Journal: JCI Insight

Article Title: Circumventing cellular immunity by miR142-mediated regulation sufficiently supports rAAV-delivered OVA expression without activating humoral immunity

doi: 10.1172/jci.insight.99052

Figure Lengend Snippet: AAV1 injections were carried out on day 0 followed by sera collection every 2 weeks for a 12-week period. (A) qPCR detection of rAAV vector genome copies (GCs) in spleen, heart, liver, and injected skeletal muscle (n = 2). (B and C) ELISA quantification of circulating OVA expression (B) and anti-OVA IgG1 (C) of mice i.m. injected with rAAV1.OVA or rAAV1.OVA.miR142BS vectors (1 × 1011 GCs/mouse, n = 10). Sera were collected throughout a 12-week period. Box plots with whiskers represent mean ± SD and maximum and minimum values (n = 5). **P < 0.01, ***P < 0.001, ANOVA with Tukey’s post hoc test.

Article Snippet: After washing 4 times, peroxidase-conjugated rabbit anti-OVA polyclonal antibody (200-4333-0100, Rockland Immunochemicals) (1:5000 diluted) was added and incubated for 1 hour at room temperature.

Techniques: Plasmid Preparation, Injection, Enzyme-linked Immunosorbent Assay, Expressing

Journal: JCI Insight

Article Title: Circumventing cellular immunity by miR142-mediated regulation sufficiently supports rAAV-delivered OVA expression without activating humoral immunity

doi: 10.1172/jci.insight.99052

Figure Lengend Snippet: Timeline for the AAV-redosing experiment (A) and the description of the rAAV serotype and OVA transgene specific for each injection (B). C57BL/6 male mice, 6 weeks old, were injected i.m. with either rAAV1.OVA or rAAV1.OVA.142BS (1 × 1011 GCs/mouse). The same mice were then redosed with either rAAV8.OVA or rAAV8.OVA.142BS (1 × 1011 GCs/mouse) i.m. 4 weeks after the first injection (n = 5). (C and D) ELISA quantification of circulating OVA expression (C) and anti-OVA IgG (D) in vector-injected mice. Sera were collected throughout a 12-week period. Box plots with whiskers correspond to mean ± SD and maximum and minimum values. P values were determined by ANOVA with Tukey’s post hoc test for pairwise comparison. Asterisks (*) denote test of significance between injection groups 1 and 3, and hashes (#) denote test of significance between injection groups 2 and 4. P values for longitudinal comparisons were determined by repeated measures with 2-way ANOVA. Daggers (†) denote test of significance. *,#P < 0.05, **,##P < 0.01, ***,###,†††P < 0.001.

Article Snippet: After washing 4 times, peroxidase-conjugated rabbit anti-OVA polyclonal antibody (200-4333-0100, Rockland Immunochemicals) (1:5000 diluted) was added and incubated for 1 hour at room temperature.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Expressing, Plasmid Preparation

Journal: Cancer Genomics & Proteomics

Article Title: Exosomes Transfer ST6GAL1-mediated Therapeutic Resistance in Rectal Cancer Cells

doi: 10.21873/cgp.20558

Figure Lengend Snippet: T6GAL1 containing exosomes are produced by human colorectal cancer cells in culture. A) Western blot of exosome lysates probed for ST6GAL1 and β-actin. B) NanoSight tracing of a representative sample of exosomes isolated from SW620 ST6GAL1 control vector (CV) cells. C) Transmission electron microscopy of a representative sample of exosomes isolated from SW620 CV cells. Purple arrows indicate some of the exosomes with a classic umbilicated appearance. D) Western blot of whole cell extract and exosome lysate for exosome negative marker Calnexin, exosome positive markers CD9, CD63, CD81, TSG101, Alix, Flotillin 1, and HSP70, and ST6GAL1. WCE: Whole cell extract; Exo: exosomes.

Article Snippet: Blots were probed with an antibody against ST6GAL1 (R&D Systems, Minneapolis, MN, USA, #AF5924) at 1:250 dilution.

Techniques: Produced, Western Blot, Isolation, Control, Plasmid Preparation, Transmission Assay, Electron Microscopy, Marker

Journal: Cancer Genomics & Proteomics

Article Title: Exosomes Transfer ST6GAL1-mediated Therapeutic Resistance in Rectal Cancer Cells

doi: 10.21873/cgp.20558

Figure Lengend Snippet: ST6GAL1 protein is transferred via exosomes to ST6GAL1 deficient cells. A ) Western blot of lysates of ST6GAL1 control vector (CV) or knockdown (KD) or ST6GAL1 KD cells treated with ST6GAL1+ exosomes for 3 h. Representative of 3 independent experiments. B) qPCR for ST6GAL1 in mRNA isolated from ST6GAL1 CV cells, ST6GAL1 KD, and KD plus ST6GAL1+ exosomes for 3 h. Normalized to GAPDH and fold change calculated relative to CV; CV vs. KD p<0.05, CV vs. KD + exo p<0.05, (analyzed using one-way ANOVA with Dunnett’s post hoc test) mean±standard deviation (SD), n=3 independent experiments. C) Immunofluorescence microscopy staining for ST6GAL1 (red) in the indicated panels, DAPI (blue) in all panels, and a Golgi marker (green) in the 2 right panels on ST6GAL1 KD cells treated with or without exosomes. Cells were treated with or without exosomes and washed prior to fixation and staining. Representative of 3 independent experiments. Exo: ST6GAL1+ exosomes isolated from SW620 cells, CV and KD refer to the ST6GAL1 genotype

Article Snippet: Blots were probed with an antibody against ST6GAL1 (R&D Systems, Minneapolis, MN, USA, #AF5924) at 1:250 dilution.

Techniques: Western Blot, Control, Plasmid Preparation, Knockdown, Isolation, Standard Deviation, Immunofluorescence, Microscopy, Staining, Marker

Journal: Cancer Genomics & Proteomics

Article Title: Exosomes Transfer ST6GAL1-mediated Therapeutic Resistance in Rectal Cancer Cells

doi: 10.21873/cgp.20558

Figure Lengend Snippet: Exosomes transfer enzymatically active ST6GAL1, which confers treatment resistance after CRT. A) Gating strategy for ST6Gal1 knockdown (KD) cells flow cytometry. 7AAD used for live-dead staining. B) Flow cytometry one parameter histogram overlay of cell surface SNA binding (specific for α2,6-sialic acid). Cells harvested 3 h after treatment with ST6GAL1+ exosomes. Representative of 3 independent experiments. C) Colony formation assay with colony count normalized to KD. Veh KD + exo vs. CRT KD + exo, p<0.01, CRT KD vs. CRT KD + exo, p<0.05, mean±standard deviation (SD), n=4 independent experiments. D) IF microscopy for cleaved caspase-3 (CC3) on CV, KD, and KD + exo. Cells were pre-treated with ST6GAL1+ exosomes for 24 h and then treated with Veh or CRT, fixed 24 h later and stained. DAPI is blue and CC3 is red. Representative images from 4 independent experiments. E) Quantitative analysis of IF for CC3 reporting signal intensity for CC3 normalized to DAPI to control for number of cells. CRT KD vs. CRT KD + exo, p<0.05, mean ± SD, n=4 independent experiments. CV and KD refer to the ST6GAL1 genotype. Exo: ST6GAL1+ exosomes; CRT: chemoradiation; IF: immunofluorescence staining; SNA: Sambucus nigra lectin; Veh: vehicle (DMSO).

Article Snippet: Blots were probed with an antibody against ST6GAL1 (R&D Systems, Minneapolis, MN, USA, #AF5924) at 1:250 dilution.

Techniques: Knockdown, Flow Cytometry, Staining, Binding Assay, Colony Assay, Standard Deviation, Microscopy, Control, Immunofluorescence

Journal: Cancer Genomics & Proteomics

Article Title: Exosomes Transfer ST6GAL1-mediated Therapeutic Resistance in Rectal Cancer Cells

doi: 10.21873/cgp.20558

Figure Lengend Snippet: More cells display ST6GAL1 protein activity than ST6GAL1 mRNA expression in a human colorectal cancer organoid. A) Gating strategy for organoid cell flow cytometry and cell sorting. 7AAD used for live-dead staining. Live dead control included with fraction of heat-killed cells. B) Flow cytometry one parameter histogram overlay of cell surface SNA binding (specific for α2,6-sialic acid). 86% of cells are positive for SNA. Green: full test panel, purple: fluorescence minus one control (excludes SNA), and gray: unstained control. C) UMAP plot of 3272 CRC organoid cells with 5 tumor clusters labeled, based on gene expression. D) Distribution of ST6GAL1 gene expression among different tumor clusters in the CRC organoid cells. 16% of cells contain ST6GAL1 mRNA. Red color represents relative gene expression. FMO: Fluorescence minus one staining control; Unst: unstained control; FSC: forward scatter; SSC: side scatter; UMAP: Uniform Manifold Approximation and Projection; SNA: Sambucus nigra lectin.

Article Snippet: Blots were probed with an antibody against ST6GAL1 (R&D Systems, Minneapolis, MN, USA, #AF5924) at 1:250 dilution.

Techniques: Activity Assay, Expressing, Flow Cytometry, FACS, Staining, Control, Binding Assay, Fluorescence, Labeling, Gene Expression

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