Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Galectin‐3 expression is abundant in human and mouse atherosclerotic lesions. (A) Ten main cell types are visualised in atherosclerotic core (AC) and proximal adjacent (PA) tissues by tSNE (t‐distributed stochastic neighbour embedding). (B) The macrophage population significantly increased in AC relative to PA. (C) Biaxial scatter plots show the expression pattern of galectin‐3 in total cell types between AC and PA. The colour scale represents expression levels in biaxial scatter plots (grey: low; pink: high). (D) Galectin‐3‐positive macrophages expanded in AC in comparison with PA. (E) Five macrophage subtypes are visualised in AC and PA tissues by tSNE. (F) My.0 and My.1 account for 34.1% and 47.6% of macrophages in AC, respectively. My.2 significantly increased in AC relative to PA. (G) Biaxial scatter plots exhibit the expression pattern of galectin‐3 in macrophage subtypes between AC and PA. (H) Galectin‐3‐positive My.0 and My.1 account for 35.8% and 47.5% of galectin‐3‐positive macrophages in AC, respectively. Galectin‐3‐positive My.2 expands in AC in comparison with PA. (I) Representative Western blots and relative quantitative analysis of galectin‐3 in human atherosclerotic lesions and peripheral normal artery. (J) Triple immunofluorescence staining for galectin‐3 (red), NLRP3 (green), CD68 (pink) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals the colocalisation of galectin‐3 and NLRP3 in CD68‐positive macrophages. Scale bar: 50 µm. (K) Representative Western blots and relative quantitative analysis of galectin‐3 in the aortas of ApoE −/− mice fed with an HFD or normal diet. (L) Triple immunofluorescence staining for galectin‐3 (red), NLRP3 (green), CD68 (pink) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals that galectin‐3 and NLRP3 are colocalised in CD68‐positive macrophages. Scale bar: 50 µm. (M) Cell lysates from ox‐LDL‐treated macrophages are immuno‐precipitated with anti‐galectin‐3 or anti‐NLRP3 antibodies, and blotted with anti‐NLRP3 or anti‐galectin‐3 antibodies. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: The whole cell lysates were sonicated and centrifuged at 12 000 × g for 15 min. Then the supernatant was incubated overnight with two antibody combinations: (i) anti‐NLRP3 (30109‐1‐AP, Proteintech) and anti‐galectin 3 (60207‐1‐Ig, Proteintech); (ii) anti‐TLR4 (19811‐1‐AP, Proteintech) and anti‐MyD88 (67969‐1‐Ig, Proteintech).

Techniques: Expressing, Comparison, Western Blot, Immunofluorescence, Staining, Derivative Assay

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

doi: 10.1016/j.alit.2021.07.012

Figure Lengend Snippet: Fig. 1. Galanin (Gal) and Gal receptor (Galr)1e3 mRNA expression in nasal mucosa. Gal and Galr1e3 expression levels were analyzed using RT-PCR. In both severe allergic rhinitis (AR) and control mice (Groups D and A, respectively), Gal and Galr2 were present in nasal mucosa (n ¼ 2) and brain tissue samples (n ¼ 1). Hypoxanthine-guanine phosphor- ibosyltransferase (Hprt) was used as a control.

Article Snippet: After washing with TBS-T, the sections were incubated for 20 min with 5% goat or donkey serum diluted with TBS-T, followed by treatment with a primary GALR2 polyclonal rabbit antibody (1:500; 26459-1-AP; Proteintech, Rosemont, IL, USA) or a GAL Please cite this article as: Kawada M et al., Involvement of galanin and International, https://doi.org/10.1016/j.alit.2021.07.012 polyclonal goat antibody (1:50; OAEB01591; Aviva Systems Biology, San Diego, CA, USA) at 4 C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

doi: 10.1016/j.alit.2021.07.012

Figure Lengend Snippet: Fig. 2. Galanin (GAL) and GAL receptor (GALR) 2 protein expression in nasal mucosa. GAL and GALR2 levels were analyzed by western blot and similar between Groups AeD (n ¼ 8/ group). NAK, NaK-ATPase membrane protein.

Article Snippet: After washing with TBS-T, the sections were incubated for 20 min with 5% goat or donkey serum diluted with TBS-T, followed by treatment with a primary GALR2 polyclonal rabbit antibody (1:500; 26459-1-AP; Proteintech, Rosemont, IL, USA) or a GAL Please cite this article as: Kawada M et al., Involvement of galanin and International, https://doi.org/10.1016/j.alit.2021.07.012 polyclonal goat antibody (1:50; OAEB01591; Aviva Systems Biology, San Diego, CA, USA) at 4 C overnight.

Techniques: Expressing, Western Blot, Membrane

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

doi: 10.1016/j.alit.2021.07.012

Figure Lengend Snippet: Fig. 3. Galanin (GAL) and GAL receptor (GALR)2 localization in nasal mucosa. (a, b) Representative photomicrograph showing immunohistochemical analysis of GAL and GALR2 localization in nasal mucosa sections in Groups AeD. A, B, C, and D indicate Group A, Group B, Group C, and Group D, respectively. Controls were only stained with (a) anti-goat and (b) anti-rabbit secondary antibody without exposure to primary antibody. Scale bars, 25 mm. (c) Representative photomicrograph showing the results of double staining for GAL (green) and GALR2 (red) in nasal mucosa sections in all groups. GAL and GALR2 were simultaneously visualized in ciliated epithelial (arrows) and submucosal gland cells (ar- rowheads). Scale bars, 50 mm. HE, hematoxylin-eosin.

Article Snippet: After washing with TBS-T, the sections were incubated for 20 min with 5% goat or donkey serum diluted with TBS-T, followed by treatment with a primary GALR2 polyclonal rabbit antibody (1:500; 26459-1-AP; Proteintech, Rosemont, IL, USA) or a GAL Please cite this article as: Kawada M et al., Involvement of galanin and International, https://doi.org/10.1016/j.alit.2021.07.012 polyclonal goat antibody (1:50; OAEB01591; Aviva Systems Biology, San Diego, CA, USA) at 4 C overnight.

Techniques: Immunohistochemical staining, Staining, Double Staining

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: Molecular Therapy. Methods & Clinical Development

Article Title: Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells

doi: 10.1016/j.omtm.2021.10.010

Figure Lengend Snippet: AAV-CAR Tregs suppress capsid-specific immune response in vivo (A) Expression of hAAT protein in the serum of animals injected i.m. with rh32.33-AAT followed by AAV-CAR Tregs (red), polyclonal Tregs (orange), or PBS (blue) as measured by ELISA (n = 6 for AAV-CAR Treg and PBS-treated groups, n = 5 for polyclonal Treg-treated group). (B) Anti-rh32.33 antibodies in the serum measured by ELISA. Anti-capsid antibodies increased in all groups over time (n = 6 for AAV-CAR Treg group, n = 7 for PBS group, n = 5 for polyclonal Treg-treated group). (C) Serum level of free active TGF-β1 measured by CBA assay over time. A significant increase in free active TGF-β1 was observed in AAV-CAR Treg group (red) at week 11 post injection compared with the PBS control group (blue). (D) Serum levels of IL-10 measured by CBA assay over time. Two-way ANOVA repeated measure with Tukey's multiple comparisons was used (n = 6 for AAV-CAR Treg and PBS-treated groups, n = 5 for polyclonal Treg treated group for C and D). (E) Quantification of number nuclei in right limb muscles of animals 2 weeks after AAV injection. Each dot represents one animal, which is the average of 10 images per animal (HPF, high-power field). One-way ANOVA, n = 3. Error bars are mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01. (F) Representative images of rh32.33-AAT muscles 2 weeks post injection stained for H&E for cellular infiltration. (Upper) scale bar, 527 μm; (lower) scale bar, 131 μm. (G) Representative images of rh32.22-AAT-injected muscles stained by immunohistochemistry for AAT protein (brown), 26 weeks after AAV injection. (Upper) scale bar, 527 μm; (lower) scale bar, 131 μm. ∗AAV-CAR Tregs compared with PBS; #AAV-CAR Tregs compared with polyclonal Tregs; ˆpolyclonal Tregs compared with PBS.

Article Snippet: After washing two times, peroxidase-conjugated rabbit anti-OVA polyclonal antibody (Rockland Immunochemicals, Limerick, PA) (1:5,000 diluted) was added and incubated for 1 h at room temperature.

Techniques: In Vivo, Expressing, Injection, Enzyme-linked Immunosorbent Assay, Control, Muscles, Staining, Immunohistochemistry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells

doi: 10.1016/j.omtm.2021.10.010

Figure Lengend Snippet: AAV-CAR Tregs bystander suppress immune response to AAV-delivered transgene (A) Expression of OVA protein from animals i.m. injected with AAV1-OVA followed by i.v. AAV-CAR Tregs (red), polyclonal Tregs (orange), or PBS (blue) as measured by ELISA. Serum levels of OVA were significantly greater in AAV-CAR Treg-treated animals compared with PBS. (B) Anti-OVA antibodies detected in the serum by ELISA. (C) Anti-AAV1 antibodies in the serum measured by ELISA. (D) Serum levels of IL-10 measured by CBA assay. (E) Serum level of free active TGF-β1 measured by CBA assay over time. Two-way repeated-measure ANOVA with Tukey's multiple comparisons was used (n = 5 for AAV-CAR Treg and polyclonal Treg-treated groups, n = 3 for the PBS treated group for A, B, C, D, and E). (F) Quantification of number of nuclei in i.m. injected muscles. Each dot represents one animal, which is the average of 10 images per animal. (G) Representative images of H&E-stained limb muscles of mice 16 weeks post i.m. injection with AAV1-OVA. Substantial cellular infiltration (blue) was observed in animals treated with PBS or polyclonal Tregs. (Upper) scale bar, 527 μm; (lower) scale bar, 131 μm. one-way ANOVA, n = 2. Error bars are mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. ∗AAV-CAR Tregs compared with PBS.-CAR Tregs compared with PBS.

Article Snippet: After washing two times, peroxidase-conjugated rabbit anti-OVA polyclonal antibody (Rockland Immunochemicals, Limerick, PA) (1:5,000 diluted) was added and incubated for 1 h at room temperature.

Techniques: Expressing, Injection, Enzyme-linked Immunosorbent Assay, Muscles, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells

doi: 10.1016/j.omtm.2021.10.010

Figure Lengend Snippet: Injection of AAV-CAR Tregs reduced IFN-γ and increased IL-10 production in muscular but not splenic immune cells after ex vivo isolation and restimulation (A) Experimental schematic. (B) Levels of IFN-γ in muscle after ex vivo isolation and restimulation measured by CBA assay. (C) Levels of IL-4 in muscle after ex vivo isolation and restimulation measured by CBA assay. (D) Levels of IL-10 in muscle after ex vivo isolation and restimulation measured by CBA assay. (E) Levels of free active TGF-β1 in muscle after ex vivo isolation and restimulation measured by CBA assay. Two-way ANOVA with Tukey's multiple comparisons was used. Error bars are mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001 (n = 3 for AAV-CAR Treg and polyclonal Treg-treated groups, n = 2 for the PBS-treated group for B, C, D and E).

Article Snippet: After washing two times, peroxidase-conjugated rabbit anti-OVA polyclonal antibody (Rockland Immunochemicals, Limerick, PA) (1:5,000 diluted) was added and incubated for 1 h at room temperature.

Techniques: Injection, Ex Vivo, Isolation