Fig 1: Comparison of autophagy markers immunolabelling among different neuromyopathies. (A-C, E, F, L) Sections of MDC1A muscle biopsy double immunolabeled with anti-pan-laminin (red channel in A-C, F, M) and 4 different autophagy markers (green channel) such as anti-Beclin-1 (A; Abcam, code ab51031), an early phase phagophore formation marker, anti-ATG5 (B; Abcam, code ab78073), intermediate phase marker, anti-LC3b (C, F; Abcam, code ab48394), late phase marker, and anti-p62/SQSTM1 (E, L; Abcam, code ab56416), a shuttle receptor of LC3 from ubiquitin pathway. These different autophagy markers localize as green stained puncta in sarcoplasms of several myofibers (arrows). (C, F-J) LC3b+ stained puncta are more frequently seen in MDC1A and IMNM than DMD, SMA, and NM. (E, L-P) p62+ stained puncta are more frequently seen in DMD and IMNM than the other neuromyopathies. (D, K, Q) No LC3 and p62 immunostainings are normally detectable in sarcoplasmic adult healthy control muscle (CM). (F-Q) Both LC3b and p62 appeared preferentially localizing in proximity to MHC-II+ endomysial cells in IMNM (G, M), but separated in the other neuromyopathies. Scale bars: A-E: 50 μm; insets horizontal side in A (27 μm), C (47 μm), E (67 μm); F, L: 2 μm; G-K, M-Q: 10 μm.
Fig 2: Angelicin promoted autophagy in cervical cancer cells. (A) Immunofluorescence micrographs demonstrating LC3B staining (green) and nucleus staining (blue) following gangelicin treatment (27.8 µM for HeLa, 36.6 µM for SiHa). (B) Western blot analysis detected angelicin-induced LC3B expression in HeLa and SiHa cells. To inhibit the degradation of LC3B-II, cells were pre-treated with 5 µM chloroquine for 6 h. (C) Western blot analysis of Atg3, Atg7 and Atg12-Atg5 protein expression in both HeLa and SiHa cells. LC3B, microtubule associated protein 1 light chain 3-β; Atg, autophagy related proteins.
Fig 3: Relative abundance of DHA (docosahexaenoic acid) and of docosanoids in retinal pigment epithelial cells (RPE/choroid) and in retina of wild -type and LC3B-/-. In retinal pigment epithelial cells (RPE/choroid): Esterified DHA (total) (A); free DHA (B); 17(S)-HDHA (C); NPD1 (D); 14(S)-HDHA (E), and Maresin-1 (F). In retina total (G) and free (H) DHA. Data obtained by LC/MS/MS lipid profiling. ∗p < 0.050. Mice were 18–20 months old. The diagram (I) depicts a phospholipid hydrolyzed by a PLA2 that releases DHA. In turn two paths for the formation of docosanoids are illustrated, one leading to NPD1 and the other to maresin-1.
Fig 4: AOA reversed the hypoxia-induced increase in mitophagy, neuronal injury, learning and memory deficits.A–F Decreased immunoreactivity of LC3B (red) and TOMM20 (green) in the EC region after AOA treatment. Blue, DAPI. G, H Fluorescence intensity of LC3B and TOMM20 in DG and EC. I–K The expression of activated caspase-3 and caspase-3 in cortex and hippocampus detected by western blotting. L–N Positive FJB signals in DG and EC in each group after hypoxia. Bar = 50 μm. n = 5/group. O Latency to the platform (Two-way ANOVA). P Frequency of platform crossings. Q Target quadrant time (%). R Opposite quadrant time (%). S Representative tracking. **P < 0.01, and ***P < 0.001, all compared with controls (One-way ANOVA). #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with that in hypoxia+saline group (One-way ANOVA with Dunnett’s T3 post-hoc test).
Fig 5: DM reversed the increased mitophagy, neuronal damage, cognitive deficits induced by hypoxia treatment.A–F Decreased immunoreactivity of LC3B (red), TOMM20 (green) in the EC after DM treatment. Blue, DAPI. G, H Fluorescence intensity of LC3B and TOMM20 in DG and EC. I–K The expression of activated caspase-3 and caspase-3 in the cortex and hippocampus detected by western blotting. L–N Positive FJB signals in DG and EC in each group. Bar = 50 μm, n = 5/group. O Latency to the platform (Two-way ANOVA). P Frequency of platform crossings. Q Target quadrant time (%), and (R) Opposite quadrant time (%). S Representative tracking. **P < 0.01, and ***P < 0.001, all compared with controls (One-way ANOVA). #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with that in hypoxia+saline group (One-way ANOVA with Dunnett’s T3 post-hoc test).
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