Journal: Cell Death Discovery

Article Title: Reduction of lymphotoxin beta receptor induces cellular senescence via the MDMX-p53 pathway

doi: 10.1038/s41420-025-02708-1

Figure Lengend Snippet: A , B A375 cells were transfected with 100 nM of siControl (control siRNA) or siLTβR (LTβR siRNA), followed by 100 ng/ml Dox treatment for 48 h. Morphological changes, relative cell number ( A ), and confocal images of a senescence green probe ( B) were analyzed. C , D B16F10 WT (LTβR WT), and B16F10 LTβR-KO (LTβR knockout) cells were treated with 100 ng/ml Dox for 48 h. Morphological changes and relative cell number ( C ), and confocal images of senescence green probe ( D) were examined. E , F Western blot images of A375 and B16F10 cells showing the indicated proteins in LTβR-depleted cells. S.E. short exposure, L.E. long exposure. Band intensities of p53, p21 and MDM2 were measured using ImageJ and normalized to β-actin. Results are presented as the mean ± SD from three separate experiments. B , D Fluorescence intensities for relative senescence green probe were quantified by ImageJ, and data are shown as mean ± SD from three independent experiments ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, using Fisher’s LSD post hoc test. n.s not significant.

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA in 0.1% TBS-T. Proteins were detected using the following specific antibodies: α-LTβR (20331-1-AP, Proteintech, Rosemont, IL, USA or PA5-88290, Invitrogen), α-p53 (10442-1-AP, Proteintech), α-p21 (556431, BD Biosciences), α-MDM2 (ab259265, Abcam), α-MDMX (17914-1-AP, Proteintech), α-IκBα (4812S, Cell Signaling Technology, Danvers, MA, USA), α-p52 (4882, Cell Signaling Technology), α-USP7 (66514-1-Ig, Proteintech), α-TRAF6 (8028S, Cell Signaling Technology), α-lamin B1 (ab16048, Abcam), α-GAPDH (AC002, Abclonal, Wuhan, China), and α-β-actin (sc-47778, Santa Cruz) were used.

Techniques: Transfection, Control, Knock-Out, Western Blot, Fluorescence

Journal: Cell Death Discovery

Article Title: Reduction of lymphotoxin beta receptor induces cellular senescence via the MDMX-p53 pathway

doi: 10.1038/s41420-025-02708-1

Figure Lengend Snippet: A – D A375 and B16F10 cells were transfected with LTβR plasmid followed by 100 ng/ml Dox treatment for 48 h. Cells were photographed for analyzing morphological change and relative cell number ( A , C ), and stained using a senescence green probe ( B , D ). Relative fluorescence intensity for the senescence green probe was quantified using ImageJ. E , F Western blot images of A375 and B16F10 cells for the indicated proteins are representative of three experiments, and the relative p53, p21, and MDM2 protein levels were measured. Bands were quantified using ImageJ software and normalized to β-actin. Graphical data are represented as mean ± SD from three independent experiments ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, using Fisher’s LSD post hoc test. n.s, not significant.

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA in 0.1% TBS-T. Proteins were detected using the following specific antibodies: α-LTβR (20331-1-AP, Proteintech, Rosemont, IL, USA or PA5-88290, Invitrogen), α-p53 (10442-1-AP, Proteintech), α-p21 (556431, BD Biosciences), α-MDM2 (ab259265, Abcam), α-MDMX (17914-1-AP, Proteintech), α-IκBα (4812S, Cell Signaling Technology, Danvers, MA, USA), α-p52 (4882, Cell Signaling Technology), α-USP7 (66514-1-Ig, Proteintech), α-TRAF6 (8028S, Cell Signaling Technology), α-lamin B1 (ab16048, Abcam), α-GAPDH (AC002, Abclonal, Wuhan, China), and α-β-actin (sc-47778, Santa Cruz) were used.

Techniques: Transfection, Plasmid Preparation, Staining, Fluorescence, Western Blot, Software

Journal: Cell Death Discovery

Article Title: Reduction of lymphotoxin beta receptor induces cellular senescence via the MDMX-p53 pathway

doi: 10.1038/s41420-025-02708-1

Figure Lengend Snippet: A Relative p53 mRNA levels in A375 cells with LTβR knockdown or LTβR-overexpressing cells determined by real-time PCR. B , C Western blot analysis of p53 and p21 protein levels in A375 cells after 20 μM of MG-132 treatment for 4 h, following siRNA ( B) or LTβR plasmid ( C) transfection. p53 and p21 relative band intensities were quantified using ImageJ software, and normalized to β-actin. D Western blot analysis of MDMX, p53, MDM2, and p21 protein expression in A375 cells after 12 h or 24 h of siControl or siLTβR transfection. E , F Cycloheximide (CHX, 100 μg/ml) chase assays were conducted to determine MDMX protein stability in LTβR-overexpressing and LTβR knockdown cells for the indicated time and quantified using ImageJ. G – J A375 cells were treated with 80 nM of BTZ for 4 h, and whole cell lysate was subjected to immunoprecipitation to confirm MDMX ubiquitination and p53 ubiquitination. Relative expression levels of ubiquitinated MDMX and p53 were measured using ImageJ software, normalized, and presented as mean ± SD from three independent experiments. Graphical data are presented as means ± SD from three independent experiments ( n = 3). n.s not significant, using an unpaired Student’s t -test ( A ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, using Fisher’s LSD post hoc test ( B , C) or Šidák’s multiple comparison test ( E , F ). n.s not significant.

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA in 0.1% TBS-T. Proteins were detected using the following specific antibodies: α-LTβR (20331-1-AP, Proteintech, Rosemont, IL, USA or PA5-88290, Invitrogen), α-p53 (10442-1-AP, Proteintech), α-p21 (556431, BD Biosciences), α-MDM2 (ab259265, Abcam), α-MDMX (17914-1-AP, Proteintech), α-IκBα (4812S, Cell Signaling Technology, Danvers, MA, USA), α-p52 (4882, Cell Signaling Technology), α-USP7 (66514-1-Ig, Proteintech), α-TRAF6 (8028S, Cell Signaling Technology), α-lamin B1 (ab16048, Abcam), α-GAPDH (AC002, Abclonal, Wuhan, China), and α-β-actin (sc-47778, Santa Cruz) were used.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Transfection, Software, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Comparison

Journal: Cell Death Discovery

Article Title: Reduction of lymphotoxin beta receptor induces cellular senescence via the MDMX-p53 pathway

doi: 10.1038/s41420-025-02708-1

Figure Lengend Snippet: A , B A375 cells were transfected with LTβR plasmid for 48 h. LTβR-overexpressing cells were subjected to immunoprecipitation with LTβR and MDMX, and PLA was performed using MDMX, Flag, and LTβR antibodies. C , D A375 cells were transfected with 100 nM of siControl or siLTβR for 48 h, and subjected to immunoprecipitation and PLA using LTβR and MDMX antibodies. E , F Immunoprecipitation and PLA of extracellular domain (ECD)-deleted LTβR-transfected cells were performed using MDMX and LTβR antibodies. G Immunoprecipitation of LTβR and immunoblotting using MDMX antibody validated the interaction in LTβR-overexpressing B16F10 cells. H B16F10 WT and B16F10 LTβR-KO cells were transfected with LTβR plasmid, and PLA was performed to determine the location of interaction between LTβR and MDMX.

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA in 0.1% TBS-T. Proteins were detected using the following specific antibodies: α-LTβR (20331-1-AP, Proteintech, Rosemont, IL, USA or PA5-88290, Invitrogen), α-p53 (10442-1-AP, Proteintech), α-p21 (556431, BD Biosciences), α-MDM2 (ab259265, Abcam), α-MDMX (17914-1-AP, Proteintech), α-IκBα (4812S, Cell Signaling Technology, Danvers, MA, USA), α-p52 (4882, Cell Signaling Technology), α-USP7 (66514-1-Ig, Proteintech), α-TRAF6 (8028S, Cell Signaling Technology), α-lamin B1 (ab16048, Abcam), α-GAPDH (AC002, Abclonal, Wuhan, China), and α-β-actin (sc-47778, Santa Cruz) were used.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Cell Death Discovery

Article Title: Reduction of lymphotoxin beta receptor induces cellular senescence via the MDMX-p53 pathway

doi: 10.1038/s41420-025-02708-1

Figure Lengend Snippet: A , B A375 cells were treated with 80 nM BTZ (bortezomib) for 4 h after siRNA or plasmid transfection. Nuclear and cytosol fractions were isolated, followed by western blotting. GAPDH (cytosol) and Lamin B1 (nuclear) were used as loading controls. C , D Confocal microscopy was used to assess MDMX localization. Nuclear MDMX band and fluorescence intensities were measured relative to cytosolic MDMX using ImageJ. E , F PLA was performed to confirm the relative interaction between MDM2 and MDMX in LTβR knockdown cells or LTβR-overexpressing cells. Relative co-localization signals of MDM2 and MDMX (shown in graph) were quantified using ImageJ. Graphical data are presented as means ± SD ( n = 3) from three independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001, using an un p aired Student’s t -test.

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA in 0.1% TBS-T. Proteins were detected using the following specific antibodies: α-LTβR (20331-1-AP, Proteintech, Rosemont, IL, USA or PA5-88290, Invitrogen), α-p53 (10442-1-AP, Proteintech), α-p21 (556431, BD Biosciences), α-MDM2 (ab259265, Abcam), α-MDMX (17914-1-AP, Proteintech), α-IκBα (4812S, Cell Signaling Technology, Danvers, MA, USA), α-p52 (4882, Cell Signaling Technology), α-USP7 (66514-1-Ig, Proteintech), α-TRAF6 (8028S, Cell Signaling Technology), α-lamin B1 (ab16048, Abcam), α-GAPDH (AC002, Abclonal, Wuhan, China), and α-β-actin (sc-47778, Santa Cruz) were used.

Techniques: Plasmid Preparation, Transfection, Isolation, Western Blot, Confocal Microscopy, Fluorescence, Knockdown

Journal: Cell Death Discovery

Article Title: Reduction of lymphotoxin beta receptor induces cellular senescence via the MDMX-p53 pathway

doi: 10.1038/s41420-025-02708-1

Figure Lengend Snippet: Tumors were established by the subcutaneously injecting of B16F10 WT and B16F10 LTβR-KO cells into mice. On day 9 after implantation, mice were administered with 4 mg Dox per kg of mouse body weight. A – C Tumors were harvested on day 16, photographed, and their weight and volume were measured ( n = 6). D Western blotting of tumor cell lysates for indicated proteins. E Paraffin-embedded tissue sections were stained for p21, and relative expression levels were measured using ImageJ. F Cryosections of tumor tissue were subjected to SA-β-Gal staining, and relative SA-β-Gal activity was measured using ImageJ. Brown pigments in the histological sections represent melanin deposits. Hematoxylin was used for counterstaining. Graphical data are presented as means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, using Fisher’s LSD post hoc test. n.s not significant.

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA in 0.1% TBS-T. Proteins were detected using the following specific antibodies: α-LTβR (20331-1-AP, Proteintech, Rosemont, IL, USA or PA5-88290, Invitrogen), α-p53 (10442-1-AP, Proteintech), α-p21 (556431, BD Biosciences), α-MDM2 (ab259265, Abcam), α-MDMX (17914-1-AP, Proteintech), α-IκBα (4812S, Cell Signaling Technology, Danvers, MA, USA), α-p52 (4882, Cell Signaling Technology), α-USP7 (66514-1-Ig, Proteintech), α-TRAF6 (8028S, Cell Signaling Technology), α-lamin B1 (ab16048, Abcam), α-GAPDH (AC002, Abclonal, Wuhan, China), and α-β-actin (sc-47778, Santa Cruz) were used.

Techniques: Western Blot, Staining, Expressing, Activity Assay

Journal: Cell Death Discovery

Article Title: Reduction of lymphotoxin beta receptor induces cellular senescence via the MDMX-p53 pathway

doi: 10.1038/s41420-025-02708-1

Figure Lengend Snippet: A – C Tumors were generated by implantating B16F10 WT and B16F10 LTβR-KO cells into mice. On day 9 after implantation, mice were injected with 20 mg nutlin-3a per kg of mouse body weight. After 7 days, tumors were collected, photographed, and their weight and volume were measured ( n = 4). D Western blot of tumor cell lysates for indicated proteins. E Paraffin-embedded tissue sections were stained for p21, and relative expression was measured. F Cryosections of tumor tissue were stained with SA-β-Gal to assess senescence activity. Brown pigments in the histological section indicate melanin deposits. Hematoxylin was used for counterstaining. Graphical data are presented as means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, using Fisher’s LSD post hoc test. n.s not significant.

Article Snippet: The membranes were blocked with 5% skim milk or 5% BSA in 0.1% TBS-T. Proteins were detected using the following specific antibodies: α-LTβR (20331-1-AP, Proteintech, Rosemont, IL, USA or PA5-88290, Invitrogen), α-p53 (10442-1-AP, Proteintech), α-p21 (556431, BD Biosciences), α-MDM2 (ab259265, Abcam), α-MDMX (17914-1-AP, Proteintech), α-IκBα (4812S, Cell Signaling Technology, Danvers, MA, USA), α-p52 (4882, Cell Signaling Technology), α-USP7 (66514-1-Ig, Proteintech), α-TRAF6 (8028S, Cell Signaling Technology), α-lamin B1 (ab16048, Abcam), α-GAPDH (AC002, Abclonal, Wuhan, China), and α-β-actin (sc-47778, Santa Cruz) were used.

Techniques: Generated, Injection, Western Blot, Staining, Expressing, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Genetic Background and Kinetics Define Wound Bed Extracellular Vesicles in a Mouse Model of Cutaneous Injury

doi: 10.3390/ijms22073551

Figure Lengend Snippet: Antibodies used for analysis of EV surface markers using vesicle flow cytometry.

Article Snippet: CD18 , M18/2 , APC , 130-104-019 , Miltenyi Biotec.

Techniques: Cytometry