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 Cell Biology

Article Title: Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin

doi: 10.1083/jcb.201304188

Figure Lengend Snippet: Ub-positive endosomes containing Salmonella or beads are targeted by autophagy. (A) HeLa cells were infected with S. Typhimurium ( Salmonella ) for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and transferrin receptor (TfR). Bar, 10 µm. (B) HeLa cells were infected with Salmonella for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and galectin3. Bar, 5 µm. (C) HeLa cells were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub (top) or LC3 and p62 (bottom). Bar, 5 µm. The percentages of LC3- or p62-positive beads per Ub-positive (Ub+) or Ub-negative (Ub−) beads were enumerated. Statistical analysis was performed by Student’s unpaired t test. *, P < 0.01. (D and E) HeLa cells stably expressing GFP-LC3 were transfected with Effectene-coated latex beads for 3 h. Bead–autophagosomes were fractionated as described in Materials and methods. The bead–autophagosome fraction was observed by confocal microscopy (D; bar, 10 µm) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies (E). In a control sample, scraped cells were mixed with Effectene-coated beads and immediately homogenized. (F) The bead–autophagosome fraction was lysed and subjected to immunoprecipitation with an anti-Ub IgG antibody (FK2) or control IgG. Co-immunoprecipitated molecules were examined by Western blotting using the indicated antibodies. (G and H) NIH3T3 cells stably expressing mStrawberry (mStr)-Gal3 and GFP-LC3, mStr-Gal3, and GFP-p62, or mStr-Gal3 and GFP-Ub were transfected with Effectene-coated latex beads for 30 min and then washed. Live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after galectin3 localization was measured for at least 30 cases for each combination (H). Statistical analysis was performed by Student’s unpaired t test. *, P < 0.05; NS, not significant.

Article Snippet: The following antibodies were used: anti–mouse Atg16L1 , anti-p62 (MBL), anti-LC3 (MBL), anti-transferrin receptor (Invitrogen), anti-poly Ub (clone FK2; BIOMOL), anti-K48 linked Ub (clone Apu2; EMD Millipore), anti-K63 linked Ub (clone Apu3; EMD Millipore) anti-galectin3 (Santa Cruz Biotechnology, Inc.), anti-Lamp1 (Santa Cruz Biotechnology, Inc.), anti-FLAG (clone M2; Sigma-Aldrich), anti-GFP (Roche), anti-Myc (clone 9E10), and anti–α-tubulin (clone B5-1-2; Sigma-Aldrich).

Techniques: Infection, Transfection, Immunocytochemistry, Stable Transfection, Expressing, Confocal Microscopy, Western Blot, Control, Immunoprecipitation, Fluorescence, Microscopy

Journal: The Journal of Cell Biology

Article Title: Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin

doi: 10.1083/jcb.201304188

Figure Lengend Snippet: Ubiquitination and recruitment of Atg proteins. (A and B) NIH3T3 cells stably expressing mStr-Ub and GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, or ULK1 were transfected with Effectene-coated latex beads for 30 min. Then, live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after Ub localization was measured for at least 30 cases for each combination. Each value represents the mean ± SD. Statistical analysis was performed by Student’s unpaired t test. *, P < 0.05; NS, not significant. (C) NIH3T3 cells stably expressing GFP-tagged Ub, LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated beads for 3 h in the presence or absence (mock) of 30 µM UBEI-41 (a ubiquitin E1–specific inhibitor) and subjected to immunocytochemistry for galectin3. The percentages of GFP-positive per galectin3-positive beads were enumerated. At least 30 beads were counted ( n = 3). The values are the mean ± SD. Statistical analysis was performed by Student’s unpaired t test. *, P < 0.05. (D and E) Parent NIH3T3 cells, Atg4B mutant overexpressing NIH3T3 cells (D), wild-type MEFs, and Atg5-KO MEFs (E) stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted ( n = 3). The values are the mean ± SD.

Article Snippet: The following antibodies were used: anti–mouse Atg16L1 , anti-p62 (MBL), anti-LC3 (MBL), anti-transferrin receptor (Invitrogen), anti-poly Ub (clone FK2; BIOMOL), anti-K48 linked Ub (clone Apu2; EMD Millipore), anti-K63 linked Ub (clone Apu3; EMD Millipore) anti-galectin3 (Santa Cruz Biotechnology, Inc.), anti-Lamp1 (Santa Cruz Biotechnology, Inc.), anti-FLAG (clone M2; Sigma-Aldrich), anti-GFP (Roche), anti-Myc (clone 9E10), and anti–α-tubulin (clone B5-1-2; Sigma-Aldrich).

Techniques: Ubiquitin Proteomics, Stable Transfection, Expressing, Transfection, Fluorescence, Microscopy, Immunocytochemistry, Mutagenesis

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