Journal: Cell Death Discovery

Article Title: Diazinon induces testicular dysfunction and testicular cell damage through increased reactive oxygen species production in mouse

doi: 10.1038/s41420-025-02399-8

Figure Lengend Snippet: A Immunostaining of SOX9, a Sertoli cell marker, in testes of DZN-treated and control mice. Scale bar = 100 µm. B The number of SOX9+ cells in testicular tubules. At least 40 tubules were scored for each testis (five to six biological replicates). Values on graph are presented as the mean ± SD. C Gene expression levels of Sox9 , Amh, and Wt1 in testis from each group. Values on graph are presented as the mean ± SD (log2 scale). D Protein expression levels of SOX9 in testes of DZN-treated and control mice. The graph shows relative protein expression levels (mean ± SD, n = 5). E Immunostaining of HSD3β1 in testes of DZN-treated and control mice. Scale bar = 100 µm. F Concentration of serum testosterone in DZN-treated and control mice. Values on graph are presented as the mean ± SD. G Expression levels of Hsd17b3 , Cyp17a1 , Hsd3b1 , and Cyp11a1 in testis from each group. Values on graph are presented as the mean ± SD (log2 scale) (n = 5). H Protein expression levels of HSD3β1 in testes of DZN-treated and control mice. The graph shows the relative protein levels (mean ± SD, n = 5). Asterisks indicate significant differences between the treatment and control groups at *p < 0.05, **p < 0.01, and ***p < 0.001. All statistical analyzes for the graphs were conducted using a t-test.

Article Snippet: Three testicular cell lines, GC-1 spermatogonia (spg), TM3 Leydig, and TM4 Sertoli cells, were purchased from the Korea Cell Bank (KCLB, Seoul, South Korea).

Techniques: Immunostaining, Marker, Control, Gene Expression, Expressing, Concentration Assay

Journal: Cell Death Discovery

Article Title: Diazinon induces testicular dysfunction and testicular cell damage through increased reactive oxygen species production in mouse

doi: 10.1038/s41420-025-02399-8

Figure Lengend Snippet: A Cell viability was assessed in GC1 spg, TM3, and TM4 cells following treatment with DZN (0–700 µM for 24 h and 48 h). DMSO was used as the control treatment. Values on graph are presented as the mean ± SD of three independent experiments. B Ki67 immunostaining of GC1 spg, TM3, and TM4 cells exposed to 0–300 µM DZN for 24 h. Scale bar = 100 µm. C Graph showed the percentage of Ki67 positive cell in GC-1 spg, TM3, and TM4 cells after DZN treatment at 24 h (n = 3). Asterisks indicate significant differences between the treatment and control groups at **p < 0.01 and ***p < 0.001.

Article Snippet: Three testicular cell lines, GC-1 spermatogonia (spg), TM3 Leydig, and TM4 Sertoli cells, were purchased from the Korea Cell Bank (KCLB, Seoul, South Korea).

Techniques: Control, Immunostaining

Journal: Cell Death Discovery

Article Title: Diazinon induces testicular dysfunction and testicular cell damage through increased reactive oxygen species production in mouse

doi: 10.1038/s41420-025-02399-8

Figure Lengend Snippet: A Detection of in situ DNA fragmentation using a TUNEL assay. TUNEL-positive nuclei (red fluorescent) increased in a dose-dependent manner in DZN-treated GC-1 spg, TM3, and TM4 cells. Scale bar = 100 µm. The graphs present Tunnel/DAPI rate analysis as a percentage (mean ± SD, n = 5). B Representative flow cytometry plots for Annexin V (FITC) and propidium iodide (PI) stained cells. The graphs present apoptosis rate analysis as a percentage of apoptotic cells number (mean ± SD, n = 5). Asterisks indicate significant differences between the treatment and control groups at **p < 0.01 and ***p < 0.001.

Article Snippet: Three testicular cell lines, GC-1 spermatogonia (spg), TM3 Leydig, and TM4 Sertoli cells, were purchased from the Korea Cell Bank (KCLB, Seoul, South Korea).

Techniques: In Situ, TUNEL Assay, Flow Cytometry, Staining, Control

Journal: Cell Death Discovery

Article Title: Diazinon induces testicular dysfunction and testicular cell damage through increased reactive oxygen species production in mouse

doi: 10.1038/s41420-025-02399-8

Figure Lengend Snippet: A Each cell was double stained with CellROX® FITC and MitoTracker Red to measure oxidative stress levels. CellROX® staining indicates oxidative stress, whereas the MitoTracker Red CMXRos signal indicates changes in mitochondrial membrane potential. Scale bar = 50 μm. B Flow cytometry dot plots for GC-1 spg, TM3, and TM4 cells stained with CellROX® for quantification. Histograms of ROS production obtained by flow cytometry after CellROX® staining of DZN-treated GC-1 spg, TM3, and TM4 cells. C Graph presents the quantitative change of CellROX in each group, and data are presented as the mean ± SD of five independent replicates. Asterisks indicate significant differences between the treatment and control groups at *p < 0.05 and ***p < 0.001.

Article Snippet: Three testicular cell lines, GC-1 spermatogonia (spg), TM3 Leydig, and TM4 Sertoli cells, were purchased from the Korea Cell Bank (KCLB, Seoul, South Korea).

Techniques: Staining, Membrane, Flow Cytometry, Control

Journal: Cell Death Discovery

Article Title: Diazinon induces testicular dysfunction and testicular cell damage through increased reactive oxygen species production in mouse

doi: 10.1038/s41420-025-02399-8

Figure Lengend Snippet: A Sod1, Sod2, Cat, Gpx, Ho-1, Nrf2 , and Nqo1 expression in GC1-spg cells treated with 300 µM DZN for 24 h. Values on graph are presented as the mean ± SD (log2 scale) (n = 5). B Protein expression levels of Bad, Cleaved-caspase 3, Caspase 3, Phospho-p53, P53, and HO-1 in GC1-spg cells exposed to 0–300 µM DZN. C Graph shows relative protein levels (mean ± SD, n = 5). D Sod1 , Sod2 , Cat , Gpx , Ho-1 , Nrf2 , and Nqo1 expression in TM3 cells before and after exposure to 300 µM DZN. Values on graph are presented as the mean ± SD (log2 scale). E Protein expression levels of Bad, Cleaved-caspase 3, Caspase 3, Phospho-p53, P53, and HO-1 in TM3 cells exposed to 0–300 µM DZN. F Graph shows relative protein levels (mean ± SD, n = 5). G Sod1 , Sod2 , Cat , Gpx , Ho-1 , Nrf2 , and Nqo1 expression in TM4 cells before and after exposure to DZN. Values on graph are presented as the mean ± SD (log2 scale). H Protein expression levels of Bad, Cleaved-caspase 3, Caspase 3, Phospho-p53, P53 and HO-1 in TM4 cells exposed to 0, 100, 200 and 300 µM DZN. I Graph shows relative protein levels (mean ± SD, n = 5). Asterisks indicate significant differences between the treatment and control groups at *p < 0.05, ** p < 0.01, and ***p < 0.001.

Article Snippet: Three testicular cell lines, GC-1 spermatogonia (spg), TM3 Leydig, and TM4 Sertoli cells, were purchased from the Korea Cell Bank (KCLB, Seoul, South Korea).

Techniques: Expressing, Control

Journal: Cell Death Discovery

Article Title: Diazinon induces testicular dysfunction and testicular cell damage through increased reactive oxygen species production in mouse

doi: 10.1038/s41420-025-02399-8

Figure Lengend Snippet: A Sod1 , Cat , Ho-1 , and Nrf2 expression in GC-1 spg cells for each treatment (control, DZN, NAC, and DZN + NAC). Values on graph are presented as the mean ± SD (log2 scale). B Protein expression levels of Bad, Cleaved-caspase 3, Caspase 3, Phospho-p53, P53, and HO-1 in GC-1 spg cells for each treatment. C Graph shows relative protein levels (mean ± SD, n = 5). D Sod1 , Cat , Ho-1 , and Nrf2 expression in TM3 cells for each treatment. Values on graph are presented as the mean ± SD (log2 scale). E Protein expression levels of Bad, Cleaved-caspase 3, Caspase 3, Phospho-p53, P53, and HO-1 in TM3 cells. F Graph shows relative protein levels (mean ± SD, n = 5). G Sod1 , Cat , Ho-1 , and Nrf2 expression in TM4 cells for each treatment. Values on graph are presented as the mean ± SD (log2 scale). H Protein expression levels of Bad, Cleaved-caspase 3, Caspase 3, Phospho-p53, P53, and HO-1 in TM4 cells for each treatment (control, DZN, NAC, and D + N). I Graph shows relative protein levels (mean ± SD, n = 5). Asterisks indicate significant differences at *p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Three testicular cell lines, GC-1 spermatogonia (spg), TM3 Leydig, and TM4 Sertoli cells, were purchased from the Korea Cell Bank (KCLB, Seoul, South Korea).

Techniques: Expressing, Control

Journal: BMC Pharmacology & Toxicology

Article Title: In vitro evaluation of cell viability and expression profile of growth factors in mouse Sertoli cells exposed to Delta-9-tetrahydrocannabinol: a mechanistic insight into the cannabinoid-induced testicular toxicity

doi: 10.1186/s40360-023-00704-8

Figure Lengend Snippet: The effect of different concentrations of THC on TM4 cell viability. The effects of different concentrations of THC on TM4 cell viability measured by MTT assay following 24-h exposure period. Each bar represents the mean ± SEM, obtained from three independent experiments ( n = 3). As the concentration of THC increased, cellular viability gradually decreased and reached approximately 75% of the control level at 50 μM of THC. Stars show the statistical significance of change among the groups. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control group (One–way ANOVA with LSD correction for multiple comparisons)

Article Snippet: The Mus musculus Sertoli cell line, TM4 (purchased from Pasteur Institute, Tehran, Iran) was cultured in 25 cm 2 tissue flasks, and grown in Dulbecco’s modified Eagle’s medium (DMEM)–F12/HEPES media (DNAbiotech, Iran) supplemented with 5% fetal bovine serum (Atocel, Austria), penicillin (50 units/mL), and streptomycin (50 units/mL).

Techniques: MTT Assay, Concentration Assay, Control

Journal: BMC Pharmacology & Toxicology

Article Title: In vitro evaluation of cell viability and expression profile of growth factors in mouse Sertoli cells exposed to Delta-9-tetrahydrocannabinol: a mechanistic insight into the cannabinoid-induced testicular toxicity

doi: 10.1186/s40360-023-00704-8

Figure Lengend Snippet: mRNA expression of caspase-3 in THC-exposed TM4 cells. The effect of THC (50 μM) for a duration exposure period of 24-h on caspase-3 gene expression level measured by RT-PCR in TM4 Sertoli cells. Caspase-3 mRNA expression was normalized to β-actin in each sample. THC significantly increased caspase-3 expression level. Data represent the mean ± SEM of three independent experiments (n = 3). Star shows the statistical significance of change between the groups. * p < 0.05 vs. control group (Mann–Whitney U-test)

Article Snippet: The Mus musculus Sertoli cell line, TM4 (purchased from Pasteur Institute, Tehran, Iran) was cultured in 25 cm 2 tissue flasks, and grown in Dulbecco’s modified Eagle’s medium (DMEM)–F12/HEPES media (DNAbiotech, Iran) supplemented with 5% fetal bovine serum (Atocel, Austria), penicillin (50 units/mL), and streptomycin (50 units/mL).

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, MANN-WHITNEY

Journal: BMC Pharmacology & Toxicology

Article Title: In vitro evaluation of cell viability and expression profile of growth factors in mouse Sertoli cells exposed to Delta-9-tetrahydrocannabinol: a mechanistic insight into the cannabinoid-induced testicular toxicity

doi: 10.1186/s40360-023-00704-8

Figure Lengend Snippet: mRNA expression of growth factors genes in THC-exposed TM4 cells. The effect of THC (50 μM) for a duration exposure period of 24-h on gene expression level of a number of key testicular growth factors VEGF ( A ), EGF ( B ), FGF ( C ), and GDNF ( D ) measured by RT-PCR in TM4 Sertoli cells. mRNA expressions were normalized to β-actin in each sample. THC significantly decreased growth factors expression levels. Data represent the mean ± SEM of three independent experiments ( n = 3). Stars show the statistical significance of change between the groups. * p < 0.05 and *** p < 0.001 vs. control groups (Mann–Whitney U-test)

Article Snippet: The Mus musculus Sertoli cell line, TM4 (purchased from Pasteur Institute, Tehran, Iran) was cultured in 25 cm 2 tissue flasks, and grown in Dulbecco’s modified Eagle’s medium (DMEM)–F12/HEPES media (DNAbiotech, Iran) supplemented with 5% fetal bovine serum (Atocel, Austria), penicillin (50 units/mL), and streptomycin (50 units/mL).

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, MANN-WHITNEY

Journal: BMC Pharmacology & Toxicology

Article Title: In vitro evaluation of cell viability and expression profile of growth factors in mouse Sertoli cells exposed to Delta-9-tetrahydrocannabinol: a mechanistic insight into the cannabinoid-induced testicular toxicity

doi: 10.1186/s40360-023-00704-8

Figure Lengend Snippet: The expression of GDNF and FGF proteins in THC-exposed TM4 cells. A Immunoblotting images of expression level of GDNF, FGF, and β-actin proteins. B and C Bar graphs represent the relative density of each band normalized to that of β-actin as an internal control. Values represent the mean ± SEM of three independent experiments. Stars show the statistical significance of change between the groups. *** p < 0.001 vs. control group (Mann–Whitney U-test)

Article Snippet: The Mus musculus Sertoli cell line, TM4 (purchased from Pasteur Institute, Tehran, Iran) was cultured in 25 cm 2 tissue flasks, and grown in Dulbecco’s modified Eagle’s medium (DMEM)–F12/HEPES media (DNAbiotech, Iran) supplemented with 5% fetal bovine serum (Atocel, Austria), penicillin (50 units/mL), and streptomycin (50 units/mL).

Techniques: Expressing, Western Blot, Control, MANN-WHITNEY

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: Pharmaceuticals

Article Title: Icariin as a Treatment Proposal in Mammalian Reproduction

doi: 10.3390/ph17091104

Figure Lengend Snippet: Summary of the main studies on the effects of ICA on male reproduction.

Article Snippet: In vivo In vitro , Male Sprague–Dawley rats Sertoli cell line TM4 , 2 and 6 mg/kg, 4 months 0.5 and 1 μM, 20 h , ICA significantly increased the testicular and epididymal weights and their indices in ageing rats. Furthermore, ICA increased sperm count and sperm viability. ICA protected against Sertoli cell injury due to age-related testicular dysfunction by upregulating the ERa/Nrf2 signalling pathway. , [ ] .

Techniques: In Vivo, Expressing, In Vitro, Activity Assay, Clinical Proteomics, Concentration Assay

Journal: International Journal of Fertility & Sterility

Article Title: Zinc Protects against MDMA-Induced Apoptosis of Sertoli Cells in Mouse via Attenuation of Caspase-3

doi: 10.22074/ijfs.2020.44410

Figure Lengend Snippet: Impact of MDMA on TM4 cells. Evaluation of MDMA effect on TM4 cells in the tested groups showed significant differences in tested groups versus controls after 24 and 48 hours. **; P<0.01, ***; P<0.001, ****; P<0.0001, and MDMA; 3,4-Methylenedioxymethamphetamine.

Article Snippet: TM4 mouse cell line (Sertoli cells) was purchased from the Pasteur Institute of Iran and used at this study.

Techniques:

Journal: International Journal of Fertility & Sterility

Article Title: Zinc Protects against MDMA-Induced Apoptosis of Sertoli Cells in Mouse via Attenuation of Caspase-3

doi: 10.22074/ijfs.2020.44410

Figure Lengend Snippet: The impact of Zinc on TM4 cells. Analysis of the Zinc effect on the tested groups showed significant differences in the tested groups versus. controls after 24 and 48 hours. *; P<0.05, ***; P<0.01, and ****; P<0.001.

Article Snippet: TM4 mouse cell line (Sertoli cells) was purchased from the Pasteur Institute of Iran and used at this study.

Techniques: