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: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: CMLD-2 effects on MAD2 protein levels in thyroid cancer cells. Panel A. MAD2 is identified as HuR target by RIP assay. Thyroid cancer cells were lysed and immunoprecipitated with anti-HuR or anti-IgG antibodies. HuR–bound mRNAs were amplified using specific MAD2 and MARCH3 (negative control) primers and analyzed by qPCR. All samples were run in triplicate. IgG-immunoprecipitate was arbitrarily set at 1.0 and the enrichment was expressed as relative expression value (Fold Enrichment). Panel B. SW1736, 8505 C, BCPAP and K1 cells were treated with DMSO or CMLD-2 35 µM for 72 h. Cells were collected and MAD2 protein levels were analyzed by Western Blot. Panel C. Densitometric analysis of MAD2 protein levels obtained with Western Blot assay. For each cell line, the results were normalized against Actin and expressed as percentage over control. Results are shown as mean ± SD. ***p < 0.001, ****p < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Immunoprecipitation, Amplification, Negative Control, Expressing, Western Blot

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: Biological effects of MAD2 silencing in thyroid cancer cells. 8505 C and BCPAP cells were transfected with vehicle (mock), non-targeting siRNA (NC, negative control) or three different siRNA (#1, #2 and #3) sequence specific to MAD2 (5 nM) for 72 h and cell viability was analyzed by MTT assay. All experiments were run in sixfold. P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Transfection, Negative Control, Sequencing, MTT Assay

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: MAD2 is involved in CMLD-2 effects in thyroid cancer cell lines. Panel A. 8505 C and BCPAP cells were treated with DMSO, CMLD-2, pCMV empty vector (NC, negative control), pCMV vector specific for MAD2 (1.3 µg), alone or in combinations. Cells were collected after 72 h treatment and MAD2 protein levels were analyzed by Western Blot. Panel B. 8505 C and BCPAP cells were treated with DMSO, CMLD-2, pCMV empty vector (NC, negative control), pCMV vector specific for MAD2 (1.3 µg), alone or in combinations and cell viability was determined by MTT assay after 72 h. NC was arbitrarily set at 1 and cell viability was expressed as relative expression value. All samples were run in sixfold. P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Plasmid Preparation, Negative Control, Western Blot, MTT Assay, Expressing