Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Temporal delay of molar tooth development in Barx1 homozygous mutants. Hematoxylin and eosin stained frontal (A–J) and sagittal (K and L) sections of lower E13.5 incisors (A and B) and first molars (C and D), E14.5 incisors (E and F), and first molars (G and H), E16.5 first molars (I and J), and postnatal day 0 (P0) first molars (K and L). At E13.5, all tooth germs have reached a bud stage both in the Barx1 homozygous mutant (B and D) and control littermate (A and C). Incisors develop normally in all Barx1 homozygous mutants, displaying a characteristic epithelial cap at E14.5 (E and F), whereas the molars of all four quadrants show a developmental delay between E13.5 and E14.5, exhibiting a bud shape instead of a cap (G and H) (n > 10). Arrowheads in E–G indicate the primary enamel knot, visualized as a bulge on the inside of the epithelial cap. At E16.5–P0, Barx1−/− molars are slightly smaller but otherwise normal (I–L). m, Meckel's cartilage; vl, vestibular lamina. (Scale bar, 100 μm in A–J and 200 μm in K and L.)

Article Snippet: C3H10T1/2 cells were sequentially immunostained with anti-Barx1 (H-55, rabbit polyclonal antibody; Santa Cruz Biotechnology) and anti-Msx1 antibody (rabbit polyclonal antibody; Santa Cruz Biotechnology).

Techniques: Staining, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Barx1 mutant molar teeth exhibit changes in cell proliferation. (A–F) BrdU staining of frontal sections of developing first molar tooth germs at E13.5 (A and B), E14.5 (C and D), and E15.5 (E and F) in a Barx1+/− (A, C, and E) and Barx1−/− (B, D, and F) lower jaw. (G) Graphs comparing the numbers of BrdU-labeled cells in the epithelium and the condensed mesenchyme of developing lower first molars at E13.5, E14.5, and E15.5. Error bars show SD.

Article Snippet: C3H10T1/2 cells were sequentially immunostained with anti-Barx1 (H-55, rabbit polyclonal antibody; Santa Cruz Biotechnology) and anti-Msx1 antibody (rabbit polyclonal antibody; Santa Cruz Biotechnology).

Techniques: Mutagenesis, BrdU Staining, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: BMP4 expression and BMP activity changes in Barx1 mutant tooth development. (A–R) Expression of Bmp4 (A–F), Shh (G–L), and distribution of phospho-Smad1/5/8 (M–R) in lower first molar tooth germs at E13.5 (A, B, G, H, M, and N), E14.5 (C, D, I, J, O, and P), and E15.5 (E, F, K, L, Q, and R) in a WT (A, C, E, G, I, K, M, O, and Q) and Barx1 homozygous mutant (B, D, F, H, J, L, N, P, and R) littermates. (S–X) Expression of Shh in lower incisor tooth germs at E13.5 (S and T), E14.5 (U and V), and E16.5 (W and X). In situ hybridization was carried out on four separate samples for each genotype at each time point and immunostaining on two separate samples for each genotype at each time point. The epithelium of molar and incisor tooth germs is outlined in white.

Article Snippet: C3H10T1/2 cells were sequentially immunostained with anti-Barx1 (H-55, rabbit polyclonal antibody; Santa Cruz Biotechnology) and anti-Msx1 antibody (rabbit polyclonal antibody; Santa Cruz Biotechnology).

Techniques: Expressing, Activity Assay, Mutagenesis, In Situ Hybridization, Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Arrest of molar tooth development associated with a lack of Bmp4 transcription in Barx1/Msx1 compound mutants. Frontal sections through upper (A, C, E, G, I, K, M, O, Q, and S) and lower (B, D, F, H, J, L, N, P, R, and T) developing first molars at E14.5 (A, B, E, F, I, J, M, N, Q, and R) and E16.5 (C, D, G, H, K, L, O, P, S, and T). Hematoxylin and eosin stained sections of Barx1+/−; Msx1+/− (A–D), Barx1+/+; Msx1−/− (E–H), and Barx1−/−; Msx1+/− (I–L). (M–T) Expression of BMP4 in the condensed mesenchyme of first molar tooth germs of Barx1+/−; Msx1+/− (M–P) and Barx1−/−; Msx1+/− (Q–T). Permanent arrest of molar tooth development was observed in three separate Barx1−/−; Msx1+/− animals and was highly penetrant. At E16.5, one molar tooth germ (n = 1/12) was occasionally observed at the cap stage (corresponding to E14.5).

Article Snippet: C3H10T1/2 cells were sequentially immunostained with anti-Barx1 (H-55, rabbit polyclonal antibody; Santa Cruz Biotechnology) and anti-Msx1 antibody (rabbit polyclonal antibody; Santa Cruz Biotechnology).

Techniques: Staining, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Msx1 interacts with molar tooth-specific transcription factor Barx1. (A) Msx1 interacts with Barx1 in living cells. C3H10T1/2 cells were cotransfected with pIRES2–Barx1–EGFP and either pCMV–FLAG–Msx1 or pCMV–FLAG–Tag2B empty control vector. Cell lysates were subjected to coimmunoprecipitations followed by Western blotting. Barx1 was detected only in the presence of FLAG–Msx1 in the IP sample. IP, immunoprecipitation; IB, immunoblotting. (B–I) Intracellular colocalization of Barx1 and Msx1 in C3H10T1/2 cells. (B–E) Intracellular colocalization of endogenously expressed Barx1 and Msx1. (B) Intracellular localization of Barx1 using anti-Barx1 (green); (C) intracellular localization of Msx1, using anti-Msx1 (red); (D) merged pictures showing intracellular colocalization of Barx1 and Msx1 (yellow); and (E) DNA staining using the fluorescence dye DRAQ5 (blue). (F–I) Intracellular colocalization of exogenously overexpressed Barx1 and Msx1 as EGFP and FLAG-tagged fusion proteins. (F) Intracellular localization of Barx1–EGFP (green); (G) intracellular localization of Msx1 using anti-Msx1 (red); (H) merged pictures show intracellular colocalization of Barx1–EGFP and FLAG–Msx1 (yellow); and (I) DNA staining using the fluorescence dye DRAQ5 (blue).

Article Snippet: C3H10T1/2 cells were sequentially immunostained with anti-Barx1 (H-55, rabbit polyclonal antibody; Santa Cruz Biotechnology) and anti-Msx1 antibody (rabbit polyclonal antibody; Santa Cruz Biotechnology).

Techniques: Plasmid Preparation, Western Blot, Immunoprecipitation, Staining, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Barx1 is expressed in all multicusped teeth with expression levels correlating with cusp numbers. (A–C) Supernumerary teeth forming in the diastema of mice homozygous for Tg737orpk display Barx1 expression levels lower than first and second molars. (A and B) Consecutive sagittal sections through the upper jaw of an E18.5 mouse homozygous for Tg737orpk showing from Left to Right a second molar (M2), first molar (M1), and supernumerary tooth (SN), the latter developing mesial to M1 in the normally toothless diastema. (A) Trichrome staining showing the premolar-like shape of the ectopic diastema tooth. (B) Radioactive in situ hybridization for Barx1. (C) Quantification of Barx1 expression level in the dental mesenchyme of Tg737orpk/Tg737orpk second molar (M2), first molar (M1), and supernumerary tooth (SN). (D–I) Level of cpBarx1 expression correlates with cusp number in shrew multicusped teeth. (D and H) Dentition of an adult shrew upper (D) and lower (H) jaw composed of molars (M), premolars (PM), unicusps (U), and incisors (I). (E and F) Radioactive in situ hybridization for cpBarx1 in shrew premolars (E) and molars (F). Developing molars and premolars (outlined in red) have reached a cap stage. (G) Quantification of Barx1 expression levels in the dental mesenchyme of shrew premolar and molar tooth primordia. Gene expression was quantified by analyzing consecutive sections spanning the whole dental papilla of each tooth using ImageJ 1.34s. The number of cusps and crests displayed by each tooth is indicated above their respective Barx1 expression level. (I) 3D reconstructions of micro-CT scans of the upper and lower first molars (M1) and premolars (PM) of a 24-d-old shrew. Teeth are viewed from a lingual side; distal is Right and proximal Left. The number of cusps of each tooth (indicated in G) was carefully assessed by rotating the 3D models.

Article Snippet: C3H10T1/2 cells were sequentially immunostained with anti-Barx1 (H-55, rabbit polyclonal antibody; Santa Cruz Biotechnology) and anti-Msx1 antibody (rabbit polyclonal antibody; Santa Cruz Biotechnology).

Techniques: Expressing, Staining, In Situ Hybridization, Micro-CT

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Temporal delay of molar tooth development in Barx1 homozygous mutants. Hematoxylin and eosin stained frontal (A–J) and sagittal (K and L) sections of lower E13.5 incisors (A and B) and first molars (C and D), E14.5 incisors (E and F), and first molars (G and H), E16.5 first molars (I and J), and postnatal day 0 (P0) first molars (K and L). At E13.5, all tooth germs have reached a bud stage both in the Barx1 homozygous mutant (B and D) and control littermate (A and C). Incisors develop normally in all Barx1 homozygous mutants, displaying a characteristic epithelial cap at E14.5 (E and F), whereas the molars of all four quadrants show a developmental delay between E13.5 and E14.5, exhibiting a bud shape instead of a cap (G and H) (n > 10). Arrowheads in E–G indicate the primary enamel knot, visualized as a bulge on the inside of the epithelial cap. At E16.5–P0, Barx1−/− molars are slightly smaller but otherwise normal (I–L). m, Meckel's cartilage; vl, vestibular lamina. (Scale bar, 100 μm in A–J and 200 μm in K and L.)

Article Snippet: For Western blotting of eluted protein, primary antibodies were used with 1:500 dilution of rabbit anti-Barx1 polyclonal antibody (Santa Cruz) or 1:1,000 dilution mouse anti-FLAG M2 monoclonal antibody (Sigma).

Techniques: Staining, Mutagenesis, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Barx1 mutant molar teeth exhibit changes in cell proliferation. (A–F) BrdU staining of frontal sections of developing first molar tooth germs at E13.5 (A and B), E14.5 (C and D), and E15.5 (E and F) in a Barx1+/− (A, C, and E) and Barx1−/− (B, D, and F) lower jaw. (G) Graphs comparing the numbers of BrdU-labeled cells in the epithelium and the condensed mesenchyme of developing lower first molars at E13.5, E14.5, and E15.5. Error bars show SD.

Article Snippet: For Western blotting of eluted protein, primary antibodies were used with 1:500 dilution of rabbit anti-Barx1 polyclonal antibody (Santa Cruz) or 1:1,000 dilution mouse anti-FLAG M2 monoclonal antibody (Sigma).

Techniques: Mutagenesis, BrdU Staining, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: BMP4 expression and BMP activity changes in Barx1 mutant tooth development. (A–R) Expression of Bmp4 (A–F), Shh (G–L), and distribution of phospho-Smad1/5/8 (M–R) in lower first molar tooth germs at E13.5 (A, B, G, H, M, and N), E14.5 (C, D, I, J, O, and P), and E15.5 (E, F, K, L, Q, and R) in a WT (A, C, E, G, I, K, M, O, and Q) and Barx1 homozygous mutant (B, D, F, H, J, L, N, P, and R) littermates. (S–X) Expression of Shh in lower incisor tooth germs at E13.5 (S and T), E14.5 (U and V), and E16.5 (W and X). In situ hybridization was carried out on four separate samples for each genotype at each time point and immunostaining on two separate samples for each genotype at each time point. The epithelium of molar and incisor tooth germs is outlined in white.

Article Snippet: For Western blotting of eluted protein, primary antibodies were used with 1:500 dilution of rabbit anti-Barx1 polyclonal antibody (Santa Cruz) or 1:1,000 dilution mouse anti-FLAG M2 monoclonal antibody (Sigma).

Techniques: Expressing, Activity Assay, Mutagenesis, In Situ Hybridization, Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Arrest of molar tooth development associated with a lack of Bmp4 transcription in Barx1/Msx1 compound mutants. Frontal sections through upper (A, C, E, G, I, K, M, O, Q, and S) and lower (B, D, F, H, J, L, N, P, R, and T) developing first molars at E14.5 (A, B, E, F, I, J, M, N, Q, and R) and E16.5 (C, D, G, H, K, L, O, P, S, and T). Hematoxylin and eosin stained sections of Barx1+/−; Msx1+/− (A–D), Barx1+/+; Msx1−/− (E–H), and Barx1−/−; Msx1+/− (I–L). (M–T) Expression of BMP4 in the condensed mesenchyme of first molar tooth germs of Barx1+/−; Msx1+/− (M–P) and Barx1−/−; Msx1+/− (Q–T). Permanent arrest of molar tooth development was observed in three separate Barx1−/−; Msx1+/− animals and was highly penetrant. At E16.5, one molar tooth germ (n = 1/12) was occasionally observed at the cap stage (corresponding to E14.5).

Article Snippet: For Western blotting of eluted protein, primary antibodies were used with 1:500 dilution of rabbit anti-Barx1 polyclonal antibody (Santa Cruz) or 1:1,000 dilution mouse anti-FLAG M2 monoclonal antibody (Sigma).

Techniques: Staining, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Msx1 interacts with molar tooth-specific transcription factor Barx1. (A) Msx1 interacts with Barx1 in living cells. C3H10T1/2 cells were cotransfected with pIRES2–Barx1–EGFP and either pCMV–FLAG–Msx1 or pCMV–FLAG–Tag2B empty control vector. Cell lysates were subjected to coimmunoprecipitations followed by Western blotting. Barx1 was detected only in the presence of FLAG–Msx1 in the IP sample. IP, immunoprecipitation; IB, immunoblotting. (B–I) Intracellular colocalization of Barx1 and Msx1 in C3H10T1/2 cells. (B–E) Intracellular colocalization of endogenously expressed Barx1 and Msx1. (B) Intracellular localization of Barx1 using anti-Barx1 (green); (C) intracellular localization of Msx1, using anti-Msx1 (red); (D) merged pictures showing intracellular colocalization of Barx1 and Msx1 (yellow); and (E) DNA staining using the fluorescence dye DRAQ5 (blue). (F–I) Intracellular colocalization of exogenously overexpressed Barx1 and Msx1 as EGFP and FLAG-tagged fusion proteins. (F) Intracellular localization of Barx1–EGFP (green); (G) intracellular localization of Msx1 using anti-Msx1 (red); (H) merged pictures show intracellular colocalization of Barx1–EGFP and FLAG–Msx1 (yellow); and (I) DNA staining using the fluorescence dye DRAQ5 (blue).

Article Snippet: For Western blotting of eluted protein, primary antibodies were used with 1:500 dilution of rabbit anti-Barx1 polyclonal antibody (Santa Cruz) or 1:1,000 dilution mouse anti-FLAG M2 monoclonal antibody (Sigma).

Techniques: Control, Plasmid Preparation, Western Blot, Immunoprecipitation, Staining, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Developmental stalling and organ-autonomous regulation of morphogenesis

doi: 10.1073/pnas.1112801108

Figure Lengend Snippet: Barx1 is expressed in all multicusped teeth with expression levels correlating with cusp numbers. (A–C) Supernumerary teeth forming in the diastema of mice homozygous for Tg737orpk display Barx1 expression levels lower than first and second molars. (A and B) Consecutive sagittal sections through the upper jaw of an E18.5 mouse homozygous for Tg737orpk showing from Left to Right a second molar (M2), first molar (M1), and supernumerary tooth (SN), the latter developing mesial to M1 in the normally toothless diastema. (A) Trichrome staining showing the premolar-like shape of the ectopic diastema tooth. (B) Radioactive in situ hybridization for Barx1. (C) Quantification of Barx1 expression level in the dental mesenchyme of Tg737orpk/Tg737orpk second molar (M2), first molar (M1), and supernumerary tooth (SN). (D–I) Level of cpBarx1 expression correlates with cusp number in shrew multicusped teeth. (D and H) Dentition of an adult shrew upper (D) and lower (H) jaw composed of molars (M), premolars (PM), unicusps (U), and incisors (I). (E and F) Radioactive in situ hybridization for cpBarx1 in shrew premolars (E) and molars (F). Developing molars and premolars (outlined in red) have reached a cap stage. (G) Quantification of Barx1 expression levels in the dental mesenchyme of shrew premolar and molar tooth primordia. Gene expression was quantified by analyzing consecutive sections spanning the whole dental papilla of each tooth using ImageJ 1.34s. The number of cusps and crests displayed by each tooth is indicated above their respective Barx1 expression level. (I) 3D reconstructions of micro-CT scans of the upper and lower first molars (M1) and premolars (PM) of a 24-d-old shrew. Teeth are viewed from a lingual side; distal is Right and proximal Left. The number of cusps of each tooth (indicated in G) was carefully assessed by rotating the 3D models.

Article Snippet: For Western blotting of eluted protein, primary antibodies were used with 1:500 dilution of rabbit anti-Barx1 polyclonal antibody (Santa Cruz) or 1:1,000 dilution mouse anti-FLAG M2 monoclonal antibody (Sigma).

Techniques: Expressing, Staining, In Situ Hybridization, Gene Expression, Micro-CT

Journal: Clinical Cancer Research

Article Title: HAND1 and BARX1 Act as Transcriptional and Anatomic Determinants of Malignancy in Gastrointestinal Stromal Tumor

doi: 10.1158/1078-0432.ccr-20-3538

Figure Lengend Snippet: Figure 1. HAND1 is associated with metastatic GIST and a distinctive transcriptional program. A, Volcano plot of RNA-seq data from localized and never recurrent GIST (“No Recurrence,”n ¼ 14) or localized and later recurrent or metastatic GIST (“Metastatic/Recurrent,” n ¼ 11). All differentially expressed TFs are labeled. The percent of genes differentially expressed is shown (n ¼ 10,000 totalexpressed transcripts). B, FPKM of HAND1 and BARX1 in No Recurrence KIT-mutant GIST (n ¼ 9),Metastatic/ Recurrent KIT-mutant GIST (n ¼ 11), PDGFRA-mutant GIST (n ¼ 5), or SDH-deficient GIST (n ¼ 10). Data were analyzed by one-way ANOVA with Tukey multiple comparison test (compared with KIT-mutant localized GIST; , P < 0.001). C, Correlation of BARX1 and HAND1 expression in all GIST samples (n ¼ 110), with the clinically annotated cohort indicated with circles and validation cohort with diamonds. The Pearson correlation is shown. D, PCA of GIST validation cohort RNA- seq (35) stratified by HAND1 positivity (n ¼ 21) and BARX1 positivity (n ¼ 39) with threshold for expression of 50 FPKM. E, Butterfly plot of all Hallmark gene sets indicating the NES and FDR q-value for the two RNA-seq data cohorts (“No Recurrence” and “Metastatic/Recurrent” GIST in purple, BARX1- or HAND1-positive GIST in yellow). The G2–M-phase checkpoint, MTORC1 signaling and EMT gene sets are indicated for each condition. F and G, GSEA showing the Hallmark G2–M-phase checkpoint gene set in the independent GIST RNA-seq datasets.

Article Snippet: Slides were incubated with HAND1 antibody (1:300, OriGene, catalog No. TA502671, RRID:AB_11125431) or BARX1 antibody (1:100, Atlas Antibodies catalog No. HPA055858, RRID: AB_2682947) for 45 minutes, washed, then incubated with either Labeled Polymer-HRP anti-mouse secondary antibody (K4007, Dako) for HAND1 or Post Primary (Leica Novolink) followed by Novolink Polymer Detection System (RE7150-K, Leica) for BARX1.

Techniques: RNA Sequencing, Labeling, Mutagenesis, Comparison, Expressing, Biomarker Discovery

Journal: Clinical Cancer Research

Article Title: HAND1 and BARX1 Act as Transcriptional and Anatomic Determinants of Malignancy in Gastrointestinal Stromal Tumor

doi: 10.1158/1078-0432.ccr-20-3538

Figure Lengend Snippet: Figure 3. IHC of GIST with standard and novel biomarkers. A, Percent positive expression in GIST subtypes including KIT-mutant (n ¼ 65), PDGFRA-mutant (n ¼ 10), and SDH- deficient (n ¼ 12) tumors for the proteins DOG1, KIT, PDGFRA, SDHB, HAND1, HOXC10, PLAGL1, and BARX1. B, IHC of KIT-mutant GIST stratified by anatomic location and localized or metastatic disease. C, IHC of KIT-mutant GIST stratified by mutational subtype. All exon 13 and compound mutation tumors were derived from metastatic disease. D, IHC of GIST with exemplary positive and negative expression of HAND1 and BARX1; scale bar indicates 20 mmol/L. E, Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of select proteins for mutational subtype.

Article Snippet: Slides were incubated with HAND1 antibody (1:300, OriGene, catalog No. TA502671, RRID:AB_11125431) or BARX1 antibody (1:100, Atlas Antibodies catalog No. HPA055858, RRID: AB_2682947) for 45 minutes, washed, then incubated with either Labeled Polymer-HRP anti-mouse secondary antibody (K4007, Dako) for HAND1 or Post Primary (Leica Novolink) followed by Novolink Polymer Detection System (RE7150-K, Leica) for BARX1.

Techniques: Expressing, Mutagenesis, Derivative Assay

Journal: Clinical Cancer Research

Article Title: HAND1 and BARX1 Act as Transcriptional and Anatomic Determinants of Malignancy in Gastrointestinal Stromal Tumor

doi: 10.1158/1078-0432.ccr-20-3538

Figure Lengend Snippet: Figure 4. Tissue-level and single-cell analysis of accessory TFs and proliferative markers from multiplexed CyCIF imaging. A, Relative intensity of signal in whole tissue (relative fluorescence units, RFU) of SDHB and PDGFRA in KIT-mutant (n ¼ 65), PDGFRA-mutant (n ¼ 10), and SDH-deficient (n ¼ 12) GIST. Data were analyzed by one-way ANOVA with Tukey multiple comparison test (compared with KIT-mutant GIST; , P < 0.001). B, Relative intensity of signal in whole tissue for HAND1 or BARX1 in KIT-mutant tumors stratified by IHC positivity or negativity for each marker, or PDGFRA-mutant and SDH-deficient GIST. C, Correlation of mitotic index and Ki-67 signal intensity for samples with available clinical annotation (n ¼ 51). D, Correlation of Ki-67 and HAND1 signal intensity, with samples distinguished by mutational subtypes (n ¼ 87). The Pearson correlation for all samples is shown. E, UMAP of all tumor samples for single tumor cells (87 tumors, n ¼ 156,036 cells), defined by the expressionofHAND1 and/orBARX1,withheatmaps showing BARX1,HAND1, Ki-67,PCNA,PDGFRA, and SDHBlog2 RFU.F,Percentofall tumor cellsthatwerenegativeor positive for Ki-67 (top) or PCNA (bottom) stratified by BARX1 (blue, n ¼ 65,340) or HAND1 (red, n ¼ 81,501) expression. Data were analyzed by Fisher exact test with P value indicated. G, UMAP of all HAND1- and/or BARX1-positive cells from localized gastric GIST (15 tumors, n ¼ 24,859 cells) showing BARX1, HAND1, Ki-67, and PCNA. H,Percent of localized gastric tumor cells that were negative or positive for Ki-67 (top) or PCNA (bottom) stratified by BARX1 (blue,n ¼ 21,586) or HAND1 (red, n ¼ 1,552) expression. I, Image showing signal for DAPI (white), BARX1 (green), HAND1 (red), and Ki-67 (blue) in a localized gastric GIST (scale bar, 5 mmol/L).

Article Snippet: Slides were incubated with HAND1 antibody (1:300, OriGene, catalog No. TA502671, RRID:AB_11125431) or BARX1 antibody (1:100, Atlas Antibodies catalog No. HPA055858, RRID: AB_2682947) for 45 minutes, washed, then incubated with either Labeled Polymer-HRP anti-mouse secondary antibody (K4007, Dako) for HAND1 or Post Primary (Leica Novolink) followed by Novolink Polymer Detection System (RE7150-K, Leica) for BARX1.

Techniques: Single-cell Analysis, Imaging, Mutagenesis, Comparison, Marker, Expressing

Journal: Clinical Cancer Research

Article Title: HAND1 and BARX1 Act as Transcriptional and Anatomic Determinants of Malignancy in Gastrointestinal Stromal Tumor

doi: 10.1158/1078-0432.ccr-20-3538

Figure Lengend Snippet: Figure 5. Expression characteristics of HAND1 and BARX1 in GIST and clinical outcomes. Expression of HAND1 and BARX1 stratified by anatomic location (A), tumor mutation (B), KIT mutation subtype (C), and disease status of KIT mutant tumors (D) across all samples (n ¼ 437). E, Frequency of coexpression of BARX1 and HAND1 across all samples (n ¼ 437). F and G, Kaplan–Meier plots of relapse-free survival following primary tumor resection stratified by risk status (F) or HAND1 and BARX1 expression (G). H, Kaplan–Meier plot of progression-free survival on first line imatinib for patients with metastatic GIST stratified by HAND1 expression.

Article Snippet: Slides were incubated with HAND1 antibody (1:300, OriGene, catalog No. TA502671, RRID:AB_11125431) or BARX1 antibody (1:100, Atlas Antibodies catalog No. HPA055858, RRID: AB_2682947) for 45 minutes, washed, then incubated with either Labeled Polymer-HRP anti-mouse secondary antibody (K4007, Dako) for HAND1 or Post Primary (Leica Novolink) followed by Novolink Polymer Detection System (RE7150-K, Leica) for BARX1.

Techniques: Expressing, Mutagenesis

Journal: Clinical Cancer Research

Article Title: HAND1 and BARX1 Act as Transcriptional and Anatomic Determinants of Malignancy in Gastrointestinal Stromal Tumor

doi: 10.1158/1078-0432.ccr-20-3538

Figure Lengend Snippet: Figure 6. Anatomic and spatial restriction of HAND1 and BARX1 expression in ICC. A, Hematoxylin and eosin staining of normaltissue with muscular layers and myenteric plexus indicated (left, scale bar 100 mmol/L) in stomach (top row) and duodenum (bottom row). KIT (brown) and HAND1 (red) IHC at the myenteric plexus (second from left, scale bar 50 mmol/L) with individual ICC indicated by an arrowhead (third from left, scale bar 20 mmol/L). KIT (red) and BARX1 (brown) IHC at the myenteric plexus (right, scale bar 20 mmol/L). B, Percentage of ICC staining positive for HAND1 or BARX1 in normal gastrointestinal tissues. Inset values indicate the numerator of positively expressing ICC over the denominator of total ICC. For quantification, 100 ICC were evaluated per section, where possible, in at least seven sections per anatomic location.

Article Snippet: Slides were incubated with HAND1 antibody (1:300, OriGene, catalog No. TA502671, RRID:AB_11125431) or BARX1 antibody (1:100, Atlas Antibodies catalog No. HPA055858, RRID: AB_2682947) for 45 minutes, washed, then incubated with either Labeled Polymer-HRP anti-mouse secondary antibody (K4007, Dako) for HAND1 or Post Primary (Leica Novolink) followed by Novolink Polymer Detection System (RE7150-K, Leica) for BARX1.

Techniques: Expressing, Staining