Journal: PLoS ONE

Article Title: Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

doi: 10.1371/journal.pone.0150143

Figure Lengend Snippet: Activation of Erk1/2 and transcription factors CREB and ATF-1 by DHEAS in TM4 cells detected by immunofluorescence: TM4 cells were incubated with or without 1 μM DHEAS for 120 min and then stained with specific antibodies against either phospho-Erk1/2 (A-B), phospho-CREB (D-E) or phospho-ATF-1 (G-H) and a secondary Alexa Fluor 488-labeled antibody as described under “Methods”. Green fluorescence indicates the phosphorylation of Erk1/2 (A-B), CREB (D-E) of ATF-1 (G-H). Nuclei of the cells were labeled with DAPI and appear blue. (A, D and G) Fluorescence of phospho-Erk1/2, phospho-CREB or phospho-ATF-1 in the absence of DHEAS. (B, E and H) Phosphorylation of Erk1/2, CREB and ATF-1 in response to DHEAS (1 μM) treatment. (C, F and I) Statistical analysis of the corresponding green fluorescence in arbitrary units (n = 90; means ±SEM; **p≤0.01).

Article Snippet: The Sertoli cell line TM4 [ ] was cultured in DMEM/F-12 high glucose with 2 mM L-glutamine (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% standardized fetal bovine serum (FBS) (Biochrom GmbH, Berlin, Germany) and 1% penicillin/streptomycin combination (5000 units penicillin and 5 mg streptomycin/ml) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Activation Assay, Immunofluorescence, Incubation, Staining, Labeling, Fluorescence, Phospho-proteomics

Journal: PLoS ONE

Article Title: Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

doi: 10.1371/journal.pone.0150143

Figure Lengend Snippet: TM4 cells were treated for 120 min with the indicated concentrations of DHEAS. Proteins in cell lysates were then separated on SDS polyacrylamide gels and subsequently probed in a western blot using a monoclonal antibody against either total Erk1/2 (t-Erk1/2) (A), as a loading control, or phosphorylated (activated) Erk1/2 (p-Erk1/2) (B). The western blots in (A) and (B) show typical results for the Erk1/2 bands of 42/44 kDa. (C) Statistical analysis of Erk1/2 activation as a function of DHEAS concentration in several identical experiments in which the chemiluminescence was quantified by gel image analysis software (n = 4; mean ±SEM; *p≤0.05; **p≤0.01). (D) Detection of total actin served as loading control in further western blots for the detection of either phosphorylated CREB or ATF-1 (E). The western blots in (D) and (E) show representative results from several identical experiments using the indicated concentrations of DHEAS; the quantification and statistical analysis of these results are shown in (F) and (G) (n = 4; mean±SEM; *p≤0.05; **p≤0.01).

Article Snippet: The Sertoli cell line TM4 [ ] was cultured in DMEM/F-12 high glucose with 2 mM L-glutamine (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% standardized fetal bovine serum (FBS) (Biochrom GmbH, Berlin, Germany) and 1% penicillin/streptomycin combination (5000 units penicillin and 5 mg streptomycin/ml) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot, Control, Activation Assay, Concentration Assay, Software

Journal: PLoS ONE

Article Title: Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

doi: 10.1371/journal.pone.0150143

Figure Lengend Snippet: (A) AR expression, indicated by the green fluorescence of the secondary antibody, in TM4 cells exposed to negative control siRNA (nc-siRNA). (B) Abrogation of AR expression after treatment of the cells with AR-specific siRNA (AR-siRNA). In both A and B, nuclei are stained blue. (C) Western blot showing that the expression of AR in the presence of AR-siRNA is reduced by 94 ± 5% (n = 3). (D) At the same time expression of actin is not affected by AR-siRNA, indicating that the reduction of AR in (C) is specific and not due to an overall suppression of protein expression. (E) Erk1/2 phosphorylation in response to DHEAS after abrogation of AR expression with AR-specific siRNA. (F) Total Erk1/2 levels after treatment of cells with either nc-siRNA or AR-siRNA. The western blot shown was generated from the western blot shown in (E) which was first stripped of the original antibodies and then reprobed with appropriate antibodies to detect total Erk1/2. (G) Statistical analysis of Erk1/2 activation in the presence of either nc-siRNA or AR-siRNA (n = 3; mean ±SEM; **p≤0.01).

Article Snippet: The Sertoli cell line TM4 [ ] was cultured in DMEM/F-12 high glucose with 2 mM L-glutamine (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% standardized fetal bovine serum (FBS) (Biochrom GmbH, Berlin, Germany) and 1% penicillin/streptomycin combination (5000 units penicillin and 5 mg streptomycin/ml) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Fluorescence, Negative Control, Staining, Western Blot, Phospho-proteomics, Generated, Activation Assay

Journal: PLoS ONE

Article Title: Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

doi: 10.1371/journal.pone.0150143

Figure Lengend Snippet: TM4 Sertoli cells were cultured to 80% confluence and were further incubated for 2 days in the absence or presence of 1 μM DHEAS. Nuclei were labeled with DAPI and appear blue; the Alexa fluor 488-labeled secondary antibody shows the localization of claudin-3 or -5. (A and D) Fluorescence in the absence of DHEAS; (B and E) Fluorescence after exposure of cells to 1 μM DHEAS. (C) Quantification and statistical analysis of results shown in panels (A) and (B); (F) Quantification and statistical analysis of results shown in panels (D) and (E) (in each case n = 45; means ±SEM; **p≤0.01).

Article Snippet: The Sertoli cell line TM4 [ ] was cultured in DMEM/F-12 high glucose with 2 mM L-glutamine (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% standardized fetal bovine serum (FBS) (Biochrom GmbH, Berlin, Germany) and 1% penicillin/streptomycin combination (5000 units penicillin and 5 mg streptomycin/ml) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Cell Culture, Incubation, Labeling, 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: 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

Journal: Molecular Metabolism

Article Title: AMPK inhibition and elevated angiogenin are associated with tRNA fragmentation in the male germline exposed to a high-fat diet

doi: 10.1016/j.molmet.2026.102350

Figure Lengend Snippet: AMPK inhibition-induced Angiogenin is accompanied by alteration of tRFs profile in vitro. A Western blot results of proteins related with AMPK-mTOR pathways in TM3, TM4, and NIH/3T3 cell lines using compound C (CC), rapamycin (Rapa), and MHY1485 (MHY) in TM3, TM4, and NIH/3T3 cell lines (left to right). The number represents the densiometric comparison of the expression of pAMPK and pmTOR proteins against AMPK and mTOR, respectively. B Angiogenin gene expression. Gene expression was quantified after 12 h of treatment. mRNA expression level was normalized to Actin . Data are represented as mean ± standard deviation. Statistical analysis was performed using One-way ANOVA with Tukey post-hoc test. Different letters are significantly different. C Northern blot results of tRF3-Pro and Val in mouse testis cell lines. Each sample RNAs were extracted from both TM3 and TM4 cells with CC treatment and overexpression of Angiogenin (Ang OE). Each intact tRNA was shown as a control. Band intensities of Northern blot are represented as bar graph after quantification using Image J software. Data are presented as mean ± standard deviation. One-way ANOVA was used for statistical analysis, and the different alphabets with Tukey post-hoc test. D Distribution of read counts based on small RNA type. E Distribution of reads assigned to GtRNAdb in tRF subtypes. The total bar height is proportional to the total read count for each group. Percentages indicate the relative proportion of each tRF subtype within the corresponding group, and n represents the actual read count number for each subtype. tRF5, reads derived from tRNA 5′ end among reads aligned to GtRNAdb; tRF3, reads derived from tRNA 3′ end among reads aligned to GtRNAdb; other, not included in the previous two groups. F Histogram showing the distribution of log 2 (Fold change) using reads aligned to GtRNAdb. Green, CC-treated versus non-treated; Yellow, ANG overexpression versus control. G Volcano plot depicting significantly changed tRFs in TM4 cell lines. Results from CC-treated versus non-treated are represented as circles, and results from ANG overexpression versus control are represented as triangles. n = 2 for each group.

Article Snippet: The mouse testicular TM3 cell line and TM4 Sertoli cell line were purchased from the Korean Cell Line Bank (Seoul, Korea).

Techniques: Inhibition, In Vitro, Western Blot, Comparison, Expressing, Gene Expression, Standard Deviation, Northern Blot, Over Expression, Control, Software, Derivative Assay