Fig 1: Risperidone represses differentiation and autophagy while enhancing apoptosis via the TNF-α/SATB2 axis in vivo. A, Western blot analysis of differentiation-related proteins (OPG, collagen I and RANKL) expression in femur tissues of mice. The band intensity was assessed. B, statistical results of alizarin red S staining in femur tissues of mice. C, Western blot analysis of apoptosis-related proteins (cleaved PARP1, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9) in femur tissues of mice. The protein band was assessed. D, apoptosis rate in femur tissues in mice assessed by TUNEL staining. E, representative images of femur tissues in mice stained by immunohistochemistry and statistical results of LC3B positive rate. F, Western blot analysis of autophagy-related proteins (LC3 II/I, Beclin1, and p62) in femur tissues of mice. The band intensity was quantified and analyzed. *p < 0.05, compared with mice introduced with lentivirus expressing si-NC. #p < 0.05, compared with mice introduced with Risperidone + lentivirus expressing si-NC. &p < 0.05, compared with mice introduced with Risperidone + lentivirus expressing si-TNF-α. The experiments are repeated 3 times n = 6
Fig 2: Increased autophagy, vacuolation, and adhesion deficiency existed in fatty liver dairy cow neutrophils. (A) Representative transmission electron micrographs of normal and fatty liver neutrophils. White arrows indicate autophagic vacuoles (AVi, AVd, GVs, and vacuoles). N (N1, N2, and N3), nucleus. Scale bars as indicated. The area ratio of autophagic vacuoles to neutrophils and the number of autophagic vacuoles in neutrophils were determined (n = 6). Data represent the mean ± s.e.m. (**p < 0.01 versus the control group; Significance calculated using t-test). (B) Autophagy levels were evaluated using confocal microscopy in neutrophils of normal and fatty liver cows. Neutrophils were stained with anti-LC3B antibody and nuclear DNA was stained with Hoechst 33258. Scale bar, 5 μm. (C) Immunoblot for LC3B, CD11b, CD18, and p62 in normal and fatty liver neutrophils. ACTB was used as a loading control (n = 3). Data represent the mean ± s.e.m. (**p < 0.01 versus the control group; significance calculated using two-way ANOVA). (D) The number of granules in neutrophils (n = 6) of normal and fatty liver cows. Data represent the mean ± s.e.m. (**p < 0.01 versus the control group; significance calculated using t-test). (E) Surface expression of CD11b and CD18 on normal and fatty liver cow neutrophils (n = 6). Surface expression of CD11b and CD18 was assessed by flow cytometry analysis (n = 6). MFI, mean fluorescence intensity. Data represent the mean ± s.e.m. (*p < 0.05 and **p < 0.01 versus the control group; significance calculated using t-test). (F) Representative fluorescence micrograph images (left) and fluorescence microplate analysis (right) of normal and fatty liver cow neutrophils (n = 6). Scale bar, 400 μm. Data represent the mean ± s.e.m. (*p < 0.05 versus the control group; significance calculated using t-test).
Fig 3: Atg8, p62, and ubiquitinated nuclear proteins accumulate in cells lacking TER94 function.a Time-course analysis of the nuclear size change and the presence of ubiquitinated proteins in Rh1 > LacZ and Rh1 > TER94K2A pupal or adult eyes stained with phalloidin (red), anti-Lamin (magenta), and anti-Ubiquitin conjugates (FK2, green) antibodies. b Pearson’s correlation analysis of nuclear cross-section size and FK2 intensity from images of 1-day-old adult retina (as shown in panel a). The number of independent nuclei measured are 88 (LacZ) and 125 (TER94K2A). r values represent Pearson’s correlation coefficient in each group. c An adult TER94K15502 clone, marked by the absence of myr.GFP (arrowhead), stained with anti-Lamin (magenta) and anti-Ubiquitin conjugates (FK2, red) antibodies. R cell boundaries are outlined in dash lines. d, e Retinas from freshly eclosed (0-day) to 5-day-old adults co-expressing GFP-Atg8a (d) or GFP-p62 (e) with LacZ (control) or TER94K2A under the control of Rh1-GAL4 stained with phalloidin (red) and anti-Lamin (magenta) antibody. The number of independent experiments performed: a 2 (LacZ), 5 (TER94K2A) for 90% pupal; 3 (LacZ), 2 (TER94K2A) for 0-day; 4 (LacZ), 6 (TER94K2A) for 1-day; 6 (LacZ), 6 (TER94K2A) for 5-day adult. c 6. d 3 (0-day), 2 (1-day), 6 (2-day), 3 (5-day). e 2 (0-day), 5 (1-day), 5 (2-day), 4 (5-day). Scale bars: 10 µm (a, d, e), 5 µm (c).
Fig 4: YQRG regulates autophagy. a Mechanisms of autophagy. b Double immunofluorescent staining was performed to determine the colocalization of LC3II (red) and α-SMA (blue) in liver tissues (Scale bar = 50 μm, n = 3). c Western blot image showing LC3I/II expression in liver tissues (n = 6). d Protein concentration analysis (n = 6). e, f mRNA expression of LC3B and ULK1 were detected by qPCR (n = 6). Data values are indicated as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. model group
Fig 5: The effect of C91 on autophagy in ST2 cells. (A) 3MA is an autophagy inhibitor. ST2 cells were pre-treated with 3MA (5 mM) for 6 hours, and then induced by C91 for 3 days. MDC staining was performed and autophagy was analyzed by fluorescence microscopy. Body formation. Red arrows indicate autophagosomes, scale bar = 25 μm. (B) Western blot detection of the expression of autophagy-related protein LC-3I/II. GAPDH was used as an internal control, and the histogram on the right is the quantification of the band on the left. (C) qRT-PCR analysis of the mRNA expression of autophagy-related gene lc3b. Results are expressed as mean ± SD (n = 3 per group). * indicates P < 0.05, compared with the DMSO group. # indicates P < 0.05, compared with the C91 group.
Supplier Page from Abcam for Anti-LC3A/B antibody