Title: Bone Morphogenetic Protein Inhibition Promotes Neurological Recovery after Intraventricular Hemorrhage

doi: 10.1523/jneurosci.0013-11.2011

Figure Lengend Snippet: Figure 3. IVH induces maturation arrest of pre-OL and affects Olig2, SOX10, and Id4 transcription factors. A, Representative double immunolabeling of the cryosections from the forebrain of pups with IVH using a combination of O4 with O1 or PDGFR antibody at day 3. Note that pre-OLs are labeled with O4, not with O1 antibody (arrows), and immature OL are labeled with both O4andO1(arrowheads).NotethatO4()cells(arrowhead)mostlydonotoverlapPDGFR()OL(arrow).InsetsshowO4,O1, and PDGFR immunolabeling under high magnification. Scale bar, 25 m. B, The data in the bar charts represent mean SEM (n5–6each).Atbothday3and7,thedensityofPDGFR()OLwascomparablebetweenpupswithandwithoutIVHincorpus callosum(CC),coronaradiata(CR),andinternalcapsule(IC).However,thepre-OLs(O4O1 )weremoreabundantintheCCand CRofpupswithIVHthanwithoutIVHatday3,butnotintheIC.Atday7,pre-OLsweremoreinnumberintheCCofIVHpupsrelative topupswithoutIVH,butnotintheCRandIC.ThedensityofimmatureOL(O4O1)wassignificantlyreducedinrabbitpupswith IVH than in pups without IVH at both day 3 and 7 in the corpus callosum and corona radiata, unlike the internal capsule. ***p 0.001,**p0.01,and*p0.05forthecomparisonbetweenpupswithandwithoutIVHincorpuscallosum. #p0.05forthe comparison between pups with and without IVH in corona radiata.

Article Snippet: The primary antibodies used in experiments included goat polyclonal BMP2 (catalog #sc-6895, Santa Cruz Biotechnology), goat polyclonal BMP4 (catalog #sc-6896, Santa Cruz Biotechnology), rabbit monoclonal Ki67 (catalog #275R-14, Cell Marque), goat polyclonal Olig2 (catalog #AF-2418, R&D Systems), rabbit polyclonal Olig1 (catalog #sc-48787, Santa Cruz Biotechnology), mouse monoclonal GFAP (catalog #G6171; Sigma-Aldrich), rat monoclonal myelin basic protein (catalog #AB7439, Abcam), mouse monoclonal caspase-3 (clone 3CSP01, catalog #MS1121-P; Thermo Scientific), goat polyclonal PDGFR (catalog #AF307 NA, R&D Systems), mouse monoclonal NG2 (catalog #5384, Millipore), mouse monoclonal Adenomatous Polyposis Coli, CC1 clone (APC) antibody (catalog #OP80, EMD Chemicals).

Techniques: Immunolabeling, Labeling, Comparison

Journal: Scientific Reports

Article Title: PDGFRA K385 mutants in myxoid glioneuronal tumors promote receptor dimerization and oncogenic signaling

doi: 10.1038/s41598-024-57859-5

Figure Lengend Snippet: K385I and K385L are oncogenic driver mutations. Ba/F3 cells were transduced with PDGFRA by retroviral infection. Receptor expression was tested by ( A ) flow cytometry or ( B ) by western blot. ( C ) Ba/F3 cell viability and proliferation after IL-3 withdrawal was assessed using CellTiter-Glo luminescent assay. Cells treated or not with PDGF-BB (50 ng/ml) 48 h after removing IL-3. The histograms represent the mean of three independent experiments with SEM, normalized to the WT condition treated with PDGF-BB. ( D ) NIH3T3 cells were transfected with WT or mutated PDGFRα and treated with G418. After approximately 2 weeks, cells were fixed in methanol and colored with crystal violet. ( E ) Foci density was quantified using Bio1D software and normalized to the WT receptor condition. The mean of three independent experiments is represented with SEM (Wilcoxon test; *p < 0.5; ***p < 0.001 compared to WT).

Article Snippet: Ba/F3 cells were harvested, washed in PBS and stained with a primary mouse anti-PDGFRα antibody (#MAB1264, R&D systems, Minneapolis, MN, USA, MAB1264) for 30 min at 4 °C.

Techniques: Transduction, Retroviral, Infection, Expressing, Flow Cytometry, Western Blot, Luminescence Assay, Transfection, Software

Journal: Scientific Reports

Article Title: PDGFRA K385 mutants in myxoid glioneuronal tumors promote receptor dimerization and oncogenic signaling

doi: 10.1038/s41598-024-57859-5

Figure Lengend Snippet: PDGFRα K385I/L receptors are sensitive to imatinib, dasatinib and avapritinib. ( A ) U-87 MG cells were co-transfected with the indicated receptor and a luciferase reporter as described above. The histograms represent the mean of three independent experiments with SEM, normalized to the WT untreated condition. ( B ) A CellTiter-Glo luminescent assay was conducted on Ba/F3 cells expressing WT or mutated PDGFRα treated with increasing concentrations of imatinib, dasatinib, or avapritinib for 24 h. The histograms represent the mean of three independent experiments with SEM, normalized to the untreated condition.

Article Snippet: Ba/F3 cells were harvested, washed in PBS and stained with a primary mouse anti-PDGFRα antibody (#MAB1264, R&D systems, Minneapolis, MN, USA, MAB1264) for 30 min at 4 °C.

Techniques: Transfection, Luciferase, Luminescence Assay, Expressing

Journal: eLife

Article Title: GATA6 regulates WNT and BMP programs to pattern precardiac mesoderm during the earliest stages of human cardiogenesis

doi: 10.7554/eLife.100797

Figure Lengend Snippet: ( A ) Western blot time course using protein lysates from GATA6 +/+ human embryonic stem cells (hESCs) probed for GATA6 with β-actin used as a loading control. ( B ) Flow cytometry quantification for % KDR and PDGFRα double-positive (%K + P + ) cells from days 3–5 of cardiac differentiation of GATA6 +/+ , GATA6 +/- , or GATA6 -/- hESCs (n ≥ 4). Asterisks indicate statistical significance comparing GATA6 -/- and WT on day 4 or 5 of cardiac differentiation. ( C ) Day 5 flow cytometry quantification for %K + P + cells (n = 7). ( D ) Representative flow cytometry plots for day 5 %K + P + cells. ( E ) Day 5 flow cytometry quantification (n = 7) for %KDR + (left) or PDGFRα + (right). ( F ) Representative flow cytometry plots for day 2 %BRACHYURY + cells (red) overlaid IgG stained controls (blue). ( G ) Quantification for day 2 or day 3 %BRACHYURY + cells (n = 4). ( H ) RT-qPCR for day 2 T , EOMES , MESP1 , and MESP2 expression levels normalized to GATA6 +/+ samples (n = 6). Data represents the mean ± SEM, with significance indicated as **p<0.01, ****p<0.0001, and ns indicating not significant by two-way ANOVA ( B, G ) or one-way ANOVA (C, E, H) with Tukey’s multiple-comparison test. The labels w4, w2 (wildtype), m2, m5 (heterozygous), and m11, m14 (homozygous) refer to the isogenic wildtype and mutant hESC clones (see ). Figure 2—source data 1. Western blots in panel A. Figure 2—source data 2. Flow cytometry in panels B, C, E and G. First tab includes KP flow cytometry in panels B, C and E. Second tab includes BRACHYURY flow cytometry in panel G. Figure 2—source data 3. RT-qPCR in panel H.

Article Snippet: Live cells were used for staining the cell surface proteins KP by incubating with FACS buffer containing PE-conjugated KDR antibody (R&D Systems FAB357P-100) and APC-conjugated PDGFRα antibody (R&D Systems FAB1264A) or IgG control antibodies (R&D Systems IC002P, IC002A) for 30 min at RT, and then stained with SYTOX Green Dead Cell Stain (Thermo Fisher Scientific S34860) in FACS buffer for 5 min at RT.

Techniques: Western Blot, Control, Flow Cytometry, Staining, Quantitative RT-PCR, Expressing, Comparison, Mutagenesis, Clone Assay

Journal: eLife

Article Title: GATA6 regulates WNT and BMP programs to pattern precardiac mesoderm during the earliest stages of human cardiogenesis

doi: 10.7554/eLife.100797

Figure Lengend Snippet: ( A ) Representative flow cytometry plots at day 4 of cardiac differentiation analyzing the % KDR and PDGFRα double-positive cells (%K + P + ) from GATA6 +/+ , GATA6 +/- , or GATA6 -/- hESCs. ( B ) Quantification of day 4 flow cytometry data for %K + P + double-positive, %KDR + single positive, or %PDGFRα + single-positive cells (n = 6). Data represent the mean ± SEM, with statistical significance indicated as *p<0.05 and ns indicating not significant by one-way repeated measures ANOVA with Holm–Šídák’s multiple-comparison test. Figure 2—figure supplement 1—source data 1. KP flow cytometry in panel B.

Article Snippet: Live cells were used for staining the cell surface proteins KP by incubating with FACS buffer containing PE-conjugated KDR antibody (R&D Systems FAB357P-100) and APC-conjugated PDGFRα antibody (R&D Systems FAB1264A) or IgG control antibodies (R&D Systems IC002P, IC002A) for 30 min at RT, and then stained with SYTOX Green Dead Cell Stain (Thermo Fisher Scientific S34860) in FACS buffer for 5 min at RT.

Techniques: Flow Cytometry, Comparison

Journal: eLife

Article Title: GATA6 regulates WNT and BMP programs to pattern precardiac mesoderm during the earliest stages of human cardiogenesis

doi: 10.7554/eLife.100797

Figure Lengend Snippet: ( A ) Schematic for treatment with DOX (days 1–4), CHIR (3 μM, days 0–2), and/or reduced BMP4 (5 ng/mL, days 0–2, indicated as ‘LB’) during CM directed differentiation using the cytokine-based protocol. ( B ) Day 5 flow cytometry quantification for %K + P + double-positive cells, %KDR + single-positive cells, or %PDGFRα + single-positive cells in WT or GATA6 -/- hESCs transduced with i LGR5 or empty vector (EV) (n = 5). Significance indicated by *p<0.05 according to one-way ANOVA and Tukey’s post hoc analysis. ( C ) %cTnT + CMs from days 13–18 of cardiac differentiation quantified by flow cytometry in WT or GATA6 -/- cells treated with CHIR LB or vehicle (DMSO) with normal BMP4 concentration treated control (n ≥ 6). ( D ) Flow cytometry at day 5 of cardiac differentiation to quantify %K + P + double-positive, %KDR + single-positive, or %PDGFRα + single-positive cells comparing WT or GATA6 -/- hESCs treated with CHIR LB with GATA6 +/+ and GATA6 -/- hESCs controls treated with vehicle and normal BMP4 concentration (n ≥ 8). ( E ) %cTnT + CMs from days 13–18 of cardiac differentiation quantified by flow cytometry in GATA6 +/- or WT hESCs treated with CHIR (3 μM) or DMSO (n ≥ 7). Data represents the mean ± SEM, significance indicated by **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Student’s t -test ( C ) and two-way ANOVA ( D, E ) with Tukey’s multiple-comparison test. Figure 6—source data 1. Flow cytometry in panels B-E. First tab includes KP flow cytometry in panels B and D. Second tab includes cTnT flow cytometry in panels C and E.

Article Snippet: Live cells were used for staining the cell surface proteins KP by incubating with FACS buffer containing PE-conjugated KDR antibody (R&D Systems FAB357P-100) and APC-conjugated PDGFRα antibody (R&D Systems FAB1264A) or IgG control antibodies (R&D Systems IC002P, IC002A) for 30 min at RT, and then stained with SYTOX Green Dead Cell Stain (Thermo Fisher Scientific S34860) in FACS buffer for 5 min at RT.

Techniques: Flow Cytometry, Transduction, Plasmid Preparation, Concentration Assay, Control, Two Tailed Test, Comparison