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: Cardiovascular Research

Article Title: Endothelial OCT4 is atheroprotective by preventing metabolic and phenotypic dysfunction

doi: 10.1093/cvr/cvac036

Figure Lengend Snippet: EC-specific conditional knockout of Oct4 exacerbates atherosclerosis in Apoe−/− mice. (A) EC-specific-lineage-tracing and conditional Oct4 knockout mouse model. Schematic of Cdh5-CreERT2; Oct4Flox/Flox; Rosa-Stop-eYFP+/+Apoe−/− mouse (designated as Oct4Flox/Flox). Mice received 10 injections of tamoxifen (TMX) between 6 and 8 weeks of age. In response to tamoxifen, Cre-recombinase under the control of the Cdh5 gene promoter excises LoxP sites (black triangles), removing Oct4 exon 1 (responsible for pluripotency) and STOP codon upstream of eYFP specifically in EC. Finally, it provides simultaneous EC-lineage-tracing and EC-specific Oct4 knockout. (B) Schematic of animal experiments. (C) Representative western blot showing protein levels of OCT4, Histone H3, and GAPDH in nuclear (Nuc) and cytoplasmic (Cyto) extracts from EC isolated from aortas of EC-specific OCT4 knockout (Oct4Δ/Δ) and wild-type (Oct4+/+) mice. L, ladder; PC, positive control, A404 precursor cells. (D) Densitometry quantification of Western blots. Values = mean ± S.E.M.. *P < 0.05 by unpaired t-test; n = 3 independent experiments. (E–H) Sudan IV en face staining and quantification of lesion area in aortas of Oct4Δ/Δ and Oct4+/+ male (E and F) and female (G and H) mice after 10 or 18 weeks of WD feeding. Values = mean ± S.E.M.. *P < 0.05, #P = 0.052 by unpaired t-test; Oct4+/+: n = 9 for 10 weeks and n = 12 for 18 weeks of WD; Oct4Δ/Δ: n = 11 (females) and n = 10 (males) for 10 weeks and n = 12 for 18 weeks of WD. (I) Representative immunostaining of LGALS3 in BCAs collected from Oct4Δ/Δ and Oct4+/+ male mice after 4 weeks of WD feeding. Scale bar = 100 µm. (J) The percentage of male mice with LGALS3-positive staining in Oct4+/+ (n = 4/11 positive BCAs) and Oct4Δ/Δ (n = 7/8 positive BCAs) BCAs. BCAs were analysed at 60 µm distance from aortic arch. *P < 0.05 by Fisher’s exact test.

Article Snippet: Immunohistochemistry was performed with antibodies for LGALS3 (Cedarlane; CL8942AP; 1:500) and TER-119 (rat anti-mouse, Santa-Cruz Biotechnology Inc.; 1:200).

Techniques: Knock-Out, Control, Western Blot, Isolation, Positive Control, Staining, Immunostaining

Journal: Cardiovascular Research

Article Title: Endothelial OCT4 is atheroprotective by preventing metabolic and phenotypic dysfunction

doi: 10.1093/cvr/cvac036

Figure Lengend Snippet: EC-specific conditional knockout of Oct4 destabilizes atherosclerotic plaque. (A–C) Assessment of collagen fibre hue variations based on PicroSirius Red staining followed by polarized microscopy. (A) PicroSirius Red staining of representative BCA cross-sections of Oct4Δ/Δ and Oct4+/+ male mice fed WD for 18 weeks. Scale bar = 100 µm. The ratio between the amount of immature (green), intermediate (yellow), and mature (red) collagen fibrils showed a significant decrease in mature fibrils within the lesion (B) and the 30 µm fibrous cap area (C) of Oct4Δ/Δ mice (n = 13) as compared with control mice (n = 14), ±S.E.M.. *P < 0.05 (for mature fibrils) by unpaired Student t-test. (D) Immunofluorescence staining of DAPI, YFP, ACTA2, and LGALS3 on representative BCA sections of Oct4+/+ and Oct4Δ/Δ mice fed WD for 18 weeks, showing a noticeable increase in LGALS3+ cells in the lesion. Scale bar = 100 µm. (E and F) Quantification of the percentages of LGALS3+ lesion area (E) and the ratio of ACTA2+ cells over LGALS3+ cells within the 30 µm protective fibrous cap area (F) of BCAs collected from Oct4Δ/Δ and Oct4+/+ male and female mice after 18 weeks of WD feeding. Values = mean ± S.E.M., *P < 0.05 determined by either unpaired t-test with Welch correction (E) or unpaired t-test (F) for Oct4+/+ (n = 12 males; 12 females) vs. Oct4Δ/Δ (n = 12 males; 11 females) mice. (G) Immunostaining for the red blood cell marker TER119. Scale bar = 100 µm. Inserts indicate magnified areas. (H) The percentage of BCAs exhibiting intraplaque haemorrhage based on TER119 staining in Oct4+/+ (n = 4/13 positive BCAs) vs. Oct4Δ/Δ (n = 10/13 positive BCAs) male mice. BCAs were analysed at 120, 420, and 720 µm distance from aortic arch. If any of these three locations had any TER119-positive staining, that animal was marked as positive for haemorrhage. *P < 0.05 by Fisher’s exact test.

Article Snippet: Immunohistochemistry was performed with antibodies for LGALS3 (Cedarlane; CL8942AP; 1:500) and TER-119 (rat anti-mouse, Santa-Cruz Biotechnology Inc.; 1:200).

Techniques: Knock-Out, Staining, Microscopy, Control, Immunofluorescence, Immunostaining, Marker