Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases germ cell apoptosis

doi: 10.1007/s00018-021-03757-2

Figure Lengend Snippet: The first wave of spermatogenesis is delayed in Lgals3−/− mice. a Testicular weight (mg) collected from Lgals3+/+ and Lgals3−/− mice at postnatal (P) days 5, 15, 20, 35, 50 and 100; (n = 4, * P < 0.05). b Representative histology of mouse testes from Lgals3+/+ (i, iii, v) and Lgals3−/− mice (ii, iv, vi) at P5 (i, ii), P15 (iii, iv) and P20 (v, vi). (C and D) The percentage of tubule cross sections was quantified according to the most differentiated germ cell stage visible (G gonocytes, L leptotene spermatocyte, P pachytene spermatocyte, RS round spermatid, S Sertoli cell, Sper spermatogonia, Z zygotene spermatocyte) at P15 (c) and P20 (d); (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). The data represent the means ± SEM

Article Snippet: Table 1 Primary antibodies Manufacturer Catalogue no Dilution Rabbit monoclonal anti-Bak Cell signaling technology, Germany 12,105 1:1000* Rabbit polyclonal anti-Bax Cell signaling technology, Germany 2772 1:1000* Rabbit monoclonal anti-Bcl-2 Cell signaling technology, Germany 2870 1:1000* Mouse monoclonal anti-β-actin Ac-15 Sigma-Aldrich, Germany A5441 1:5000* Rabbit monoclonal anti-galectin 3 GeneTex, USA GTX62084 1:500** Rat monoclonal anti-germ cell specific antigen (TRA98) Abcam, UK ab82527 1:1000** Rabbit polyclonal anti-HSD11B1 Abcam, UK ab39364 1:100** Rabbit polyclonal anti-Insl3 Biorbyt Ltd, UK orb648755 1:100* Rabbit polyclonal anti-PCNA Abcam, UK ab18197 1:500** Rabbit polyclonal anti-phospho-histone H3 (Ser10) Cell signaling technology, Germany 9701 1:200** Rabbit monoclonal anti-Sox9 Abcam, UK ab185230 1:250** Rabbit monoclonal anti—StAR Cell signaling technology, Germany 8449 1:1000* Secondary antibodies Manufacturer Catalogue No Dilution Alexa Fluor 448 goat anti-rabbit IgG Thermo fisher scientific, USA A11008 1:1000** Alexa Fluor 546 goat anti-rat IgG Thermo fisher scientific, USA A11081 1:1000** HRP-labelled goat anti-rabbit IgG MP biomedicals, USA 855,676 1:10,000* HRP-labelled sheep anti-mouse IgG Sigma-Aldrich, Germany A5906 1:10,000* Open in a separate window Antibody dilution used for *Western blotting or **Immunofluorescence/Immunohistochemistry.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases germ cell apoptosis

doi: 10.1007/s00018-021-03757-2

Figure Lengend Snippet: Testicular germ cell numbers are reduced in neonatal Lgals3−/− testes. a–d Identification of germ cell numbers in Lgals3+/+ and Lgals3−/− testes using Tra98 immunofluorescence staining (red) at P5 (a–d). Phosphorylated histone 3 staining (PH3, green; b, c, d) and Tra98 staining were used to distinguish between dividing germ and somatic cells (a, c, d). Part d shows the magnified areas identified by the white boxes in part (c). The thin arrow indicates germ cells; the thick arrow indicates somatic cells, both positive for PH3 (a cell proliferation marker). Nuclei were counterstained with Topro 3 (blue). e–g The numbers of germ cells (e), PH3-positive germ cells (f) and somatic cells (g) were normalized to the total cross-section area (mm2) in both Lgals3+/+ and Lgals3−/− testes. h Ddx4 mRNA expression (germ cell marker) of P5 and P15 testes was normalized to Actb and Gapdh; (n = 3–4, *P < 0.05). The data represent the means ± SEM

Article Snippet: Table 1 Primary antibodies Manufacturer Catalogue no Dilution Rabbit monoclonal anti-Bak Cell signaling technology, Germany 12,105 1:1000* Rabbit polyclonal anti-Bax Cell signaling technology, Germany 2772 1:1000* Rabbit monoclonal anti-Bcl-2 Cell signaling technology, Germany 2870 1:1000* Mouse monoclonal anti-β-actin Ac-15 Sigma-Aldrich, Germany A5441 1:5000* Rabbit monoclonal anti-galectin 3 GeneTex, USA GTX62084 1:500** Rat monoclonal anti-germ cell specific antigen (TRA98) Abcam, UK ab82527 1:1000** Rabbit polyclonal anti-HSD11B1 Abcam, UK ab39364 1:100** Rabbit polyclonal anti-Insl3 Biorbyt Ltd, UK orb648755 1:100* Rabbit polyclonal anti-PCNA Abcam, UK ab18197 1:500** Rabbit polyclonal anti-phospho-histone H3 (Ser10) Cell signaling technology, Germany 9701 1:200** Rabbit monoclonal anti-Sox9 Abcam, UK ab185230 1:250** Rabbit monoclonal anti—StAR Cell signaling technology, Germany 8449 1:1000* Secondary antibodies Manufacturer Catalogue No Dilution Alexa Fluor 448 goat anti-rabbit IgG Thermo fisher scientific, USA A11008 1:1000** Alexa Fluor 546 goat anti-rat IgG Thermo fisher scientific, USA A11081 1:1000** HRP-labelled goat anti-rabbit IgG MP biomedicals, USA 855,676 1:10,000* HRP-labelled sheep anti-mouse IgG Sigma-Aldrich, Germany A5906 1:10,000* Open in a separate window Antibody dilution used for *Western blotting or **Immunofluorescence/Immunohistochemistry.

Techniques: Immunofluorescence, Staining, Marker, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases germ cell apoptosis

doi: 10.1007/s00018-021-03757-2

Figure Lengend Snippet: Testes from Lgals3−/− mice exhibit a higher number of seminiferous tubules without germ cells and altered maturation of Sertoli cells. a–d Representative immunofluorescent labeling of germ cells with Tra98 (red; a, c, d) and Sertoli cells with Sox9 (green; b–d) in testes from Lgals3+/+ and Lgals3−/− mice at P5. Part d shows the magnified areas identified by the white boxes in part (c). The white stars indicate the seminiferous tubules without germ cells. The enumeration of tubules containing germ cells was performed on P5, P15 and P20 (i). At least 100 seminiferous tubules were counted in each testis; (n = 3, *P < 0.05). e Representative HE staining of testes from Lgals3+/+ and Lgals3−/− mice at P15. The lower panel (iii, iv, v, vi) shows the magnified areas from (i) and (ii) containing seminiferous tubules with (iii, v) or without (iv, vi) a lumen in Lgals3+/+ (i) and Lgals3−/− (ii) testes, respectively. f–g The percentage of seminiferous tubules without (f) and with (g) a lumen was quantified by counting at least 100 seminiferous tubule cross sections in each testis. (H) Relative Amh mRNA expression in testes from Lgals3+/+ and Lgals3−/− mice at P5, P15, P20 and P35 was normalized to Actb and Gapdh; (n = 3, *P < 0.05, **P < 0.01). The data represent the means ± SEM

Article Snippet: Table 1 Primary antibodies Manufacturer Catalogue no Dilution Rabbit monoclonal anti-Bak Cell signaling technology, Germany 12,105 1:1000* Rabbit polyclonal anti-Bax Cell signaling technology, Germany 2772 1:1000* Rabbit monoclonal anti-Bcl-2 Cell signaling technology, Germany 2870 1:1000* Mouse monoclonal anti-β-actin Ac-15 Sigma-Aldrich, Germany A5441 1:5000* Rabbit monoclonal anti-galectin 3 GeneTex, USA GTX62084 1:500** Rat monoclonal anti-germ cell specific antigen (TRA98) Abcam, UK ab82527 1:1000** Rabbit polyclonal anti-HSD11B1 Abcam, UK ab39364 1:100** Rabbit polyclonal anti-Insl3 Biorbyt Ltd, UK orb648755 1:100* Rabbit polyclonal anti-PCNA Abcam, UK ab18197 1:500** Rabbit polyclonal anti-phospho-histone H3 (Ser10) Cell signaling technology, Germany 9701 1:200** Rabbit monoclonal anti-Sox9 Abcam, UK ab185230 1:250** Rabbit monoclonal anti—StAR Cell signaling technology, Germany 8449 1:1000* Secondary antibodies Manufacturer Catalogue No Dilution Alexa Fluor 448 goat anti-rabbit IgG Thermo fisher scientific, USA A11008 1:1000** Alexa Fluor 546 goat anti-rat IgG Thermo fisher scientific, USA A11081 1:1000** HRP-labelled goat anti-rabbit IgG MP biomedicals, USA 855,676 1:10,000* HRP-labelled sheep anti-mouse IgG Sigma-Aldrich, Germany A5906 1:10,000* Open in a separate window Antibody dilution used for *Western blotting or **Immunofluorescence/Immunohistochemistry.

Techniques: Labeling, Staining, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases germ cell apoptosis

doi: 10.1007/s00018-021-03757-2

Figure Lengend Snippet: Testicular cells in Lgals3−/− mice experience increased apoptosis due to an imbalance of anti- and pro-apoptotic proteins. a, b TUNEL staining (arrows) of testicular sections from Lgals3+/+ mice and Lgals3−/− mice at P50. c The numbers of apoptotic germ cells were quantified in 100 tubular cross sections in testes from Lgals3+/+ and Lgals3−/− mice at P5, P15, P35, P50 and P100. d A scheme is illustrating how galectin 3 can stabilize the mitochondrial membrane by binding to Bcl-2 and inhibiting cytochrome c release. Bcl-2 inhibits activity of pro-apoptotic proteins Bak and Bax. Western blot e and densitometric analysis of Bcl-2 f Bak g and Bax h expression in testes from Lgals3+/+ and Lgals3−/− mice at P50 is shown; (n = 3, *P < 0.05; **P < 0.01; ***P < 0.001). β-actin was used as a loading control. The data represent the means ± SEM

Article Snippet: Table 1 Primary antibodies Manufacturer Catalogue no Dilution Rabbit monoclonal anti-Bak Cell signaling technology, Germany 12,105 1:1000* Rabbit polyclonal anti-Bax Cell signaling technology, Germany 2772 1:1000* Rabbit monoclonal anti-Bcl-2 Cell signaling technology, Germany 2870 1:1000* Mouse monoclonal anti-β-actin Ac-15 Sigma-Aldrich, Germany A5441 1:5000* Rabbit monoclonal anti-galectin 3 GeneTex, USA GTX62084 1:500** Rat monoclonal anti-germ cell specific antigen (TRA98) Abcam, UK ab82527 1:1000** Rabbit polyclonal anti-HSD11B1 Abcam, UK ab39364 1:100** Rabbit polyclonal anti-Insl3 Biorbyt Ltd, UK orb648755 1:100* Rabbit polyclonal anti-PCNA Abcam, UK ab18197 1:500** Rabbit polyclonal anti-phospho-histone H3 (Ser10) Cell signaling technology, Germany 9701 1:200** Rabbit monoclonal anti-Sox9 Abcam, UK ab185230 1:250** Rabbit monoclonal anti—StAR Cell signaling technology, Germany 8449 1:1000* Secondary antibodies Manufacturer Catalogue No Dilution Alexa Fluor 448 goat anti-rabbit IgG Thermo fisher scientific, USA A11008 1:1000** Alexa Fluor 546 goat anti-rat IgG Thermo fisher scientific, USA A11081 1:1000** HRP-labelled goat anti-rabbit IgG MP biomedicals, USA 855,676 1:10,000* HRP-labelled sheep anti-mouse IgG Sigma-Aldrich, Germany A5906 1:10,000* Open in a separate window Antibody dilution used for *Western blotting or **Immunofluorescence/Immunohistochemistry.

Techniques: TUNEL Assay, Staining, Membrane, Binding Assay, Activity Assay, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases germ cell apoptosis

doi: 10.1007/s00018-021-03757-2

Figure Lengend Snippet: Expression of steroidogenic enzymes and Insl3 is upregulated in Lgals3−/− testes. a–b The relative mRNA expression of Cyp11a1, Hsd3b1, Cyp17a1 (A) and Insl3 b in Lgals3+/+ and Lgals3−/− testes was analyzed by real-time RT-PCR at P35, P50 and P100. The right panel shows the main pathway of steroidogenesis in rodents, with the corresponding enzymes highlighted by a dotted frame. c Western blot analysis of StAR and Insl3 protein expression in testes from Lgals3+/+ and Lgals3−/− mice at P50; (n = 3, *P < 0.05, ***P < 0.001, ****P < 0.0001). β-actin was used as a loading control. (D) Testosterone, androstandiol-3a, 17b, and dihydrotestosterone (DHT) concentrations in the sera from Lgals3+/+ and Lgals3−/− mice at P50, determined by GC–MS/MS; (n = 3–4, *P < 0.05, **P < 0.01). e–f StAR expression and testosterone levels in galectin 3-depleted MLTC-1 cells stimulated with 1 IU/ml hCG for 24 h. StAR protein expression in the cell lysates e and the testosterone concentrations in conditioned media (F) were determined by Western blotting and ELISA, respectively. Representative data are shown; (n = 3–4, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The data represent the means ± SEM

Article Snippet: Table 1 Primary antibodies Manufacturer Catalogue no Dilution Rabbit monoclonal anti-Bak Cell signaling technology, Germany 12,105 1:1000* Rabbit polyclonal anti-Bax Cell signaling technology, Germany 2772 1:1000* Rabbit monoclonal anti-Bcl-2 Cell signaling technology, Germany 2870 1:1000* Mouse monoclonal anti-β-actin Ac-15 Sigma-Aldrich, Germany A5441 1:5000* Rabbit monoclonal anti-galectin 3 GeneTex, USA GTX62084 1:500** Rat monoclonal anti-germ cell specific antigen (TRA98) Abcam, UK ab82527 1:1000** Rabbit polyclonal anti-HSD11B1 Abcam, UK ab39364 1:100** Rabbit polyclonal anti-Insl3 Biorbyt Ltd, UK orb648755 1:100* Rabbit polyclonal anti-PCNA Abcam, UK ab18197 1:500** Rabbit polyclonal anti-phospho-histone H3 (Ser10) Cell signaling technology, Germany 9701 1:200** Rabbit monoclonal anti-Sox9 Abcam, UK ab185230 1:250** Rabbit monoclonal anti—StAR Cell signaling technology, Germany 8449 1:1000* Secondary antibodies Manufacturer Catalogue No Dilution Alexa Fluor 448 goat anti-rabbit IgG Thermo fisher scientific, USA A11008 1:1000** Alexa Fluor 546 goat anti-rat IgG Thermo fisher scientific, USA A11081 1:1000** HRP-labelled goat anti-rabbit IgG MP biomedicals, USA 855,676 1:10,000* HRP-labelled sheep anti-mouse IgG Sigma-Aldrich, Germany A5906 1:10,000* Open in a separate window Antibody dilution used for *Western blotting or **Immunofluorescence/Immunohistochemistry.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Gas Chromatography-Mass Spectrometry, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases germ cell apoptosis

doi: 10.1007/s00018-021-03757-2

Figure Lengend Snippet: The percentage of testicular macrophages in the population of CD45+ leukocytes is decreased in Lgals3−/− mice. a–b Testicular macrophage frequency was assessed by flow cytometry in Lgals3+/+ and Lgals3−/− testes at P50. A representative dot plot a and the corresponding quantification b is shown; (n = 4–6, *P < 0.05). The data represent the means ± SEM

Article Snippet: Table 1 Primary antibodies Manufacturer Catalogue no Dilution Rabbit monoclonal anti-Bak Cell signaling technology, Germany 12,105 1:1000* Rabbit polyclonal anti-Bax Cell signaling technology, Germany 2772 1:1000* Rabbit monoclonal anti-Bcl-2 Cell signaling technology, Germany 2870 1:1000* Mouse monoclonal anti-β-actin Ac-15 Sigma-Aldrich, Germany A5441 1:5000* Rabbit monoclonal anti-galectin 3 GeneTex, USA GTX62084 1:500** Rat monoclonal anti-germ cell specific antigen (TRA98) Abcam, UK ab82527 1:1000** Rabbit polyclonal anti-HSD11B1 Abcam, UK ab39364 1:100** Rabbit polyclonal anti-Insl3 Biorbyt Ltd, UK orb648755 1:100* Rabbit polyclonal anti-PCNA Abcam, UK ab18197 1:500** Rabbit polyclonal anti-phospho-histone H3 (Ser10) Cell signaling technology, Germany 9701 1:200** Rabbit monoclonal anti-Sox9 Abcam, UK ab185230 1:250** Rabbit monoclonal anti—StAR Cell signaling technology, Germany 8449 1:1000* Secondary antibodies Manufacturer Catalogue No Dilution Alexa Fluor 448 goat anti-rabbit IgG Thermo fisher scientific, USA A11008 1:1000** Alexa Fluor 546 goat anti-rat IgG Thermo fisher scientific, USA A11081 1:1000** HRP-labelled goat anti-rabbit IgG MP biomedicals, USA 855,676 1:10,000* HRP-labelled sheep anti-mouse IgG Sigma-Aldrich, Germany A5906 1:10,000* Open in a separate window Antibody dilution used for *Western blotting or **Immunofluorescence/Immunohistochemistry.

Techniques: Flow Cytometry

Journal: PLOS Neglected Tropical Diseases

Article Title: Distinct features of the host-parasite interactions between nonadherent and adherent Trichomonas vaginalis isolates

doi: 10.1371/journal.pntd.0011016

Figure Lengend Snippet: A and B. Surface expression or secretion of galectins. CFSE-labeled TH17 trophozoites (green) were co-incubated with h VECs (moi 1:3) were sampled at intervals for the detection of galectin-1 (red) and galectin-3 (cyan) by IFA (A), while these galectins in the conditioned medium were detected by ELISA (B). PI, post-infection. Adherent trophozoites in A are indicated by white triangles. C, D, and E. T1 or TH17 trophozoites were incubated in fresh or conditioned KSFM (D) from h VECs cultures for 30 min. Cells were fixed for detection of galectin-1 or -3 by IFA without permeation for (C) or with permeation for (D and E) before immune detection. E. IFA co-staining with anti-galectin-3 antibody and LysoTracker. F and G. T1 or TH17 trophozoites were incubated with FITC-conjugated recombinant galectin-1 or galectin-3 and analyzed by flow cytometry (F) or fluorescence microscopy (G). The bar graph is shown by mean ± SD. All assays were repeated three times and the representative data are shown here. Differences were statistically analyzed by Student’s t-test, with P <0.01(**) and P <0.05(*).

Article Snippet: In brief, standards and serial-diluted supernatants recovered from human cultures challenged with T . vaginalis were incubated in 96-well plates precoated with 50 μl of goat anti-human galectin-1 (1 μg/ml, R&D Systems AF1152) or rabbit anti-human galectin-3 antibody (1 μg/ml, GeneTex) at room temperature for 1 hr.

Techniques: Expressing, Labeling, Incubation, Enzyme-linked Immunosorbent Assay, Infection, Staining, Recombinant, Flow Cytometry, Fluorescence, Microscopy

Journal: PLOS Neglected Tropical Diseases

Article Title: Distinct features of the host-parasite interactions between nonadherent and adherent Trichomonas vaginalis isolates

doi: 10.1371/journal.pntd.0011016

Figure Lengend Snippet: On infection, T . vaginalis may trigger the surface expression and secretion of galectins from h VECs. The extracellular galectins may initially bind on the parasite surface, then galectin-3 is internalized and enclosed in the lysosomes inside the trophozoites of adherent isolate. The extracellular galectins bound to the parasite might trigger the aggregation of trophozoites on h VECs, as this is diminished in the presence of lactose. In the adherent isolate, the predominant flagellate-amoeboid transition, as well as cytoadherence, were simultaneously suppressed by CytD, speculating that actin cytoskeleton-based behavior may mediate cytoadherence. When axostyle microtubule assembly was disturbed by TPI, the axostyle anchoring was abolished with the reduced cytoadherence, suggesting that axostyle anchoring might be an early event required for cytoadherence. For T . vaginalis , it is speculated that the cytoskeleton and cell surface adhesion molecules may coordinate their cytoadherence, so these factors should be considered in the study of host-parasite interactions and provide new insights into the host colonization of T . vaginalis .

Article Snippet: In brief, standards and serial-diluted supernatants recovered from human cultures challenged with T . vaginalis were incubated in 96-well plates precoated with 50 μl of goat anti-human galectin-1 (1 μg/ml, R&D Systems AF1152) or rabbit anti-human galectin-3 antibody (1 μg/ml, GeneTex) at room temperature for 1 hr.

Techniques: Infection, Expressing