lc3 a b  (Cell Signaling Technology Inc)

Journal: Discover Oncology

Article Title: Interaction between Maslinic acid and HSF1 enhances the ubiquitin degradation of HSF1, resulting in the inhibitory effect of pancreatic cancer

doi: 10.1007/s12672-025-02786-8

Figure Lengend Snippet: HSF1 expression was important for MA-induced autophagy in pancreatic cancer cells. (A) Pancreatic cancer cells were infected with adenovirus harboring tandem fluo-rescent mRFP-GFP-LC3 at 1,000 multiplicity. After incubation for another 24 h, the cells were treated without or with HSF1 siRNA (Panc-28/siR), scramble siRNA (Panc-28/NC) or the combination of HSF1 siRNA and MA (Panc-28/siR + MA) as well as the combination of scramble siRNA and MA (Panc-28/NC + MA). After cultured for 48 h, the cells were photographed using a laser confocal microscope (Nikon, Tokyo, Japan), and the red and green fluorescence spot was merged. (C) Quantitative of the results of ( A ), the autophagy flow. (B) The expression of autophagy-related proteins. Wild-type pancreatic cancer cells and HSF1 knock-down cancer cells were treated with MA, the expression of autophagy-related proteins were detected by Western Blot and the gray value was calculated by Image J software (D) ; Mean ± SD, n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001 vs Control group

Article Snippet: Non-specific binds were blocked with 5% non—fat dry milk in 1 × TBST buffer (1 × TBS and 0.1% Tween 20) for 1 h at room temperature and incubated with the corresponding primary antibodies overnight at 4℃, including mTOR (Cell Signaling Technology, 1:1,000), HSF1(Cell Signaling Technology, 1:1,000), p-mTOR(Cell Signaling Technology, 1:1,000), Atg7(Proteintech, 1:1,000), ULK1(Cell Signaling Technology, 1:1,000), Atg5 (Cell Signaling Technology, 1:1,000), Beclin-1(Proteintech, 1:1,000), LC3 A/B(Cell Signaling Technology, 1:1,000), Atg3(Proteintech, 1:1,000), and β-Actin (Cell Signaling Technology, 1:1,000).

Techniques: Expressing, Infection, Incubation, Cell Culture, Microscopy, Fluorescence, Knockdown, Western Blot, Software, Control

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Chloroquine induces ATG16L1 independent recruitment of LC3 to small puncta located close to 505 swollen lysosomes. Panel A. Control (WT) and ATG16L1-/- MEFs were incubated for 2 hours in nutrient media (i and iv), HBSS (ii & v) or nutrient media containing chloroquine (100μM iii and vi) as indicated. Cells were fixed andimmunostained for LC3. Scale bar 5μm. WT MEFs (panel B) or ATG16L1-/- MEFs (panel D) were incubated for 2 hours in media containing chloroquine (100μM). Cells were fixed and immunostained for LC3 and LAMP1. Regions of interest indicated in panels B and D were used to generate line profiles to follow distribution of LC3 and LAMP in small puncta and swollen lysosomes (panel C and E).

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Control, Incubation

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Formation of LC3 puncta and LC3II independently of ATG16L1 requires ATG3, ATG5 and ATG7. Panel A. ATG3-/-, ATG5-/- and ATG7 -/- MEFs were incubated for 2 hours in chloroquine (100 µM) fixed cells were immunostained for LC3. Panel B. LC3 puncta in the indicated cells were counted using Imaris software. Panel C. Cells were incubated as described above and generation of LC3II was assessed by western blot for LC3. Panel D. Densitometric calculation of LC3II/LC3 ratios from duplicate blots.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Incubation, Software, Western Blot

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Vacuole swelling in response to osmotic stress induces formation of LC3 puncta and LC3II in ATG16L1-/- cells. Panel A. Atg16L1-/- MEFs were incubated with chloroquine (100 µM) for 2 hours in the presence of wortmannin (100 nM iv-vi) DPI (20 µM vii-ix), phloretin (180 µM x-xii) or bafilomycin (100 nM xiii-xv) as indicated. Cells were fixed and immunostained for LC3 (green) and LAMP1 (red). Panel B. Numbers of LC3 puncta from more than 30 cells incubated as described in panel A were quantified by Imaris TM , P-values were calculated using multiple t-test (**** P < 0.0001). Panel C. Cells were incubated as described in panel A and generation of LC3II was assessed by western blot for LC3. Panel D. Cells were incubated as described and cross-sectional area (um 2 ) of lysosomes containing fluorescent dextran were determined by Fiji. Scale bar 5 μm.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Incubation, Western Blot

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Osmotic stress induces recruitment of galectin-3 to LC3 puncta generated in the absence of ATG16L1. Control (Panel A) and Atg16L1-/- MEFs (Panel B) were incubated for 2 hours in media containing chloroquine (100 µM i-iv), LLOMes (1 mM v-viii) or monensin (100 µM ix-xii). Fixed cells were immunostained for LC3 (green) and galectin 3 (red). Scale bar 5 mm. Panel C. distribution of LC3 (green) and galectin 3 (red) assessed from line profiles. Panel D. Numbers of small (1.0 um dia.) LC3 puncta counted from 20 cells by Imaris. Panel E. LC3II assessed by western blot of cell lysates. Panel F. LC3II/LC3 ratio calculated by densitometry of duplicate blots.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Generated, Control, Incubation, Western Blot

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Conjugation of LC3 to ruptured lysosomes in the absence of ATG16L1. Control and ATG16L1-/- MEFs were incubated chloroquine (100 µM) for 2 hours as indicated, scale bar 5 um. Panel A. Immunostaining for LC3 (green) and V-ATPase (red). Panel B. distribution of LC3 (green and V-ATPase (red) assessed from line profiles. Panel C. Images of ATG16L1-/- MEFS where small puncta of LC3 co-localise with V-ATPase. Panel D. Co-localisation of LC3 and V-ATPase quantified by Imaris TM. Panel E. Atg16L1-/- MEFs immunostained for PI4P (green), galectin-3 (red) and ATG5 (far red purple). Panel F. Co-localisation of PI4P (green), galectin-3 (red) and ATG5 (purple) quantified by Imaris TM . Panel G. immunostaining for p62 (red) and galectin-3 or LAMP1 as indicated. Panel H. Distribution of p62 (red) and galectin-3 or LAMP1 (green) assessed from line profiles.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Conjugation Assay, Control, Incubation, Immunostaining

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Conjugation of LC3 to damaged lysosomes in the absence of ATG16L1 requires TECPR1. Panel A. Domain map of TECPR-1 showing site of insertion of stop codon by custom CRISPR gRNA. ATG16L1-/- MEFs and gene edited ATG16L1-/- MEFS expressing truncated TECPR1* were incubated in nutrient media with chloroquine (100 µM, panel B) or LLOMes (1 mM panel C). Cells were fixed and immunostained for LC3(green) and galectin-3 (red). Rendered images (v and vi, xi and xii) were used to count puncta. Higher magnification images of boxed ROI are shown in iv and x. The graphs in panels D and F compare numbers of LC3 puncta co-localised with galectin-3 counted from rendered images of 30 cells incubated with chloroquine (panel D) or LLOMEs (panel F), P-values were calculated using multiple t-test (****P < 0.0001). Panels E and G show assessment of LC3II by western blot for LC3 for chloroquine (panel E) or LLOMes (panel G). Scale bar 5 μm.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Conjugation Assay, CRISPR, Expressing, Incubation, Western Blot

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: TECPR-1 facilitates recruitment of LC3 and ATG5 to damaged lysosomes in the absence of ATG16L1. Control (WT), Atg16L1-/- and ATG16L1-/- MEFS expressing truncated TECPR1* were incubated for 2 hours in nutrient media containing chloroquine (100 µM). Panel A. Cells were fixed and immunostained for ATG5 (red) and galectin-3 (green). Panel B. Cells were fixed and immunostained for ATG5 (red) and LC3 (green). Panel C. Quantification and composition of puncta positive for ATG5 and galectin 3. Panel D. Quantification and composition of puncta positive for ATG5 and LC3. n ≥ 30 cells were quantified by Imaris, P-values were calculated using multiple t-test (****P < 0.0001). Scale bar 5 μm. Panel E. ATG16L1-/- MEFS expressing GFP-lysenin (green) and TECPR1-RFP (red) were incubated in nutrient media containing chloroquine (200 µM) for 30 minutes. Scale bar 5 μm.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Control, Expressing, Incubation

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Recruitment of galectin-3 and GFP-lysenin to lysosomes correlates with release of dextran. ATG16L1-/- MEFS expressing GFP-lysenin were cultured in Dextran-Red R (300 mg/ml) to label lysosomes and incubated with chloroquine (200 µM) for 10 or 30 minutes as indicated. Images show distribution of GFP-lysenin, Dextran-Red R and galectin-3 (far red). Asterisks indicate puncta positive for lysenin and galectin-3 that are negative for dextran.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Expressing, Cell Culture, Incubation

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Ultrastructure of LC3 puncta analsyed by CLEM. MEFs growing on grid dishes were transduced with LC3-GFP adenovirus, incubated with chloroquine (100 µM) and observed by live cell imaging. Fixed cells permeabilised with saponin were incubated with rabbit antibody against GFP followed by antirabbit nanogold and silver enhancement. Panel A. ATG16L1-/- MEFs of interest were identified by correlating the grid and cell pattern with previously acquired brightfield images. Panel B gallery of images generated from WT (i-iii) and ATG16L1-/- MEFs (iv-vi).

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Transduction, Incubation, Live Cell Imaging, Generated

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Analysis of LC3 and galectin-3 distribution on damaged lysosomes by structured illumination microscopy: MEFs were incubated nutrient media containing chloroquine (100 µM) for 2 hours and then stained with DAPI (cyan) and immunostained for LC3 (yellow) and galectin3 (magenta) and imaged by 3D-SIM. Panel A: control MEFS. A swollen vacuole (I) recruits LC3 (I-i, maximum intensity projection-MIP) but not galactin3. To visualise this better, using Imaris, a 3D rendered surface was applied (I-ii) and then bisected (I-iii) showing the hollow vacuole interior. Analysis of small dense puncta (II) shows that LC3 (II-i, yellow MIP) and galectin3 (II-ii, magenta MIP) are recruited together as seen in the coloured overlay (II-iii). A 3D rendered surface (II-iv) bisected (II-v) shows the densely packed interior of this puncta. Scale bar = 1 µm. Panel B: ATG16L1-/- MEFs. Analysis of small dense puncta shows that LC3 (i, yellow MIP) and galectin3 (ii, magenta MIP) are recruited together as seen in the coloured overlay (iii). A 3D rendered surface (iv) bisected (v) shows the densely packed interior of the puncta. Scale bar = 1 µm.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Microscopy, Incubation, Staining, Control

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: Role played by the LIR motif, PH domain of TECPR-1 during conjugation of LC3 to damaged lysosomes. Gene edited ATG16L1-/- MEFS expressing truncated TECPR1* were transfected with plasmids expressing full length TECPR-1-RFP (i-iii), TECPR-1 lacking the LIR within the N-terminal TECPR domain (TECPR1-∆LIR-GFP pseudo red, iv-vi) or TECPR-1 lacking the central PH domain (TECPR1-∆PH-GFP pseudo red, vii-ix). Cells were incubated with chloroquine (100 µM) for 2 hours. Panel A shows counter staining for LC3 (green) and panel B counter staining for galectin 3 (green). Panel C and D. Western blot analysis and relative quantification of LC3II. ATG16L1-/- TECPR*/*MEFS transfected with indicated plasmids were incubated in nutrient media containing chloroquine (100 µM) for 2 hours. Data represent mean ± S.E. of three independent experiments (***P < 0.001). Scale bar 5 μm.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Conjugation Assay, Expressing, Transfection, Incubation, Staining, Western Blot, Quantitative Proteomics

Journal: Autophagy Reports

Article Title: The TECPR1:ATG5-ATG12 complex conjugates LC3/ATG8 to damaged lysosomes that expose luminal glycans in response to osmotic imbalance

doi: 10.1080/27694127.2025.2476218

Figure Lengend Snippet: TECPR1-dependent conjugation of LC3 to lysosomes in response to osmotic imbalance. i). Dysferlin domains of TECPR1 bind sphingomyelin exposed early during osmotic stress. ii). TECPR1 binding is enhanced by TR1 dependent tethering to the lysosome and binding of the PH domain to PI4P. iii). The AIR and LIR domains of TECPR1 recruit the ATG5-ATG12 conjugate and LC3, but conjugation of LC3 is inefficient. iv). Lysosome rupture exposes luminal galactose residues to recruit galectin-3 to facilitate conjugation of LC3 by the TECPR1:ATG5-ATG12 complex. v). Ruptured lysosome membranes containing LC3II, p62 and galectin 3 may be removed by lysophagy.

Article Snippet: Panel B. LC3 puncta in the indicated cells were counted using Imaris software.

Techniques: Conjugation Assay, Binding Assay