LC3B Antibody - #AF4650

Fig. 2. Effects of different concentrations of AGEs on mitochondrial function and mitophagy of BMSCs. The BMSCs were treated with AGEs (50–200 μg/mL) or BSA for 24–72 h. (A) Representative fluorescence images with DCF (green) staining in BMSCs stimulated with AGEs. Scale bar: 50 μm. (B) Representative fluorescence images with Mito-SOX (red) and Mito-Tracker (green) double-staining in BMSCs stimulated with AGEs. Scale bar: 50 μm. (C) The MMP was detected through JC-1 staining in BMSCs stimulated with AGEs. Scale bar: 50 μm. (D) Representative fluorescence images with Mtphagy Dye (red) and Mito-Tracker (green) double-staining in BMSCs stimulated with AGEs. Scale bar: 50 μm. (E) Representative fluorescence images with LC3B (red) and Mito-Tracker (green) double-staining in BMSCs stimulated with AGEs. Scale bar: 50 μm. (F) Representative Western blotting assay and quantitation of the level of LC3B, P62, Parkin, Sirt3. **p < 0.01 versus BSA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 10 Immunofluorescence images of Beclin-1 (A), p62 (B), and LC3(C) were captured under fluorescence microscopy, and the relative fluorescence intensity was calculated. All images were obtained at identical magnification, ×200, scale bar = 50 μm. Data are represented as mean ± SEM (n = 3). **P

Figure 5 The representative western blot images of LC3β, HO-1, Beclin-1, p-AMPK, p62, p-mTOR and immunohistochemical image of LC3β showed that LDOP-1 induced autophagy via AMPK/mTOR signaling way in vivo. (A) The expression of LC3β, HO-1, Beclin-1, p-AMPK, p62, p-mTOR detected by Western blot. (B-G) Statistical analysis on LC3β, HO-1, Beclin-1, p-AMPK, p62, p-mTOR. Immunohistochemical image of LC3β (H) measured by immunohistochemical analysis (400×, brown yellow granules indicate positive reaction). Data are expressed as the mean  ±  SD of three independent experiments. #P < 0.05, ##P < 0.01, ###P < 0.001 compare the control group; *P < 0.05 and **P < 0.01 compare model group. LDOP-L stood for LDOP-1-L, LDOP-H stood for LDOP-1-H.

Figure 5 The representative western blot images of LC3β, HO-1, Beclin-1, p-AMPK, p62, p-mTOR and immunohistochemical image of LC3β showed that LDOP-1 induced autophagy via AMPK/mTOR signaling way in vivo. (A) The expression of LC3β, HO-1, Beclin-1, p-AMPK, p62, p-mTOR detected by Western blot. (B-G) Statistical analysis on LC3β, HO-1, Beclin-1, p-AMPK, p62, p-mTOR. Immunohistochemical image of LC3β (H) measured by immunohistochemical analysis (400×, brown yellow granules indicate positive reaction). Data are expressed as the mean  ±  SD of three independent experiments. #P < 0.05, ##P < 0.01, ###P < 0.001 compare the control group; *P < 0.05 and **P < 0.01 compare model group. LDOP-L stood for LDOP-1-L, LDOP-H stood for LDOP-1-H.

Figure 9 The expression of LC3β when treated with 2 %, 4 %, 6 %, 8 % concentration ethanol for 0.5 h, 1 h, 1.5 h, 2 h and effects of LDOP-1 on it. (A) The expression of LC3β at the ethanol at concentrations of 2 %, 4 %, 6 %, 8 %. (B) The expression of LC3β for 0.5 h, 1 h, 1.5 h. (C) Immunofluorescence image of LC3β measured by immunofluorescence technique (GES-1 were treated with 8% ethanol for 2 h after pretreatment with LDOP-1). Data are expressed as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 compare to 2 % concentrations ethanol or 0.5 h group. LDOP stood for LDOP-1.

FIGURE 1 Depletion of geminin suppresses autophagy activity, enhances ROS production, and induces the apoptosis of HGSOC cells. (A) Immunoblotting analysis of geminin protein levels in A2780 cells as indicated. shNC, A2780 cells transfected with shNC; sh-GMNN, geminin-knockout A2780 cells transfected with shRNA. Quantification of three independent experiments was performed, normalized to tubulin, and expressed as a ratio of NC, mean ± SEM, Student’s unpaired t-test, ***p < 0.001. (B) IB analysis of apoptosis-related proteins’ expression levels in A2780 cells as indicated. Mean ± SEM, n = 3, Student’s unpaired t-test, **p < 0.01, ***p < 0.001, ns—non-significant. (C) Apoptosis analysis of A2780 cells under geminin depletion. The percentage of apoptotic cells was analyzed with flow cytometry. Mean ± SEM, n = 3, Student’s unpaired t-test, ***p < 0.001. (D) Representative phase-contrast images of bioreactor expanded A2780 cells at indicated days of cultivation. Scale bar 20 μm. (E) Graphical illustration of the average aggregate size measured at the indicated day of cultivation: Day 8, shNC n = 116, sh-GMNN n = 108; Day 16, shNC n = 130, sh-GMNN n = 161. Images were analyzed using Nikon NIS Elements D software, mean ± SD, Student’s unpaired t-test, ***p < 0.001. (F) The protein–protein associations about geminin protein. Data from STRING. (G) ROS analysis of A2780 cells under geminin depletion. The percentage of apoptosis cells was analyzed with flow cytometry. Mean ± SEM, n = 3, Student’s unpaired t-test, ***p < 0.001. (H) IB analysis of LC3B protein levels in A2780 cells as indicated. Mean ± SEM, n = 3, Student’s unpaired t-test, ***p < 0.001.