Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 expression is substantially increased in patients with colorectal cancer (CRC) resistant to neoadjuvant therapy. ST6GAL1 expression was measured in 14 surgically removed fresh CRC tumor tissues and matched adjacent normal tissues using RT‒qPCR, WB and IHC after. The correlation between ST6GAL1 expression and sensitivity to neoadjuvant therapy was analyzed in combination with the clinical information. ( A ) ST6GAL1 mRNA expression was quantified by RT‒qPCR in tumor tissues compared with adjacent normal tissues from 14 clinical CRC patients. ( B ) Statistical analysis of the S T6GAL1 mRNA level in the CRC patients in A. ( C ) ST6GAL1 protein levels were measured in 14 clinical samples from CRC patients by WB. ( D ) Statistical analysis of the ST6GAL1 protein levels in CRC patients in C. ( E ) Representative images of HE staining and IHC staining of ST6GAL1 in colorectal tumor tissue and adjacent normal colorectal tissue from CRC patients (scale bars, 625 μm; scale bars, 50 μm). ( F ) Statistical results for the relative intensity of ST6GAL1 staining shown in E. ( G ) Statistical analysis of the correlation between ST6GAL1 mRNA expression and sensitivity to neoadjuvant therapy. ( H ) Representative images of IHC staining for ST6AGL1 in colorectal tumor tissue from neoadjuvant therapy-sensitive and nonsensitive CRC patients (scale bars, 625 μm; scale bars, 50 μm). ( I ) Statistical results for the relative intensity of ST6GAL1 staining shown in H. Statistical significance was determined using Student’s t test ( B , D , F and G )

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 expression is negatively correlated with the immune response in CRC. scRNA-seq data from public CRC databases were used to analyze ST6GAL1 expression in different cell subsets and its correlation with immune response pathways. ( A ) Uniform manifold approximation and projection (UMAP) plots showing the clusters of cells in the tumor tissue. ( B ) ST6GAL1 expression in different cell clusters. ( C ) GSEA of immune response-associated pathways between ST6GAL1 + cells and ST6GAL1 − cells in tumor tissues. ST6GAL1 expression and IFNG sensitivity in different CRC cell lines were further verified by RT‒qPCR and WB. ( D ) ST6GAL1 mRNA expression in the different CRC cell lines was quantified by RT‒qPCR. ( E ) ST6GAL1 protein levels were measured in 4 CRC cell lines using WB. ( F ) The mRNA levels of molecules associated with the IFNG response were measured by RT‒qPCR in different CRC cell lines treated with 100 ng/µl IFNG for 24 h

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Expressing

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 expression decreases IFNG sensitivity in CRC cell lines. After ST6GAL1 overexpression (OE) in the SW48 cell line (SW48-OE) and the HT29 cell line (HT29-OE) and ST6GAL1 knockdown (KD) in the Caco2 cell line (Caco2-KD) and the HT29 cell line (HT29-KD), as well as the construction of the related empty virus (EV) control cell lines SW48-EV, Caco2-KD and HT29-EV, the levels of molecules associated with the IFNG response were measured. ( A , B , C and D ) The mRNA levels of IFNG response-associated molecules were measured by RT‒qPCR in the SW48-EV and SW48-OE cell lines ( A ), the HT29-EV and HT29-OE cell lines ( B ), the Caco2-EV and Caco2-KD cell lines ( C ), and the HT29-EV and HT29-KD cell lines ( D ) after treatment with 100 ng/µl IFNG for 24 h. ( E , F , and G ) GBP1 and IRF1 protein levels in the SW48-EV and SW48-OE cell lines ( E ), the Caco2-EV and Caco2-KD cell lines ( F ), and the HT29-EV, HT29-OE and HT29-KD cell lines ( G ) were measured by WB after treatment with 100 ng/µl IFNG for 12 and 24 h

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Expressing, Over Expression, Knockdown, Virus, Control

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 overexpression promotes the immune escape of CRC tumor cells in vivo. An ST6GAL1 -OE MC38 cell line (MC38-OE) and the related empty virus (EV)-expressing control cell line MC38-EV were constructed. We used MC38-EV ( n = 4) and MC38-OE ( n = 4) cells to construct an animal model of subcutaneous tumorigenesis in wild-type C57BL/6 mice. When the tumor grew to an appropriate size, the tumor was removed, and the change in cell count was detected by flow cytometry. ( A ) Statistical analysis of the differences in the numbers of cells in tumors, draining lymph nodes and PBMCs between the ST6GAL1-EV and ST6GAL1-OE groups. ( B ) Flow cytometry analyses of CD3 + T, CD4 + T, CD8 + T and Treg cells in CD45-gated cells from tumor tissues from the ST6GAL1-EV and ST6GAL1-OE groups. ( C , D and E ) Statistical results for CD3 + T-cell numbers, CD4 + and CD8 + T-cell numbers ( D ) and Treg numbers ( E ) between the ST6GAL1-EV and ST6GAL1-OE groups. ( F , H and J ) Flow cytometry analyses of GZMB from CD8 + T cells ( F ), IFNG from CD4 + T cells ( H ) and IFNG from CD8 + T cells ( J ) in tumor tissues from the ST6GAL1-EV and ST6GAL1-OE groups. ( G , I and K ) Statistical results for GZMB from CD8 + T cells ( G ), IFNG from CD4 + T cells ( I ) and IFNG from CD8 + T cells ( K ) in tumor tissues from the ST6GAL1-EV and ST6GAL1-OE groups. Statistical significance was determined using Student’s t test ( A , C , D , E , G , I and K )

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Over Expression, In Vivo, Virus, Expressing, Control, Construct, Animal Model, Cell Counting, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: Multiomics analysis revealed that ST6GAL1 inhibits the activity of the IFNG signaling pathway. SW48-EV ( n = 3) and SW48-OE ( n = 3) cell lines were subjected to RNA-sequencing (RNA-seq) after treatment with 100 ng/µl IFNG for 24 h ( A , B and C ). ( A ) Volcano plot showing all genes expressed in the SW48-EV and SW48-OE cell lines. The x-axis shows the log 2 fold change (SW48-OE vs. SW48-EV cells), and the y-axis shows the -log 10 P value, which represents the threshold values in log transformation. Each dot represents a differentially expressed gene (DEG). The red dots indicate significantly upregulated DEGs, the blue dots indicate significantly downregulated DEGs, and the gray dots represent DEGs whose differential expression was not statistically significant. ( B ) Heatmap showing the relative expression of selected genes in three different functional modules (immune-related genes, inflammatory factor-related genes and IFNG response-related genes) in the S6TGAL1-OE cell line. ( C ) Gene set enrichment analysis (GSEA) of the SW48-OE cell line revealed an upregulated signaling pathway compared with that of the SW48-EV cell line. Kinase phosphorylation array detection of the SW48-EV and SW48-OE cell lines was subsequently performed ( D and E ). ( D ) Results of the IFNGR1-pTyr457 measurement via kinase phosphorylation array in the SW48-EV and SW48-OE cell lines. ( E ) KEGG pathway analysis of the differentially phosphorylated proteins in SW48-EV and SW48-OE cell lines identified via kinase phosphorylation array detection

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Activity Assay, RNA Sequencing, Transformation Assay, Quantitative Proteomics, Expressing, Functional Assay, Phospho-proteomics

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: ST6GAL1 regulates the IFNGR1/JAK1/STAT1 signaling pathway by affecting IFNGR1 and BICD2 binding. ( A , C and E ) WBs showing the levels of the pIFNGR1, IFNGR1, and pSTAT1 proteins in the SW48-EV and SW48-OE cell lines ( A ), the Caco2-EV and Caco2-KD cell lines ( C ), and the HT29-EV, HT29-OE and HT29-KD cell lines ( E ) after treatment with 100 ng/µl IFNG for 15 and 30 min ( B , D , and F ) Statistical results for the gray values shown in A ( B ), C ( D ) and E ( F ). ( G ) IP results for the HT29-EV and HT29-OE cell lines. ( H ) Immunofluorescence staining for SNA-FITC (green) and BICD2 (red) in the Caco2-EV and Caco2-KD cell lines. Nuclei were counterstained with DAPI (blue). Scale bars, 20 μm

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Binding Assay, Immunofluorescence, Staining

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: ST6GAL1-mediated sialylation inhibits the antitumor immune response in colorectal cancer

doi: 10.1007/s13402-025-01118-w

Figure Lengend Snippet: The mechanism by which ST6GAL1 promotes tumor immune evasion. ST6GAL1, an enzyme that adds sialic acid to proteins, is abundantly localized in the tumor microenvironment, where it inhibits IFNGR1 phosphorylation through the sialylation of BICD2, thereby suppressing the activation of the IFNGR1/JAK1/STAT1 signaling pathway. This interaction leads to resistance to the antitumor immune response and CRC development. *BICD2, BICD cargo adaptor 2; GBP1, guanylate binding protein 1; IDO1, indoleamine 2,3-dioxygenase 1; ISGs, IFN-stimulating genes; IRF, interferon regulatory factor; IFNG, interferon gamma; IFNGR, interferon gamma receptor; JAK, Janus kinase; NK, natural killer; STAT1, signal transducer and activator of transcription 1; ST6GAL1, ST6 β-galactoside α2,6-sialyltransferase; TH1, helper T

Article Snippet: Paraffin-embedded tissue sections were stained with antibodies against ST6GAL1 (AF5924; R&D Systems) and visualized using the Dako REALTM EnVisionTM detection system (Dako, K5007).

Techniques: Phospho-proteomics, Activation Assay, Binding Assay

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: Awd expression in tracheal cells. (A) Affinity-purified rabbit polyclonal antibody against Drosophila Awd recognizes a single band by Western blotting in whole embryo and Drosophila S2 cell lysates. (B) The same antibody was used to detect Awd expression (red) in vivo. The genotypes of the embryos are 1-eve-1 (wt); y, w; 1-eve-1, awdj2A4 (awd); and y, w; btl-GAL4, UAS-awd; 1-eve-1, awdj2A4 (UAS-awd + awd). Tracheal cells were visualized by mouse monoclonal anti-β-Gal staining (green). Arrows in the top left panel mark the anterior (a) and dorsal (d) sides of all the embryos shown. The top two rows of images are projections of five 1.5-μm confocal sections and the close-up images are single sections. The bottom two rows are projections of five 2-μm sections. The two channels were recorded sequentially to avoid bleed-through.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Expressing, Affinity Purification, Western Blot, In Vivo, Staining

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: awd mutant alleles display distinct tracheal phenotypes. Embryos were collected at 25°C and double stained using the 2A12 monoclonal antibody against a lumen antigen and a FITC-conjugated polyclonal antibody against β-Gal (red) (A–I), or double stained using the rat polyclonal anti-Trh antibody (green) and mouse monoclonal anti-β-GAL antibody (J–H). btl and the three awd mutants are homozygotes (see Materials and Methods). Stage 15–16 embryos are shown with anterior to the left. (A) Lateral view of a y, w embryo, representing wild type. Branches relevant to the following figures are marked. (DT) Dorsal trunk; (LT) lateral trunk; (DB) dorsal branch. (B) Dorsal view of a y, w embryo. (C) Lateral view of a btl embryo. There is very little tubule formation. (D) Lateral view of an awdj2A4 embryo. There is a general disruption of the tracheal network with ectopic branches emanating from the lateral trunk (marked between arrows in the enlarged section). Also, additional branches are seen sprouting out of the lateral region (bracket). (E) Dorsal–lateral view of an awdj2A4 embryo. Two ectopic branches are shown (arrowheads). (F) Lateral view of an awdKRS6 embryo. An ectopic “bulge” is indicated (arrowhead). (G) Dorsal–lateral view of an awdKRS6 embryo. The region exhibiting abnormal branching and looping is highlighted. (H) Dorsal–lateral view of an awdKRB embryo. One dorsal branch (DB) shows an ectopic loop (arrowhead) and two other dorsal branches form abnormal axial connection (arrow). (I) Dorsal–lateral view of an awdKRB embryo. The region exhibits ectopic branching and looping is indicated by a bracket. Also the dorsal trunk (DT) shows an abnormal 90° turn (arrow). (J) Lateral view of a y, w embryo showing nuclear expression of the Trh protein in all tracheal cells. (K) Lateral view of an awdj2A4 embryo. Arrows point to dorsal trunk (DT) cells that migrate ectopically (misallocated), resulting in gaps in dorsal trunk. (L) Lateral view of an awdKRS6 embryo. Arrows point to misallocated dorsal trunk (DT) cells and ectopic connection of two dorsal branches (DB). White bars, 50 μm; yellow bars, 20 μm.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Mutagenesis, Staining, Expressing

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: awd mutant exhibits ectopic tracheal cell migration in contrast to the btl phenotype. All embryos were collected at 25°C and stained with mouse monoclonal anti-β-Gal antibody. Identification of homozygous and heterozygous awd and btl is described in Materials and Methods. Unless noted, anterior is to the left and dorsal side is up. (A) Lateral view of a wild-type stage 14 embryo. One tracheal subunit is highlighted. Three tracheal branches relevant in the following figures are marked. (DB) Dorsal branches; (DT) dorsal trunk; (TC) transverse connectives. (B) Dorsal–lateral view of a wild-type stage 17 embryo showing fully extended tracheal tubes. (C) A dorsal close-up view of a wild-type stage 16 embryo showing connection of two dorsal branches across the dorsal midline (arrow). (D) Dorsal view of a stage 16 homozygous awdj2A4 embryo showing disrupted dorsal trunk (DT) and sprouts of ectopic branches (sharp arrows). The first compartment of the properly formed midgut can be seen in this view (the light, rounded internal structure in the middle). (E) Dorsal–lateral view of a stage 17 heterozygous awdj2A4 embryo. A segment of the dorsal trunk moves ventrally into the position of transverse connective (bracket) instead of connecting with the lateral neighbor. Ectopic small cellular processes are seen sprouting from the abnormal branches (sharp arrows in the insert). (F) Dorsal–lateral view of a stage 16 heterozygous awdj2A4 embryo. The highlighted region shows two dorsal branches fused at the base but separate at the tip (asterisk) as well as misconnection of two neighboring dorsal branches (sharp arrow), instead of crossing the dorsal midline. (G) Close-up view of dorsal branches of a wild-type embryo. Arrows point to the few filopodia extending from the tip cells of the dorsal branches. (H) Close-up view of dorsal branches of a heterozygous awdj2A4 embryo. Multiple, random cellular projections extend from the tip cells (sharp arrows). (I) Lateral view of a stage 14 homozygous btlH82Δ3 embryo. There is very little tubule migration as compared to the wild type. (J) Dorsal–lateral view of a stage 16 heterozygous btlH82Δ3 embryo. The highlighted region shows lack of dorsal branch formation. (K) Quantitation of the recorded phenotypes as shown in A–J. The awd and btl alleles are awdj2A4 and btlH82Δ3. Note that to avoid complications from early embryonic patterning defects, only embryos older than stage 13 that showed proper germband retraction and, after stage 15, proper gut formation, were tabulated. Construction of the btl-GAL4, UAS-awd rescue chromosome is described in Materials and Methods.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Mutagenesis, Migration, Staining, Quantitation Assay

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: shi mutant shows migration phenotype similar to awd. Progenies of shi1 homozygous females crossed with 1-eve-1 males (A–D) and of shi1 homozygous females crossed with 1-eve-1, awdj2A4/TM3, P{ry + t7.2 = HZ2.7}DB2, Sb males (E). The embryos were collected at 25°C for 7 h, then incubated for 7 h at 34°C and stained with anti-β-Gal antibody. Unless indicated, anterior is to the left and dorsal side is up. (A) Dorsal–lateral view. A sharp arrow points to the ectopic branching event. Also, abnormal cellular projections are seen extending from the dorsal branch tip cells (arrows in the inset). (B) Lateral view. A segment of the dorsal trunk is seen moving ventrally (bracket) into the position normally occupied by transverse connectives, instead of anteriorly to connect with the lateral neighbor. (C) Dorsal–lateral view. A group of tracheal cells move dorsally (bracket) at the expense of forming a proper dorsal trunk connection. Ectopic cellular extensions are also visible (arrow). (D) Lateral view. An extra loop of dorsal trunk is formed (bracket) with ectopic small projections (arrows). (E) Dorsal view of severely disrupted trachea. The enlarged section shows ectopic cellular projections and randomly migrating tracheal cells (arrows). (F) Quantitation of the recorded phenotypes as shown in A–E. There is a nearly 30-fold increase in the most severe phenotype (E) when shi is combined with heterozygous awd mutant. Only embryos older than stage 13 that appear normal in segmental patterns were tabulated as described in Figure 3K.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Mutagenesis, Migration, Incubation, Staining, Quantitation Assay

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: Overaccumulation of Btl/FGFR in awd mutant. The genotypes of the indicated embryos are y, w; btl-GAL4/+; 1-eve-1, UAS-btl-GFP/+ (wt) and y, w; btl-GAL4/+; 1-eve-1, UAS-btl-GFP, awdj4A2/+ (awd/+). Embryos were double stained with anti-β-Gal (red) and anti-GFP (green) antibodies. The two channels were recorded sequentially. Anterior is to the left. (A) Dorsal–lateral view of a wild-type stage 14 embryo. A projection of five 2-μm confocal sections. The Btl-GFP chimeric protein is barely detectable. In the close-up image (single confocal section), circles mark individual tracheal cells and sharp arrows point to examples of internalized Btl-GFP. (B) Lateral view of portion of a wild-type stage 12 tracheal subunit. Circles mark individual tracheal cells and sharp arrows point to examples of internalized Btl-GFP. Single confocal section. (C) Single confocal section of a wild-type stage 15 dorsal branch tip cell. Sharp arrows point to examples of internalized Btl-GFP. (D) Stage 13 awd/+ tracheal subunits. A projection of five 1-μm confocal sections. In the close-up image, arrows point to examples of large aggregates of Btl-GFP. (E) Lateral view of three dorsal branches from a stage 15 awd/+ embryo. A projection of five 1-μm confocal sections. Abnormal migration (sharp arrows), ectopic cellular projections (asterisk), and high level of Btl-GFP are seen. In the close-up images (single confocal section), Btl-GFP is seen outlining a very fine cellular projection (bracket). Note that the close-up section is only partially included in the projected image. (F) Dorsal view of a stage 17 awd/+ embryo. A projection of five 2-μm confocal sections. The total disruption of the tracheal system is rarely seen in the awd heterozygotes. The properly formed midgut can also be seen beneath the abnormal tracheal branches (red auto-fluorescence). The enlarged view (GFP stain only) shows high level of Btl-GFP expression and ectopic cellular projections (arrows). (G) Western blot quantitation of the Btl-GFP accumulation. Embryonic extracts from awd/+ and wt embryos as described above were Western blotted and probed with antibodies indicated. Two independent lines of 1-eve-1, UAS-btl-GFP, awdj2A4 were examined. Because the btl-GFP and the 1-eve-1 transgenes are located on the same chromosome, β-Gal was used as a loading control. Btl-GFP is up-regulated by approximately fivefold whereas there is no concomitant increase in EGFR (a slight decrease, in fact) or the total MAPK levels in the awd mutant.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Mutagenesis, Staining, Migration, Fluorescence, Expressing, Western Blot, Quantitation Assay

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: Ectopic activation of MAPK in awd mutant. 1-eve-1 (wt) and awdj2A4, 1-eve-1/1-eve-1 (awd/+) stage 12 and stage 15 embryos were double stained with anti-β-Gal-FITC (green) and anti-dp-MAPK (red) antibodies. The two channels were recorded sequentially. Anterior is to the left. (A) Tracheal subunits of a stage 12 wild-type embryo. A projection of five 1.5-μm confocal sections. Within the tracheal placodes, activated MAPK are detected in the two tips cells marked by circles. Lighter MAPK activation is also detected on the opposite tip (arrow). Activated MAPK-expressing cells surrounding the tracheal placodes are of mesodermal origin, including cardiac precursors. (B) One dorsal branch of a wild-type stage 15 embryo. A single confocal section. Only one cell at the tip contains activated MAPK. (C) Tracheal subunits of a stage 12 awd heterozygote. A projection of five 1.5-μm confocal sections. Multiple cells besides the migrating tips within the tracheal placodes express activated MAPK (arrows). (D) Two laterally fused dorsal branches of a stage 15 awd heterozygote. A projection of five 1.5-μm confocal sections. The stalks (brackets) ectopically express activated MAPK. Bars, 10 μm.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Activation Assay, Mutagenesis, Staining, Expressing