Journal: Autophagy

Article Title: Periplocin suppresses the growth of colorectal cancer cells by triggering LGALS3 (galectin 3)-mediated lysophagy.

doi: 10.1080/15548627.2023.2239042

Figure Lengend Snippet: Figure 6. Periplocin binds and prevents ubiquitin-mediated degradation of LGALS3 in CRC cells. (A) Immunoblotting analysis of LGALS3 in cells treated with periplocin for 24 h at the indicated concentrations. (B and C) Representative images (B) and quantitative analysis (C) of immunohistochemical staining for LGALS3 in SW480 ×enografts from vehicle- or periplocin-treated mice. Scale bar: 50 μm. (D) Immunoblotting analysis of LGALS3 in cells treated with 0.50 μM periplocin for 24 h in the presence or absence of cycloheximide (CHX, 50 μg/mL). (E) Quantitation of LGALS3 protein level in (D). (F) Immunoblotting analysis of LGALS3 in cells treated with 0.50 μM periplocin for 24 h in the presence or absence of MG132 (25 μM, 6 h). (G) Quantitation of LGALS3 protein level in (F). (H) FLAG-LGALS3 was co-expressed

Article Snippet: LGALS3 (MAB1154) was purchased from R&D Systems.

Techniques: Ubiquitin Proteomics, Western Blot, Immunohistochemical staining, Staining, Quantitation Assay

Journal: Autophagy

Article Title: Periplocin suppresses the growth of colorectal cancer cells by triggering LGALS3 (galectin 3)-mediated lysophagy.

doi: 10.1080/15548627.2023.2239042

Figure Lengend Snippet: Figure 7. Periplocin induces lethal lysophagy by upregulating LGALS3 in CRC cells. (A and B) Immunoblotting analysis of LC3B turnover in cells transfected with siNC or siLGALS3 followed by 0.50 μM periplocin treatment for 24 h. (C and D) Immunoblotting analysis of LC3B turnover in parental or lgals3 KO cells followed by 0.50 μM periplocin treatment for 24 h. (E and F) Representative images (E) and quantitative analysis (F) for immunofluorescent staining of endogenous LC3B puncta in cells transfected with siNC or siLGALS3 followed by 0.50 μM periplocin treatment for 24 h. Scale bar: 10 μm. (G and H) MTT assay of CRC cells with or without LGALS3 knockout (G, lgals3 KO#1; H, lgals3 KO#2) in response to 0.50 μM periplocin treatment for 24 h. (I) Colony formation assay of parental or lgals3 KO cells treated with or without 0.50 μM periplocin for 24 h. (J) Quantification of clone numbers in (I). (K and L) DLD-1 parental or lgals3 KO cells were subcutaneously inoculated into nude mice. Mice were injected with vehicle or periplocin (15 mg/kg/day) for two weeks. Image (K) and weight (L) of tumor xenografts were shown. Results in A-J are representative of three independent experiments. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant.

Article Snippet: LGALS3 (MAB1154) was purchased from R&D Systems.

Techniques: Western Blot, Transfection, Staining, MTT Assay, Knock-Out, Colony Assay, Injection

Journal: The Journal of Clinical Investigation

Article Title: ADAMTS7 promotes smooth muscle foam cell expansion in atherosclerosis

doi: 10.1172/JCI187451

Figure Lengend Snippet: ( A ) Schematic outlining experimental design and the generation of the SMC transgenic ADAMTS7 mouse. Created with BioRender.com. ( B ) ORO staining of the en face aorta and quantification of lesion area. n = 9. ( C ) Representative images of H&E-stained aortic root sections, quantification of plaque areas ( n = 6). Scale bar: 500 μm. ( D ) Representative images of H&E-stained aortic root sections with necrotic core outlined in dotted line ( n = 6 mice). Scale bar: 300 μm. ( E ) Representative images of aortic root sections stained against α-SMA (Cy3; red), MAC2 (Alexa Fluor 488 green), and cell nuclei (DAPI blue) with their subsequent quantification relative to lesion area ( n = 6). Scale bar: 300 μm. ( F ) Representative Picrosirius red staining of aortic root lesions with bars indicating fibrous cap thickness and quantification of fibrous cap thickness ( n = 6). Scale bar: 300 μm. ( G ) Schematic outlining experimental design and the generation of the EC transgenic ADAMTS7 mouse. Created with BioRender.com. ( H ) ORO staining of the en face aorta and quantification of lesion area. n = 3–4. ( I ) Representative images of H&E-stained aortic root sections and quantification of plaque areas ( n = 5–6). Scale bar: 500 μm. **** P < 0.0001, * P < 0.05. Statistics were analyzed using a 2-tailed Student’s t test.

Article Snippet: The sections then were stained using the following Abs and dilutions: 1:1,000 α-SMA-Cy3 (MilliporeSigma, C6198) and 1:500 MAC2 (Cedarlane, CL8942AP).

Techniques: Transgenic Assay, Staining

Journal: Journal of the American Heart Association

Article Title: Smooth Muscle Cell‐Derived Fibronectin Promotes an Atheroprotective Smooth Muscle Cell Phenotype Associated With Altered NO‐cGMP Signaling

doi: 10.1161/jaha.124.040395

Figure Lengend Snippet: Figure 3. Analysis of modulated SMCs in atherosclerotic aortae. A, UMAP visualization of selected cell clusters (SMC 2, mod-SMC, and MΦ) from atherosclerotic aortae of control and Fn-smko mice shown in Figure 2A. B, Distribution of cells expressing Gucy1a1 (left bar graph) and Gucy1b1 (right bar graph) mRNA across the 3 clusters shown in A. C, Representative Western blot detection (left) and semiquantitative analysis (right bar graphs) of Fn1, Gucy1b1, Prkg1, and Acta2 in atherosclerotic aortae from 1-year-old control (n=3) and Fn-smko (n=3) mice on chow diet. Gapdh was used as a loading control. The position of molecular weight markers in kDa is shown next to the Western blots. D, Immunostaining of Gucy1b1 of aortic arch sections from mice fed 18 weeks with HFD (left, black arrows indicate examples of positive cells) and quantification of Gucy1b1+ cells in the lesion core (right bar graph) (control n=12 lesions from 4 mice; Fn-smko n=16 lesions from 6 mice). E, Immunostaining of Lgals3 in parallel sections of those shown in (D) (left, black arrows indicate some positive cells) and quantification of Lgals3+ cells in the lesion core (right bar graph) (control n=23 lesions from 6 mice; Fn-smko n=27 lesions from 7 mice). Scale bars in (D) and (E), 100 μm. Black lines in (D) and (E) indicate the lesion with the fibrous cap outlined as the 30-μm layer of the lesion adjacent to the vascular lumen. F, Plaque stability determined by the ratio of Acta2+ over Lgals3+ lesion area measured in parallel sections (control n=25 lesions from 6 mice; Fn-smko n=27 lesions from 7 mice). Note that the control group contained 1 data point >5. This data point is not shown in the bar graph but included in statistical analysis. Data are shown as mean±SEM. *P<0.05 by unpaired t test (C) or Mann–Whitney U test (D through F). Acta2 indicates alpha smooth muscle actin; C, core; Ctrl, control; FC, fibrous cap; Fn-smko, smooth muscle-specific deletion of fibronectin; Gucy1a1, alpha1 subunit of NO-GC; Gucy1b1, beta1 subunit of NO-GC; HFD, high-fat diet; Lgals3, galectin 3; MΦ, macrophage; mod-SMC, modulated SMC; Prkg1, cGMP-dependent protein kinase I; SMC, smooth muscle cell; and UMAP, uniform manifold approximation and projection.

Article Snippet: Paraffin- embedded sections of left anterior descending arteries from previously described autopsy material38 were stained with hematoxylin/ eosin or immunostained with antibodies recognizing human LGALS3 (R&D Systems, AF1154, from goat) or GUCY1B1 (Abcam, ab24824, from rabbit).

Techniques: Control, Expressing, Western Blot, Molecular Weight, Immunostaining, MANN-WHITNEY

Journal: Journal of the American Heart Association

Article Title: Smooth Muscle Cell‐Derived Fibronectin Promotes an Atheroprotective Smooth Muscle Cell Phenotype Associated With Altered NO‐cGMP Signaling

doi: 10.1161/jaha.124.040395

Figure Lengend Snippet: Figure 4. NO-GC expression in modulated SMCs of murine atherosclerotic plaques. A, Immunofluorescence staining of Gucy1b1 (red) and Lgals3 (green) in an atherosclerotic lesion. Scale bars, 100 μm. The framed area is shown at higher magnification in the adjacent image on the right. White arrowheads indicate Gucy1b1/Lgals3 coexpressing cells (yellow). Scale bar, 10 μm. B, Lineage tracing experiment (for experimental details, see Methods section). SMCs in the aortic media were genetically pulse labeled before the onset of atherosclerosis resulting in permanent expression of β-galactosidase in the originally labeled cells as well as in their progeny. After the development of atherosclerotic plaques, SMC-derived cells were visualized by X-Gal staining of the aorta (blue). Lgals3 (brown, left) and Gucy1b1 (brown, right) were detected by immunohistochemical staining of X-Gal-stained plaque sections. Scale bars, 100 μm. The framed areas are shown at higher magnification in the adjacent images on the right. Examples of SMC-derived cells expressing Lgals3 or Gucy1b1 (blue and brown) are indicated by arrowheads. Scale bars, 25 μm. Atherosclerotic aortae analyzed in (A) and (B) were from- 52-week-old apolipoprotein E-deficient mice fed with a normal chow. Shown immunostainings are representative for 3 independent experiments. C, UMAP visualization of modulated SMCs isolated from atherosclerotic aortae shown in Figure 2A. Upper sections show cells with detectable Gucy1a1 (red) and Gucy1b1 (green) transcripts. Middle sections show cells expressing Gucy1a1 (red) and Lgals3 (green) mRNA, and lower sections show cells expressing Gucy1b1 (red) and Lgals3 (green) mRNA. Overlays (right) show coexpressing cells in yellow. C indicates core; FC, fibrous cap (30-μm layer of the lesion adjacent to the vascular lumen); Gucy1a1, alpha1 subunit of NO-GC; Gucy1b1, beta1 subunit of NO-GC; Lgals3, galectin 3; SMC, smooth muscle cell; and UMAP, uniform manifold approximation and projection.

Article Snippet: Paraffin- embedded sections of left anterior descending arteries from previously described autopsy material38 were stained with hematoxylin/ eosin or immunostained with antibodies recognizing human LGALS3 (R&D Systems, AF1154, from goat) or GUCY1B1 (Abcam, ab24824, from rabbit).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Derivative Assay, Immunohistochemical staining, Isolation