Journal: Advanced Science

Article Title: A Novel FGFR3‐Targeting Antibody‐Drug Conjugate Induces Tumor Cell Apoptosis through the cGAS–STING Pathway in Bladder Cancer

doi: 10.1002/advs.202509933

Figure Lengend Snippet: A2 induces apoptosis in BC cells by targeting MAD2L1. A) Reaction scheme for biotinylation at the 20th hydroxyl group of A2. B) Overview of differentially expressed proteins identified by MS. C) Top 10 proteins ranked by the A2‐biotin/biotin abundance ratio. D) Western blot analysis of MAD2L1 expression in pull‐down products. E) Western blot detection of soluble MAD2L1 in T24 cells treated with A2 at different temperatures ( n = 3). F) SPR analysis of A2‐MAD2L1 binding kinetics and KD value. G) Representative immunofluorescence images showing co‐localization of A2‐biotin and MAD2L1 in T24 cells after 3 h of A2‐biotin treatment. Red: Anti‐MAD2L1, green: A2‐biotin, blue: DAPI, scale bar = 20 µm. H) Western blot analysis of MAD2L1 expression in T24 and UMUC‐3 cells treated with different concentrations of A2 for 24 h. I) Western blot validation of MAD2L1 OE in T24 and UMUC‐3 cells. J) Western blot validation of MAD2L1 KD in T24 and UMUC‐3 cells. K) Colony formation capacity of MAD2L1‐OE and control cells treated with different concentrations of A2 for 48 h. L) Apoptotic effects in MAD2L1‐OE and control cells treated with different concentrations of A2 for 48 h. ( n = 3). M) Apoptotic effects in MAD2L1‐KD and control cells treated with different concentrations of A2 for 48 h ( n = 3). Data are presented as mean ± SD. **** P < 0.0001. Statistical significance was determined by two‐way ANOVA followed by Tukey's multiple comparison test (L,M).

Article Snippet: SPR experiments were performed using a CM5 chip with amine coupling to immobilize MAD2L1 protein (HY‐ P71540 , MCE, USA).

Techniques: Western Blot, Expressing, Binding Assay, Immunofluorescence, Biomarker Discovery, Control, Comparison

Journal: Advanced Science

Article Title: A Novel FGFR3‐Targeting Antibody‐Drug Conjugate Induces Tumor Cell Apoptosis through the cGAS–STING Pathway in Bladder Cancer

doi: 10.1002/advs.202509933

Figure Lengend Snippet: A2 specifically binds to the Lys73 site of MAD2L1. A) Molecular docking simulation of the interaction between A2 and MAD2L1. B) Western blot validation of MAD2L1 expression levels in control, MAD2L1‐KD, and mutant T24 cells. C) Western blot analysis of MAD2L1 expression in pull‐down products from each group. D–F CETSA evaluating the binding of A2 to MAD2L1 in MAD2L1‐KD T24 cells reconstituted with MAD2L1 WT (D), MAD2L1V55A+I62A+V69A (E), and MAD2L1K73A (F) ( n = 3). Data are presented as mean ± SD.

Article Snippet: SPR experiments were performed using a CM5 chip with amine coupling to immobilize MAD2L1 protein (HY‐ P71540 , MCE, USA).

Techniques: Western Blot, Biomarker Discovery, Expressing, Control, Mutagenesis, Binding Assay

Journal: Advanced Science

Article Title: A Novel FGFR3‐Targeting Antibody‐Drug Conjugate Induces Tumor Cell Apoptosis through the cGAS–STING Pathway in Bladder Cancer

doi: 10.1002/advs.202509933

Figure Lengend Snippet: A2 targets MAD2L1 to activate the cGAS‐STING pathway. A) Effect of A2 on aneuploidy formation in T24 and UMUC‐3 cells. B) Representative images showing cGAS subcellular distribution and micronuclei formation (indicated by arrows) in T24 and UMUC‐3 cells treated with 0.064 µ m A2 for 48 h. Red: cGAS, green: PicoGreen (DNA stain), Scale bar = 20 µm. C) Quantification of cytosolic DNA in T24 and UMUC‐3 cells treated with 0.32 µ m A2 for 48 h ( n = 3). D) Western blot analysis of cGAS‐STING pathway protein expression in T24 and UMUC‐3 cells treated with different concentrations of A2 for 48 h. E) Western blot analysis of cGAS‐STING pathway protein expression in MAD2L1‐OE and control cells treated with different concentrations of A2 for 48 h. F) Western blot analysis of cGAS‐STING pathway protein expression in MAD2L1‐KD and control cells treated with different concentrations of A2 for 48 h. G) Apoptotic effects in T24 and UMUC‐3 cells treated with 1.6 µ m A2 after STING inhibitor H151‐mediated blockade of the cGAS‐STING pathway. Data are presented as mean ± SD. **** P < 0.0001. Statistical significance was determined by a two‐tailed Student's t‐ test (C).

Article Snippet: SPR experiments were performed using a CM5 chip with amine coupling to immobilize MAD2L1 protein (HY‐ P71540 , MCE, USA).

Techniques: Staining, Western Blot, Expressing, Control, Two Tailed Test

Journal: Advanced Science

Article Title: A Novel FGFR3‐Targeting Antibody‐Drug Conjugate Induces Tumor Cell Apoptosis through the cGAS–STING Pathway in Bladder Cancer

doi: 10.1002/advs.202509933

Figure Lengend Snippet: Cytotoxicity of LZU‐WZLYCS01 against BC cells and PDOs, and validation of the bystander effect. A) Cell viability of WT and FGFR3‐KO T24 and UMUC‐3 cells treated with different concentrations of LZU‐WZLYCS01, A2, or FGFR3 antibody for 72 h. B) Apoptotic effects in WT and FGFR3‐KO T24 and UMUC‐3 cells treated with different concentrations of LZU‐WZLYCS01 for 48 h ( n = 3). C) Colony formation capacity of T24 and UMUC‐3 cells treated with LZU‐WZLYCS01 for 48 h. D) Schematic diagram of the co‐culture model for assessing the bystander effects. E) Effect of LZU‐WZLYCS01 on the viability of FGFR3‐KO T24 and UMUC‐3 cells in the co‐culture system ( n = 3). F) Representative bright‐field and AM/PI‐stained images showing LZU‐WZLYCS01‐induced cytotoxicity in PDOs. Green: viable cells, red: dead cells, scale bar = 50 µm. G) Representative H&E staining of PDOs and matched parental tumor tissues. Scale bar = 25 µm. H) Representative IHC staining of UPK2, FGFR3, and MAD2L1 in PDOs. Scale bar = 25 µm. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. Statistical significance was determined by two‐way ANOVA followed by Tukey's multiple comparison test (B) and a two‐tailed Student's t‐ test (E).

Article Snippet: SPR experiments were performed using a CM5 chip with amine coupling to immobilize MAD2L1 protein (HY‐ P71540 , MCE, USA).

Techniques: Biomarker Discovery, Co-Culture Assay, Staining, Immunohistochemistry, Comparison, Two Tailed Test

Journal: Advanced Science

Article Title: A Novel FGFR3‐Targeting Antibody‐Drug Conjugate Induces Tumor Cell Apoptosis through the cGAS–STING Pathway in Bladder Cancer

doi: 10.1002/advs.202509933

Figure Lengend Snippet: Mechanism of action and tumor‐targeting capability of LZU‐WZLYCS01. A) Flow cytometry analysis of LZU‐WZLYCS01 binding and internalization in T24 and UMUC‐3 cells at 37 and 4 °C. B) Representative images showing LZU‐WZLYCS01 binding, internalization, and lysosomal co‐localization in T24 and UMUC‐3 cells at 37 and 4 °C. Red: LZU‐WZLYCS01, green: anti‐LAMP2, blue: DAPI, scale bar = 10 µm. C) Western blot analysis of cGAS‐STING pathway protein expression in T24 and UMUC‐3 cells treated with different concentrations of LZU‐WZLYCS01 for 48 h. D) Western blot analysis of cGAS‐STING pathway protein expression in MAD2L1‐OE and control cells treated with different concentrations of LZU‐WZLYCS01 for 48 h. E) In vivo fluorescence imaging of UMUC‐3 xenograft models 24 h after intravenous injection of LZU‐WZLYCS01–Cy5 ( n = 3). F) Ex vivo fluorescence imaging of tumors and major organs (heart, liver, spleen, lungs, kidneys, and brain) collected 24 h post‐injection.

Article Snippet: SPR experiments were performed using a CM5 chip with amine coupling to immobilize MAD2L1 protein (HY‐ P71540 , MCE, USA).

Techniques: Flow Cytometry, Binding Assay, Western Blot, Expressing, Control, In Vivo, Fluorescence, Imaging, Injection, Ex Vivo

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A New Method for Chromosomes Preparation by ATP-Competitive Inhibitor SP600125 via Enhancement of Endomitosis in Fish

doi: 10.3389/fbioe.2020.606496

Figure Lengend Snippet: Effects of SP600125 on the expression levels of spindle assembly checkpoint genes. (A) qRT-PCR analysis for the differentially expressed BUB1, MAD2 , and CDC20 genes between the control group and the SP600125-treated cells. (B) Western blot analysis of expression levels of BUB1, MAD2, and CDC20 upon SP600125 treatment, where “1” represents the cells in the control group (without SP600125), “2” stands for the cells treated by SP600125 for 48 h, and “3” represents the cells first being treated by SP600125 for 48 h, then by SP600125-free culture for the next 12 h, and further by SP600125 treatment for another 48 h. Each bar represents the mean ± SD of three independent experiments (* P < 0.05).

Article Snippet: The membrane was blocked with 5% (w/v) non-fat milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C and then incubated with primary antibodies, including anti-p53 (Abcam, USA), anti-MDM2 (Bioworld, USA), anti-p21 (Bioworld, USA), anti-Mad2L1 (Bioworld, USA), anti-CDC20 (Bioworld, USA), anti-BUB1 (Proteintech, USA), anti-Flag-tag (Proteintech, USA), and anti-β-ACTIN (Abcam, USA), overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A New Method for Chromosomes Preparation by ATP-Competitive Inhibitor SP600125 via Enhancement of Endomitosis in Fish

doi: 10.3389/fbioe.2020.606496

Figure Lengend Snippet: Analysis of cell proliferation by flow cytometry after transfection with pAd-MAD2. Lane 1 represents the normal cultured group. Lane 2 represents the negative control group. Lanes from 3 to 6 represent the overexpressed MAD2 groups (OE-MAD2): the cells being transfected with pAd-mCMV-MAD2-GFP-3-Flag-pA for 72 h (Lane 3), the cells being treated with SP600125 for the next 48 h (Lane 4), the cells continued to be further cultured in SP600125-free medium for 12 h (Lane 5), and the cells being further cultured using SP600125 treatment for another 48 h (Lane 6).

Article Snippet: The membrane was blocked with 5% (w/v) non-fat milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C and then incubated with primary antibodies, including anti-p53 (Abcam, USA), anti-MDM2 (Bioworld, USA), anti-p21 (Bioworld, USA), anti-Mad2L1 (Bioworld, USA), anti-CDC20 (Bioworld, USA), anti-BUB1 (Proteintech, USA), anti-Flag-tag (Proteintech, USA), and anti-β-ACTIN (Abcam, USA), overnight.

Techniques: Flow Cytometry, Transfection, Cell Culture, Negative Control

Journal: iScience

Article Title: Distinct effects of sacituzumab govitecan and berzosertib on DNA damage response in ovarian cancer

doi: 10.1016/j.isci.2024.111283

Figure Lengend Snippet:

Article Snippet: Rabbit anti- MAD2L2/REV7 , ProteinTech , Cat #12683-1-AP; RRID: AB_2139530.

Techniques: Recombinant, Staining, Western Blot, Stripping, Control, SYBR Green Assay, cDNA Synthesis, Gene Expression, Software, Microscopy, Imaging

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Establishment of a prognostic hypoxic gene signature in osteosarcoma in the TARGET cohort. (A) The chromosomal locations of 197 hypoxic genes. (B) The relationships between the number of trees and error rates. (C) The rank of relative importance of the top five variables. (D) Univariate cox regression analysis of TES, SDC3, MXI1, CAVIN1, and EGFR in osteosarcoma. (E) Heat map visualizing the up- (red) and down-regulated (green) genes in high- and low-risk groups. (F) The distributions and proportions of survival status (alive: green; dead: red). (G) Kaplan-Meier curves of OS between high- and low-risk specimens. The differences were compared by log-rank tests.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Univariate cox regression analysis for prognosis-related hypoxic genes in the TARGET cohort.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: The relative importance of prognosis-related hypoxic genes.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Prognostic values of hypoxic genes from the gene signature in osteosarcoma. Kaplan-Meier curves for overall survival of osteosarcoma patients in the high and low (A) CAVIN1, (B) EGFR, (C) SCD3, (D) TES, and (E) MXI1 expression groups from the TARGET cohort. The survival differences were assessed via log-rank tests.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Verification of the expression of hypoxic genes from the gene signature. (A–E) RT-qPCR for detecting the expression of SCD3, EGFR, MXI1, CAVIN1, and TES in U2OS osteosarcoma cells and hFOB1.19 normal cells. (F, G) Western blotting for the expression of the above genes in U2OS osteosarcoma cells and hFOB1.19 normal cells. (H, I) Western blotting for the expression of the above genes in Saos-2 osteosarcoma cells and hFOB1.19 normal cells. (J–N) RT-qPCR for the expression of the above genes in tumors with Saos-2 osteosarcoma cells and hFOB1.19 normal cells. ** p < 0.01; *** p < 0.001; **** p < 0.0001. U2OS osteosarcoma cells compared with hFOB1.19 normal cells.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Silencing MXI1 attenuates proliferation, migration, and invasion of osteosarcoma cells. (A, B) RT-qPCR for verifying the expression of MXI1 in U2OS and Saos-2 cells following transfection with its specific siRNAs. (C, D) Colony formation for measuring the proliferative capacity of U2OS and Saos-2 cells after silencing MXI1 expression. (E–H) Transwell assays for detecting the (E, F) migration and (G, H) invasion of U2OS and Saos-2 cells with MXI1 knockdown. ** p < 0.01; **** p < 0.0001.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, Knockdown

Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Hec1 S69 is phosphorylated throughout mitosis. (A) Amino acid sequences of the human and PtK1 cell Hec1 N-terminal tail domain. Shown in yellow is the human peptide sequence that was used to generate the S69 phosphospecific antibody. The arrow points to S69 in the human sequence and the corresponding serine residue in the PtK1 sequence. Asterisks indicate all other mapped Aurora B kinase sites in the human Hec1 tail domain ( ; ). (B) Immunofluorescence images of HeLa cells stained with phosphospecific antibodies to Hec1 pS69. Depletion of Hec1 (bottom) results in loss of pS69 staining at kinetochores. Cells are also immunostained with antibody 9G3 (pan-Hec1 antibody) and an anticentromere antibody (ACA) derived from human calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum. (C) Immunofluorescence images of HeLa cells demonstrating kinetochore localization of pS69 during mitosis. Quantification is shown on the right. For each phase shown, ≥400 kinetochores from ≥30 cells were measured. (D) Immunofluorescence images of a HeLa cell stained with antibodies to pS69 and Mad2. For the cell shown, most chromosomes are aligned at the spindle equator, and one chromosome remains near a spindle pole (arrows). A schematic illustrating examples of pole-proximal chromosomes is shown on the left. (E) Immunofluorescence images of HeLa cells depleted of CENP-E to increase the number of pole-proximal chromosomes (arrows) and stained with Hec1 phosphospecific antibodies. Quantification is shown on the right from one representative experiment. n values are as follows: pS69, 20 polar kinetochores and 40 aligned kinetochores; pS55, 17 polar kinetochores and 57 aligned kinetochores; and pS44, 13 polar kinetochores and 29 aligned kinetochores. Error bars indicate SD. Bars: (B, C, and E) 10 µm; (D) 3 µm.

Article Snippet: Two rabbits were immunized with the Mad2 protein (Rockland Immunochemicals Inc), and antisera from the two injected rabbits were affinity purified against full-length Mad2 protein using a HiTrap-NHS column.

Techniques: Sequencing, Residue, Immunofluorescence, Staining, Derivative Assay