Journal: International Journal of Biological Sciences

Article Title: The Remodeling of Mitochondrial-Endoplasmic Reticulum Contacts by Omega-3 Fatty Acids Mitigates Dietary Advanced Glycation End Product-Driven Sertoli Cell Senescence and Oligoasthenozoospermia

doi: 10.7150/ijbs.117091

Figure Lengend Snippet: Omega-3 mitigates AGEs effects on spermatogenesis and testicular senescence. (A) Animal Experimental Design: All animals were randomly divided into three groups: a control group (CTL), an advanced glycation end product-rich diet group (dAGEs) for 16 weeks, and a group fed an AGEs-rich diet supplemented with omega-3 PUFAs for the last 4 weeks (dAGEs + Omega-3). (B, C) Concentrations of AGE in sera(B) and testis(C) were analyzed by ELISA. (D-F) The mRNA levels (D) and the protein expression(E) of RAGE in mice testis. Protein expression was quantified by ImageJ and normalized (F). (G, H) Sperm concentration (G) and sperm progressive motility (PR) (H) of the mice after 16 weeks of treatment. (I) Representative photographs of testicular (upper and middle) and epididymal tail (lower) morphology with HE staining. Scale bars: for testis pictures, 100 μm for the original picture and 25 μm for the enlarged picture; for epididymal tail picture, 50 μm. Spermatogonia (SPG), Spermatocytes (SPC), Round Spermatids (RS), Elongated Spermatids (ES) and Sertoli Cells (SC) are indicated. (J-K) Diameter assessment and epithelial thickness quantification of each seminiferous tubule across different groups. (L-N) The expression of P16 (upper) and P21 (lower) proteins in mouse spermatogenic tubules was assessed by immunohistochemistry. Scale bars: 50 μm (M, N) Quantitative analysis using Average Optical Density (AOD) to assess the expression levels of proteins in tissue sections and normalized. (O) The levels of SASP factors in mouse testes were displayed using a heatmap. (P, Q) Representative images of ROS staining (P) and TUNEL staining (Q) in mice testicular spermatogenic tubules. Scale bars: 50 μm. Statistical analysis between multiple groups was performed by one-way ANOVA. A two-sided p-value < 0.05 was considered to be statistically significant. The level of significance defined as p < 0.05 (*), p < 0.01 (**), p < 0.005(***), p < 0.001 (****).

Article Snippet: After drawing circles using a histochemical pen around the tissues to prevent the leakage of antibodies, the ROS staining solution (Servicebio, Wuhan, China) was applied within the circles and the slides were incubated at 37°C in the dark for 30 minutes.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay, Staining, Immunohistochemistry, TUNEL Assay

Journal: International Journal of Biological Sciences

Article Title: The Remodeling of Mitochondrial-Endoplasmic Reticulum Contacts by Omega-3 Fatty Acids Mitigates Dietary Advanced Glycation End Product-Driven Sertoli Cell Senescence and Oligoasthenozoospermia

doi: 10.7150/ijbs.117091

Figure Lengend Snippet: Omega-3 alleviates AGE-induced senescence and dysfunction in TM4 cells in vitro. (A) Analysis of TM4 cell survival following treatment with AGEs at a concentration of 200 μg/mL, and the rescuing effects of Omega-3, as determined by the CCK-8 assay. (B-C) The protein expression of RAGE in TM4 cells was detected using Western blotting following treatment with AGEs and an Omega-3 supplement, and quantified by ImageJ (C). (D, F) SA-β-gal activity detection usingβ-galactosidase staining, and statistical analysis of the proportion of positive cells. Scale bar: 50 μm. (E, G) ROS detection using DCFH-DA staining and the fluorescence densities were calculated using ImageJ. Scale bar: 50 μm. (H, I) Analysis of apoptosis using Annexin V-FITC staining by flow cytometry. The late apoptotic rates were calculated and presented in the right panel. (J) The mRNA levels of SASP factors in TM4 cells. (K)Assessment of TM4 cell barrier integrity in vitro . After cell barrier formation, cells were treated and TER detection were used to analyze the integrity of TM4 cell barriers. (L, M) Tight junction protein levels in TM4 cells were examined by western blotting, and quantified by ImageJ (M). (N) Representative images of the formation of cytoskeleton in TM4 cells by F-actin staining immunofluorescence. Scale bar: 20 μm. (O) The mRNA levels of cell adhesion function-related genes in TM4 cells. Statistical analysis between multiple groups was performed by one-way ANOVA. A two-sided p-value < 0.05 was considered to be statistically significant. The level of significance defined as p < 0.05 (*), p < 0.01 (**), p < 0.005(***), p < 0.001 (****).

Article Snippet: After drawing circles using a histochemical pen around the tissues to prevent the leakage of antibodies, the ROS staining solution (Servicebio, Wuhan, China) was applied within the circles and the slides were incubated at 37°C in the dark for 30 minutes.

Techniques: In Vitro, Concentration Assay, CCK-8 Assay, Expressing, Western Blot, Activity Assay, Staining, Fluorescence, Flow Cytometry, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: The Remodeling of Mitochondrial-Endoplasmic Reticulum Contacts by Omega-3 Fatty Acids Mitigates Dietary Advanced Glycation End Product-Driven Sertoli Cell Senescence and Oligoasthenozoospermia

doi: 10.7150/ijbs.117091

Figure Lengend Snippet: RAGE is a central mediator of AGE-induced Sertoli cell dysfunction. (A-C) Representative immunostaining pictures of colocalization between mitochondria and ER in AGEs treated TM4 with or without the RAGE inhibitor FPS-ZM1(A). Scale bar: 10 μm. Line intensity profile analysis of Mito-tracker and ER-tracker (B). Intensity values were measured along a path indicated in lowest pictures (A). The Pearson's correlation between ER-tracker and Mito-tracker was analyzed (C). (D, E) MERCs protein levels in treated TM4 cells were examined by western blotting. (F, G) Representative immunostaining pictures of mitochondrial Ca 2+ detection in treated TM4 (F) and the fluorescence densities were calculated using ImageJ and normalized (G). (H, I) SA-β-gal activity detection usingβ-galactosidase staining, and statistical analysis of the proportion of positive cells. Scale bar: 50 μm. (J, K) ROS detection using DCFH-DA staining and the fluorescence densities were calculated using ImageJ. Scale bar: 50 μm. (L) Analysis of TM4 cell survival following treatment with AGEs with or without FPS-ZM1, as determined by the CCK-8 assay. (M) Assessment of TM4 cell barrier integrity in vitro . After cell barrier formation, cells were treated and TER detection were used to analyze the integrity of TM4 cell barriers. (N) Representative images of the formation of cytoskeleton in TM4 cells by F-actin staining immunofluorescence. Scale bar = 20 μm. (O, P) The adhesion function of Sertoli cells was assessed using fluorescence detection (O) after co-culturing mCherry-labeled germ cells with Sertoli cells, and the resulting ratio of germ cells (GCs) to Sertoli cells (SCs) was quantified (P). (Q) The mRNA levels of cytokines which influence germ cells in TM4 cells. (R) The mRNA levels of cell adhesion function-related genes in TM4 cells. Statistical analysis between multiple groups was performed by one-way ANOVA. A two-sided p-value < 0.05 was considered to be statistically significant. The level of significance defined as p < 0.05 (*), p < 0.01 (**), p < 0.005(***), p < 0.001 (****).

Article Snippet: After drawing circles using a histochemical pen around the tissues to prevent the leakage of antibodies, the ROS staining solution (Servicebio, Wuhan, China) was applied within the circles and the slides were incubated at 37°C in the dark for 30 minutes.

Techniques: Immunostaining, Western Blot, Fluorescence, Activity Assay, Staining, CCK-8 Assay, In Vitro, Immunofluorescence, Labeling

Journal: NPJ Science of Food

Article Title: Haematococcus pluvialis ameliorates renal fibrosis by restoring mitophagy via PINK1-Parkin-p62-LC3 signaling

doi: 10.1038/s41538-025-00654-x

Figure Lengend Snippet: A Blue: Nuclei. Purple: Mitochondrial. Red: PINK1/Parkin/p62/LC3B. IF staining was performed to detect PINK1, Parkin, p62, and LC3B protein levels (magnification: 100×, scale bar: 15 μm). B Colocalization analysis of p62, LC3B, and mitochondria was performed using ImageJ software. C Colocalization Pearson correlation coefficient analysis were obtained based on IF images of p62 and LC3B ( n = 6). D Western blot analysis of PINK1 and LC3B expression in renal cortical tissues from different experimental groups. Quantification of gray values for Western blotting results in ( D ) was performed using ImageJ ( n = 3). E Blue: Nuclei. Red: E-cadherin/N-cadherin/Vimentin. IF assay was performed to detect E-cadherin, N-cadherin and Vimentin protein levels (magnification: 100×, scale bar: 15 μm). F Quantitative analysis of fluorescence intensity using ImageJ software ( n = 6). G ELISA quantification of CTGF and PDGF secretion in HK-2 cells ( n = 3). H Western blot analysis of E-cadherin, N-cadherin and Vimentin expression in TGF-β1-induced HK-2 cells. Quantification of gray values for Western blotting results in ( H ) was performed using ImageJ ( n = 3). I Representative flow cytometry plots showing the detection of MMP. J Quantitative analysis of flow cytometry fluorescence intensity of ROS ( n = 3). * p < 0.05 vs. Con group, # p < 0.05 vs. TGF-β1 group. a p < 0.05 vs. HP group, b p < 0.05 vs. AST group.

Article Snippet: The cells were then resuspended in 1× JC-1 (C2003S, Beyotime, Shanghai, China) staining solution or 50 μM ROS staining solution (KeyGen Biotech Co., Ltd., Jiangsu, China), and incubated at 37 °C in the dark for 20 and 30 min followed by two washing steps with HBSS.

Techniques: Staining, Software, Western Blot, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry