murine leydig cell line tm3  (ATCC)

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: Fluoride impairs male reproductive capacity by differentially regulating autophagy of testicular somatic cells in mice. (A to F) Six-week-old male mice freely drank distilled water with or without 100 mg/l NaF for 18 weeks. (A) Representative images of H&E staining on testis. Scale bars, 100 μm (left) and 50 μm (right). (B) Representative microscopic views of sperm morphology. Scale bars, 50 μm. (C) Sperm quality. (D and E) Representative transmission electron microscopy images of autophagosomes in (D) Leydig cells and (E) Sertoli cells [scale bars, 2 μm (bottom) and 1 μm (top)], with red arrows indicating autophagosomes. (F) Immunohistochemistry was used to detect the protein expression of LC3B and p62 in Leydig cells and Sertoli cells. (G and H) Testicular somatic cells were treated with various concentrations of NaF for 24 h. The relative expression of LC3B and p62 proteins in (G) TM3 cells, (H) TM4 cells, and (I) TM4 cells cotreated with 20 μM chloroquine. Cell viability of (J) TM3 and (K) TM4 cells that were treated with 0.25 mM NaF and 20 μM chloroquine for 24 h or pretreated with 20 nM rapamycin for 1 h. All values in the figure are means ± SEM. n ≥ 3. * P < 0.05, ** P < 0.01 versus control; # P < 0.05, ## P < 0.01 versus chloroquine.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Staining, Transmission Assay, Electron Microscopy, Immunohistochemistry, Expressing, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: miR-34a-5p was selected as the key miRNA in the differential regulation of autophagy in testicular somatic cells by fluoride. (A) Network toxicological analysis results of miRNAs related to fluoride-induced male reproductive injury. (B) HAMDB database analysis results of autophagy-related candidate miRNAs. (C and D) qRT-PCR revealed autophagy-related candidate miRNA expression changes in (C) TM3 and (D) TM4 cells after fluoride treatment. (E) FISH of miR-34a-5p in the testis of control and fluoride-treated mice (dashed areas indicate Leydig cells, and white arrows indicate Sertoli cells). All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Quantitative RT-PCR, Expressing, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: Fluoride differentially regulates miR-34a-5p to modulate autophagy in testicular somatic cells. (A to F) Testicular somatic cells were treated with lentiviruses expressing miR-34a-5p/miR-NC and Inhibitor-miR-34a-5p/Inhibitor-NC. (A and D) Relative expression of miR-34a-5p. (B, C, E, and F) Relative expression of LC3B and p62 proteins. (G to I) miR-34a-5p overexpression rescued fluoride-treated TM3 cells. (G) Relative expression of miR-34a-5p. (H and I) Relative expression of LC3B and p62 proteins. (J to L) miR-34a-5p knockdown rescued fluoride-treated TM4 cells. (J) Relative expression of miR-34a-5p. (K and L) Relative expression of LC3B and p62 proteins. All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01 versus control, miR-NC, or Inhibitor-NC; # P < 0.05, ## P < 0.01 versus 0.25 mM NaF.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Expressing, Over Expression, Knockdown, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: miR-34a-5p targets REST in testicular somatic cells exposed to fluoride. (A) Expression changes of p62 mRNA in TM3 and TM4 cells after fluoride treatment and overexpression or knockdown of miR-34a-5p. (B) Prediction results of miR-34a-5p target genes by miRWalk combined with ChEA3 database. (C and F) Differential expression of the top 15 predicted target genes after fluoride treatment in (C) TM3 and (F) TM4 cells. (D, E, G, and H) Relative mRNA expression of Elf1 , Foxo1 , Foxp1 , Klf10 , Mef2a , Rest , Smad5 , and Yy1 . (I) Schematic showing the targeted binding sites of miR-34a-5p to FOXO1 and REST 3′ UTR. (J and K) The histogram shows the relative fluorescence intensity of each group. (L) FISH of miR-34a-5p and Rest mRNA in testicular tissue from control mice (white arrow indicates the positive area within the Sertoli cells or Leydig cells). (M and N) Differential expression of REST protein in testicular somatic cells after overexpression or knockdown of miR-34a-5p. All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Expressing, Over Expression, Knockdown, Quantitative Proteomics, Binding Assay, Fluorescence, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: miR-34a-5p targets REST to regulate autophagy in testicular somatic cells. (A to F) TM3 and TM4 cells were treated with lentivirus expressing OE-REST, si-REST, OE-NC, or si-NC. (A and D) Relative Rest mRNA expression. (B, C, E, and F) Relative LC3B, p62, and REST protein expression. (G to K) REST overexpression rescued TM3 and TM4 cells overexpressing miR-34a-5p. (G and H) Relative miR-34a-5p, Rest , and p62 mRNA expression. (I to K) Relative REST, LC3B, and p62 protein levels. All values in the figure are means ± SEM. n = 3. ns indicates not significant; * P < 0.05, ** P < 0.01 versus control; # P < 0.05, ## P < 0.01 versus miR-34a-5p.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Expressing, Over Expression, Control

Journal: Research

Article Title: Environmental Fluoride Compromises Male Fertility: Differentially Modulated miR-34a-5p Targets REST to Regulate Autophagy in Testicular Somatic Cells

doi: 10.34133/research.1113

Figure Lengend Snippet: Fluoride differentially regulates REST to modulate autophagy in testicular somatic cells. (A) Representative image of Rest mRNA FISH in testes from control and fluoride-treated mice. (B) Representative immunofluorescence staining of REST proteins in testes from control and fluoride-treated mice. Dashed areas indicate Leydig cells, and white arrows indicate Sertoli cells. (C and D) Relative REST protein expression in testicular somatic cells treated with fluoride. (E to G) REST knockdown rescued fluoride-treated TM3 cells. (E) Relative expression of Rest mRNA. (F and G) Relative expression of REST, p62, and LC3B proteins. (H to J) REST overexpression rescued fluoride-treated TM4 cells. (H) Relative expression of Rest mRNA. (I and J) Relative expression of REST, p62, and LC3B proteins. All values in the figure are means ± SEM. n = 3. * P < 0.05, ** P < 0.01 versus control, si-NC, or OE-NC; # P < 0.05, ## P < 0.01 versus 0.25 mM NaF.

Article Snippet: Murine Leydig cell line (TM3) and murine Sertoli cell line (TM4) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).

Techniques: Control, Immunofluorescence, Staining, Expressing, Knockdown, Over Expression

Journal: Materials Today Bio

Article Title: Generating tolerance through in situ recruitment of regulatory T cells for allogeneic cell transplantation in a bioengineered lymphoid platform

doi: 10.1016/j.mtbio.2025.102469

Figure Lengend Snippet: Immunomodulatory agents do not affect viability of TM3-Venus-Akaluc cells. MTT cytotoxicity assay assessing viability of TM3-Venus-Akaluc cells following incubation with low or high concentrations of individual immunomodulatory agents at either 48 or 72 h post treatment: (A) CCL22 (low: 3 μg/mL, high: 15 μg/mL), (B) IL-2 (low: 0.6 μg/mL, high: 3 μg/mL), (C) JES6 (low: 3 μg/mL, high: 1.5 μg/mL), (D) MR1 (low: 3 μg/mL, high: 15 μg/mL), (E) anti-CD8 (low: 6 μg/mL, high: 30 μg/mL), (F) combined cocktail of all agents at the concentrations listed above. ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.001. Data represent mean ± SD from six biological replicates. Statistical significance determined via two-way ANOVA.

Article Snippet: TM3 murine Leydig cell line was purchased from ATCC (Virginia, USA).

Techniques: Cytotoxicity Assay, Incubation

Journal: Materials Today Bio

Article Title: Generating tolerance through in situ recruitment of regulatory T cells for allogeneic cell transplantation in a bioengineered lymphoid platform

doi: 10.1016/j.mtbio.2025.102469

Figure Lengend Snippet: NanoLymph delivery of Treg-promoting immunomodulatory agents prolongs persistence of transplanted payload. (A) Representative in vivo bioluminescent IVIS images of TM3-Venus-Akaluc cells transplanted into the NanoLymph device, with the drug reservoir loaded with PBS or various combinations of immunomodulatory agents, tracked over a 39-day period. (B) Normalized radiance efficiency over time and (C) Transplant survival probability, defined as bioluminescent signal retention above 25 % of initial signal intensity, across treatment groups over 39 days. (D) Flow cytometric analysis of immune cell populations within the NanoLymph device on day 31: CD25 + FoxP3 + CCR4 + cells among CD4 + T cells, CD8 + T cells among CD45 + cells, and the ratio of CD25 + FoxP3 + Tregs to CD8 + T cells, and CD4 + T cells among CD45 + cells (E) Corresponding flow cytometric quantification in the spleen on day 31: CD8 + T cells among CD45 + cells and the Treg-to-CD8 + T cell ratio. ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.001. Data represent mean ± SD from four to ten biological replicates. Statistical significance determined via one-way ANOVA.

Article Snippet: TM3 murine Leydig cell line was purchased from ATCC (Virginia, USA).

Techniques: In Vivo