Journal: Redox Biology

Article Title: Protein palmitoylation-mediated palmitic acid sensing causes blood-testis barrier damage via inducing ER stress

doi: 10.1016/j.redox.2022.102380

Figure Lengend Snippet: PA damages cell barrier and induces ER stress in Sertoli cell. (A) TER detection of primary Sertoli cell barriers. The cells were incubated with (PA) or without (Control) 0.4 mM PA for 3 days after barriers were formed on day 4 (n = 5). (B) FITC-dextran permeability assessment of primary Sertoli cell barriers. The cells were treated with (PA) or without (Control) 0.4 mM PA for 24 h after cell barriers were formed (n = 5). (C, D) Subcellular localization of PA. Fluorescently marked PA (BODIPY® FL C16) colocalized with (C) ER (ER-Tracker Red), but not (D) mitochondria (MitoRed). The nuclei were stained with Hoechst. Scale bar: 5 μm. White arrowheads, PA and ER colocalization. (E) Ultrastructural changes in the ER of TM4 cells treated with (PA) or without (Control) 0.4 mM PA for 24 h were observed by transmission electron microscopy. The lower panels show magnifications of the boxed areas in the relevant upper panels, revealing ribosomes lining the ER membranes. Scale bar: 1 μm. (F, G) Observation of ER distribution in TM4 Sertoli cells by ER-Tracker Green staining. The cells were incubated with (PA) or without (Control) 0.4 mM PA for 30 min (F) or 24 h (G). The nuclei were stained with Hoechst. Scale bar: 10 μm. White arrowheads, reticular structures at the periphery of nuclei. Time-lapse observations of ER distribution were shown in the form of videos in Supplementary files ( and ). (H) Translational expression levels of ER stress-related genes were analyzed using western blotting. The relative intensities of bands were quantified by ImageJ and normalized to β-actin levels (n = 3). (I) ROS detection using DCFH-DA staining. The fluorescence densities were calculated using ImageJ (n = 3). Scale bar: 50 μm. In Panels G and H, TM4 cells were incubated with (PA) or without (Control) 0.4 mM PA for 24 h. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. Control group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mouse TM4 Sertoli cells (iCell Bioscience, Inc., Shanghai, China) were cultured in DMEM/F12 supplemented with 10% FBS at 37 °C under a 5% CO 2 atmosphere.

Techniques: Incubation, Control, Permeability, Staining, Transmission Assay, Electron Microscopy, Expressing, Western Blot, Fluorescence

Journal: Redox Biology

Article Title: Protein palmitoylation-mediated palmitic acid sensing causes blood-testis barrier damage via inducing ER stress

doi: 10.1016/j.redox.2022.102380

Figure Lengend Snippet: Inhibition of protein palmitoylation ameliorates PA induced Sertoli cell dysfunction. (A) Analysis of the palmitoylation levels of proteins extracted from the testes of mice administered or not administered the PA injection, with or without gavage of 2-BP (n = 6 for Control, n = 7 for PA and 2-BP + PA). (B) Analysis of the palmitoylation levels of proteins extracted from primary Sertoli cells, Leydig cells and germ cells, which were treated with or without 0.4 mM PA (n = 3). (C) Inhibition of palmitoylation by 2-BP suppressed PA-induced ER stress in Sertoli cells. Translational expression levels of ER stress-related genes were analyzed using Western blotting (n = 3). (D) ROS detection using DCFH-DA staining. The fluorescence densities were calculated using ImageJ (n = 3). Scale bar: 50 μm. (E) TER detection of primary Sertoli cell barriers. The cells were incubated with PA (PA), with PA combined with 2-BP (2-BP + PA), or with the vehicle (Control) for 3 days after barriers were formed on day 4 (n = 5). (F) FITC-dextran permeability assessment of primary Sertoli cell barriers. The cells were treated PA (PA), with PA combined with 2-BP (2-BP + PA), or with the vehicle (Control) for 24 h after cell barriers were formed (n = 5). (G) Tight junction protein levels were examined by western blotting in TM4 Sertoli cells (n = 3). The relative intensities of bands in western blotting results were quantified by ImageJ and normalized to β-actin levels. Data are presented as mean ± SD. n. s., no significant difference vs. Control group. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. Control group. # P < 0.05, ## P < 0.01 and ### P < 0.001 vs. PA group.

Article Snippet: Mouse TM4 Sertoli cells (iCell Bioscience, Inc., Shanghai, China) were cultured in DMEM/F12 supplemented with 10% FBS at 37 °C under a 5% CO 2 atmosphere.

Techniques: Inhibition, Injection, Control, Expressing, Western Blot, Staining, Fluorescence, Incubation, Permeability

Journal: Redox Biology

Article Title: Protein palmitoylation-mediated palmitic acid sensing causes blood-testis barrier damage via inducing ER stress

doi: 10.1016/j.redox.2022.102380

Figure Lengend Snippet: Identification of palmitoylated proteins regulated by PA. (A) The flowchart illustrating the mass spectrum analysis of palmitoylated proteins regulated by PA. (B – D) GO (B), COG (C) and KEGG pathway (D) analysis of proteins whose palmitoylation levels were up-regulated after PA treatment in TM4 cells. (E) Detection of palmitoylation levels of ER proteins predicted to be regulated by PA. TM4 cells were treated by PA, with or without 2-BP pretreatment.

Article Snippet: Mouse TM4 Sertoli cells (iCell Bioscience, Inc., Shanghai, China) were cultured in DMEM/F12 supplemented with 10% FBS at 37 °C under a 5% CO 2 atmosphere.

Techniques:

Journal: Redox Biology

Article Title: Protein palmitoylation-mediated palmitic acid sensing causes blood-testis barrier damage via inducing ER stress

doi: 10.1016/j.redox.2022.102380

Figure Lengend Snippet: The palmitoylation of calnexin is involved in PA-induced cell barrier disruption in Sertoli cells. (A) Sequences of wild type (WT) and mutated calnexin fragments. The predicted palmitoylation sites are marked with red color, and the mutated sites are marked with green color. (B) The palmitoylation of exogenous calnexin was validated, and sites 8, 503 and 504 were found to be its palmitoylation target sites. (C) The palmitoylation of exogenous calnexin was up-regulated by PA, while the mutation of all three target sites diminished palmitoylation. (D) Western blotting results indicated that mutation of all three palmitoylation target sites in calnexin alleviated PA-induced upregulation of CHOP (n = 3). (E) Mutation of all three palmitoylation target sites in calnexin alleviated PA-induced ROS production in Sertoli cells. ROS production was detected using DCFH-DA staining. The fluorescence densities were calculated using ImageJ (n = 3). Scale bar: 50 μm. (F, G) Mutation of all three palmitoylation target sites in calnexin ameliorated PA-damaged Sertoli cell barrier. TER detection (F, n = 7) and FITC-dextran permeability assays (G, n = 5) were used to detect the cell barrier integrity. (H) Tight junction protein levels were examined by western blotting. The relative intensities of bands were quantified by ImageJ and normalized to β-actin levels (n = 3). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. Control group; # P < 0.05 and ## P < 0.01 vs. PA group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mouse TM4 Sertoli cells (iCell Bioscience, Inc., Shanghai, China) were cultured in DMEM/F12 supplemented with 10% FBS at 37 °C under a 5% CO 2 atmosphere.

Techniques: Disruption, Mutagenesis, Western Blot, Staining, Fluorescence, Permeability, Control

Journal: Redox Biology

Article Title: Protein palmitoylation-mediated palmitic acid sensing causes blood-testis barrier damage via inducing ER stress

doi: 10.1016/j.redox.2022.102380

Figure Lengend Snippet: ω-3 PUFAs ameliorate PA-induced Sertoli cell dysfunction and protein over-palmitoylation. (A) ω-3 PUFAs suppressed PA-induced ER stress in Sertoli cells. TM4 cells were treated by PA, with or without a pretreatment of ω-3 PUFAs. Translational expression levels of ER stress-related genes were analyzed using Western blotting. The relative intensities of bands were quantified by ImageJ and normalized to β-actin level (n = 3). (B) ROS detection using DCFH-DA staining. The fluorescence densities were calculated using ImageJ (n = 3). Scale bar: 50 μm. (C, D) Assessment of cell barrier integrity in vitro . After cell barrier formation, cells were incubated with PA (PA), with PA combined with ω-3 (ω-3 + PA), or with the vehicle (Control). TER detection (C, n = 8) and FITC-dextran permeability assays (D, n = 5) were used to analyze the integrity of primary Sertoli cell barriers. (E) Tight junction protein levels in TM4 Sertoli cells were examined by western blotting. The relative intensities of bands were quantified by ImageJ and normalized to β-actin levels (n = 3). (F) Analysis of the palmitoylation levels of proteins extracted from PA-treated TM4 cells with or without a pretreatment with ω-3 PUFAs. The relative intensities of bands were quantified by ImageJ and normalized to β-actin levels (n = 3). (G) Detection of palmitoylation levels of CNX in TM4 cells treated by PA, with or without pretreatment of ω-3 PUFAs. Data are presented as mean ± SD. ** P < 0.01 and *** P < 0.001 vs. Control group. # P < 0.05, ## P < 0.01 and ### P < 0.001 vs. PA group.

Article Snippet: Mouse TM4 Sertoli cells (iCell Bioscience, Inc., Shanghai, China) were cultured in DMEM/F12 supplemented with 10% FBS at 37 °C under a 5% CO 2 atmosphere.

Techniques: Expressing, Western Blot, Staining, Fluorescence, In Vitro, Incubation, Control, Permeability

Journal: Redox Biology

Article Title: Protein palmitoylation-mediated palmitic acid sensing causes blood-testis barrier damage via inducing ER stress

doi: 10.1016/j.redox.2022.102380

Figure Lengend Snippet: Mechanism of Sertoli cell barrier disruption induced by PA. PA enters Sertoli cells, penetrates ER, and palmitoylates ER proteins. Palmitoylated CNX and other ER proteins activate ER stress, especially PERK pathway, promote cell apoptosis and down-regulate tight junction proteins by inducing CHOP expression, and finally disrupt Sertoli cell barrier. On the other hand, ω-3 PUFAs alleviates the palmitoylation of ER proteins and Sertoli cell dysfunction induced by PA.

Article Snippet: Mouse TM4 Sertoli cells (iCell Bioscience, Inc., Shanghai, China) were cultured in DMEM/F12 supplemented with 10% FBS at 37 °C under a 5% CO 2 atmosphere.

Techniques: Disruption, Expressing