Journal: Cell Death and Differentiation

Article Title: Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation

doi: 10.1038/s41418-021-00861-5

Figure Lengend Snippet: A Immunoprecipitation with control IgG and Gm364 antibody was performed and followed by SDS-PAGE and silver staining. Then distinct bands were sent for MALDI. TTC37, MIB2, and GRAMD1A were identified as Gm364-interacting proteins. B RT-PCR showed that within oocytes, Notch2 is the most abundant among Notch family members 1–4. C Immunofluorescence showed that Notch2 was enriched on the oocyte membrane. DNA in blue, Notch2 in green. D Western blot showed that Notch2 is more abundant in oocytes than in granular cells. E Co-IP and blots showed that Gm364 interacts with Notch2 in oocytes. F . Western blot showed that NICD2 was more abundant in oocytes than in granular cells. G Blot showed that NICD2 protein levels decreased gradually during oocyte meiosis. H – J Immunofluorescence and blot showed that Gm364 knockout significantly decreased the NICD2 protein level. DNA in blue, NICD2 in green. K Blot showed that γ-secretase inhibition significantly decreased NICD2 levels. L. NICD2 reduction by γ-secretase inhibition greatly decreased the percentage of MII oocytes. M , N Immunofluorescence of in vitro fertilized oocytes and quantification showed that inhibiting γ-secretase significantly decreased the percentage of fertilized oocytes and the percentage of 2-PN (two pronucleus). PNs in the control oocyte or chromosomes in the γ-secretase-inhibited (γ-secretase(-)) oocytes were delineated with red dot-line circle; polar bodies (pbs) were labeled with arrows. DNA in blue, tubulin in green. β-actin or α-tubulin was used as a loading control. Scale bar, 20 μm. *Indicates p < 0.05.

Article Snippet: Primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti-β-Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti-β-Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti‐alpha Tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss, Beijing, China); Human anti-centromere CREST antibody (cat#: 15-234; Antibodies Incorporated, USA); rabbit polyclonal anti-Notch2 (cat#: ab8926, Abcam, Cambridge, UK); Rabbit polyclonal anti-cleaved Notch2 (Asp 1733) antibody (cat#:AF5255; Affinity biosciences, OH, USA); Anti-DLL3 rabbit polyclonal antibody (cat#: Ab103102; Abcam, Cambridge, UK); Anti-MIB2 rabbit polyclonal antibody (cat#: A17829; ABclonal, MA, USA); anti-Phosphorylation RICTOR (Thr1135) (cat#: D30A3, Cell Signaling Technology, MA, USA); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phospho AKT (Thr308, Cat#: 13038, Cell Signaling Technology); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science).

Techniques: Immunoprecipitation, SDS Page, Silver Staining, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Co-Immunoprecipitation Assay, Knock-Out, Inhibition, In Vitro, Labeling

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting SKAP2 restores sperm motility and morphology through modulating mitochondrial organization and cytoskeletal remodeling

doi: 10.1038/s41392-025-02513-3

Figure Lengend Snippet: Disruption of acrosome, nuclear, and manchette assembly in KI mouse sperm. a PAS (left) and PNA (right) staining of seminiferous tubules from WT and KI mice at different stages of spermatogenesis. Green arrowheads (PAS) and red arrowheads (PNA) indicate aberrant acrosomal morphology in KI spermatids. M: Metaphase. Scale bars, 10 μm. b Transmission electron microscopy (TEM) of sperm head development in WT and KI spermatids. Nu, nucleus; g, Golgi apparatus; av, proacrosomal vesicles; apx, acroplaxome; ac, acrosome. Red arrows indicate malformed acrosomes; blue asterisks denote abnormal nuclei. Scale bars, 1 μm. c Representative TEM images of manchette morphology during spermatid elongation in WT and KI mice. White double-headed arrows highlight the asymmetric manchette in KI spermatids. Abbreviations: Nu, nucleus; PR, perinuclear ring; M, manchette. Scale bars, 1 μm. d Immunofluorescence staining of tubulin in spermatozoa from adult WT and KI mice. Tubulin marks the manchette structure; DNA was counterstained with DAPI. Scale bars, 5 μm. e Quantification of manchette microtubule lengths from ( d ). For each group, 40 spermatozoa were randomly selected per mouse, and average values were calculated for every 10 sperms. Data are presented as mean ± SD. P -values were determined using a two-sided Student’s t -test. n = 36 per group

Article Snippet: We extend our gratitude to Bioss for the antibody of alpha Tubulin (bsm-33039M) and alpha Tubulin Antibody from MedChemExpress (Monmouth Junction, NJ, USA; Cat# HY- P86200 ).

Techniques: Disruption, Staining, Transmission Assay, Electron Microscopy, Immunofluorescence

Journal: Cells

Article Title: New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms

doi: 10.3390/cells9122531

Figure Lengend Snippet: uPAR interactions with FPR1 and β1 integrins are involved in migration of prostate carcinoma PC3 cells. PC3 cells were lysed and 50 μg of cell extract was analyzed by Western blot with indicated antibodies; membranes were reprobed with an anti-tubulin mouse antibody for loading control ( A ). PC3 cells were pre-incubated with diluent (−) or 5 nM W Peptide (W Pep) or 50 µM P25 ( B ) or with 5 µg/mL nonimmune Ig (NI) or an antibody directed to 84–95 uPAR residues ( C ) or with diluent (−) or 10 µM Rho kinase inhibitor or 20 µM Rac1 inhibitor ( D ). Cells were then plated in Boyden chambers and allowed to migrate towards 10% fetal bovine serum (FBS). Migrated cells were fixed, stained with hematoxylin, and counted (( B – D ) left panels). Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (( B – D ) right panels). The values are the mean + SD of three experiments performed in triplicate. (*) p < 0.05, as determined by the Student’s t -test.

Article Snippet: The mAb anti-tubulin antibody (bsm-50179M) was from Bioss antibodies (Woburn, MA, USA).

Techniques: Migration, Western Blot, Incubation, Staining

Journal: Cells

Article Title: New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms

doi: 10.3390/cells9122531

Figure Lengend Snippet: Removal of uPAR GPI-anchor impairs uPAR-controlled mechanism of cell migration. PC3 ( A ) and uPAR-293 ( B ) cells were treated with 1 U/mL of phosphatidylinositol-specific phospholipase C (PI-PLC) or with diluent. Cells were then plated in Boyden chambers and allowed to migrate towards 10% fetal bovine serum (FBS). Migrated cells were fixed, stained with hematoxylin, and counted (left graphs). Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right graphs). The values are the mean ± SD of three experiments performed in triplicate. (*) p ≤ 0.05, as determined by the Student’s t -test. PC3 and uPAR-293 cells treated with or without PI-PLC were also lysed and analyzed by Western blot with an anti-uPAR monoclonal antibody to assess PI-PLC effect; membranes were then reprobed with anti-tubulin or anti-GAPDH rabbit antibodies for loading control (( A , B ), right panels).

Article Snippet: The mAb anti-tubulin antibody (bsm-50179M) was from Bioss antibodies (Woburn, MA, USA).

Techniques: Migration, Staining, Western Blot

Journal: Cells

Article Title: New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms

doi: 10.3390/cells9122531

Figure Lengend Snippet: Disruption of lipid rafts impairs uPAR-controlled cell migration. PC3 ( A ) or uPAR-293 ( B ) cells were treated with or without 10 mM methyl-beta-cyclodextrin (MCD). Cells were then plated in Boyden chambers and allowed to migrate towards 10% fetal bovine serum (FBS). MCD treated or untreated uPAR-293 cells were also incubated with 5 μg/mL polyclonal anti-uPAR antibody or nonimmune Ig (NI) before migration ( C ). Migrated cells were fixed, stained with hematoxylin, and counted (left panels). Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right panels). The values are the mean + SD of three experiments performed in triplicate. (*) p < 0.05, as determined by the Student’s t -test. PC3 and uPAR-293 cells treated with or without MCD were also lysed in buffer containing 1% Triton X-100. Cell lysates (1 mg) were loaded onto a sucrose density gradient and subjected to ultracentrifugation. Then, 4.5 mL were harvested as 0.45 mL fractions. Equal volumes of each fraction were analyzed by Western blot with an anti-uPAR monoclonal antibody, or with anti-tubulin or anti-caveolin antibodies, as controls of non-raft associated proteins or raft associated proteins, respectively ( D ).

Article Snippet: The mAb anti-tubulin antibody (bsm-50179M) was from Bioss antibodies (Woburn, MA, USA).

Techniques: Migration, Incubation, Staining, Western Blot

Journal: Cells

Article Title: New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms

doi: 10.3390/cells9122531

Figure Lengend Snippet: uPAR drives its signaling partners to lipid rafts. Cultured PC3 cells ( A ) or PC3 cells treated with 50 μM P25 or with its scrambled version (−) ( B ) were lysed in buffer containing 1% Triton X-100. Cell lysates were loaded onto a sucrose density gradient and subjected to ultracentrifugation. Then, 4.5 mL were harvested as 0.45 mL fractions; 45 μL of each fraction was analyzed by Western blot with the indicated antibodies; tubulin and caveolin were used as controls of non-raft associated proteins or raft associated proteins, respectively. ( C ) PC3 cells were pre-incubated with or without 50 μM C6 compound, then plated in Boyden chambers and allowed to migrate toward 100 ng/mL EGF Epidermal Growth Factor (EGF). Migrated cells were fixed, stained with hematoxylin, and counted (left panel). Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right panels). The values are the mean + SD of three experiments performed in triplicate. (*) p < 0.05, as determined by the Student’s t -test. C6 treated or untreated cells were also lysed in buffer containing 1% Triton X-100, loaded onto a sucrose density gradient and subjected to ultracentrifugation. Then, 45 μL of each gradient fraction was analyzed by Western blot with the indicated antibodies; tubulin and caveolin were used as control of non-raft associated proteins and raft associated proteins, respectively ( D ).

Article Snippet: The mAb anti-tubulin antibody (bsm-50179M) was from Bioss antibodies (Woburn, MA, USA).

Techniques: Cell Culture, Western Blot, Incubation, Staining, Migration