l ap ythdf3 proteintech  (Proteintech)

Journal: Molecular Cancer

Article Title: CircZFR/YTHDF3 axis drives lymph node metastasis in cervical cancer via FASN translation

doi: 10.1186/s12943-025-02424-5

Figure Lengend Snippet: CircZFR Binds to YTHDF3 and Enhances FASN Expression in Cervical Cancer Cells. A Western blot analysis of in vitro circZFR pulldown assay indicated that circZFR could bind with YTHDF3 protein. B RIP-qPCR showing the association of YTHDF3 protein with circZFR in HeLa cells. C Left: Simulated model of the ternary complex formation between circZFR and YTHDF3 protein. Right: Schematic diagram highlighting the predicted binding regions between YTHDF3 and circZFR. D Schematic workflow of circZFR-YTHDF3 downstream target analysis. E. Western blot analysis of FASN protein levels in HeLa, C33A, and Caski cells with circZFR overexpression and knockdown. F. Western blot analysis of FASN protein levels in HeLa cells transfected with either wild-type or mutant circZFR plasmids after circZFR knockdown. Data in E and F are mean value ± standard error of the mean (SEM) of three biological replicates, student t test, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Cell lysates were incubated with beads conjugated to YTHDF3 or eIF4A3 antibodies (Proteintech).

Techniques: Expressing, Western Blot, In Vitro, Binding Assay, Over Expression, Knockdown, Transfection, Mutagenesis

Journal: Molecular Cancer

Article Title: CircZFR/YTHDF3 axis drives lymph node metastasis in cervical cancer via FASN translation

doi: 10.1186/s12943-025-02424-5

Figure Lengend Snippet: Elevated FASN Levels Correlate with Lymph Node Metastasis and Poor Survival Outcomes in Cervical Cancer. A Kaplan-Meier survival analysis of TCGA CESC data showed that the cervical cancer patients with higher FASN mRNA expression ( n = 146) had shorter survival time than the cases with lower FASN mRNA expression ( n = 146). Median value of FASN mRNA expression was set as cut-off. Left: survival curve of DFS (HR = 2, Log-rank P = 0.021); Right: survival curve of overall survival (HR = 2, Log-rank P = 0.0033). B Analysis of TCGA CESC data showed that the advanced stage of cervical cancer had higher FASN mRNA expression ( n = 292, P = 0.0289). C and E. Immunohistochemistry of FASN and YTHDF3 protein levels in the primary tumors of cervical patients with long or short DFS. The cut-off value is 2 years. Left: representative immunohistochemistry (three samples in each group) of FASN and YTHDF3 protein levels. Right: histograms of FASN and YTHDF3 protein levels in the patients with DFS < 2 years ( n = 19) and DFS > 2 years ( n = 21). Lines show mean value and SD, student t test, *** P < 0.001. D and F. Kaplan-Meier survival analysis showed significant differences in DFS between the patients with high FASN protein expression (IHC score ≥ 8, n = 23) and low FASN protein expression (IHC score < 8, n = 17). And the patients with high YTHDF3 protein expression (IHC score ≥ 8, n = 22) had shorter DFS than low YTHDF3 protein expression (IHC score < 8, n = 18). G. The level of FASN and YTHDF3 protein in lymph nodes positive patients was higher than lymph nodes negative cases. Left: representative western blot of FASN and YTHDF3 protein levels in the primary tumors of the patients with ( n = 6) or without ( n = 6) lymph nodes metastasis. Right: histograms of FASN and YTHDF3 protein relative intensity in the patients with ( n = 39) or without ( n = 65) lymph nodes metastasis. Lines show mean value and SD, student t test, *** P < 0.001. H. Positive correlation existed between FASN and YTHDF3 proteins. N = 104, Pearson r = 0.664, P < 0.001

Article Snippet: Cell lysates were incubated with beads conjugated to YTHDF3 or eIF4A3 antibodies (Proteintech).

Techniques: Expressing, Immunohistochemistry, Western Blot

Journal: Molecular Cancer

Article Title: CircZFR/YTHDF3 axis drives lymph node metastasis in cervical cancer via FASN translation

doi: 10.1186/s12943-025-02424-5

Figure Lengend Snippet: CircZFR-YTHDF3 Axis Promotes Proliferation, Migration, and Invasion in Cervical Cancer Cells. A RT-qPCR analysis of circZFR expression in HeLa and C33A cells with circZFR stable overexpression. circZFR-OE, circZFR-overexpression. B RT-qPCR analysis of YTHDF3 mRNA expression in HeLa and C33A cells treated with two independent siRNAs against YTHDF3. siCtrl, Si-Control. C Western blot analysis of YTHDF3 protein expression in HeLa and C33A cells treated with two independent siRNAs against YTHDF3. D Cell counting kit-8 (CCK-8) assay of HeLa and C33A cells treated with siRNAs against YTHDF3. E EdU assay of HeLa, C33A, and Caski cells treated with siRNAs against YTHDF3. Left: representative immunofluorescence of Fluro594 (red), DAPI (blue), and merge. Scale bars: 50 μm. Right: percentages of EdU-incorporated Hela, C33A, and Caski cells. F Migratory and invasive ability assessed by Transwell assay of HeLa and C33A cells treated with siRNAs against YTHDF3. Scale bars: 50 μm. G Representative western blot of EMT markers protein levels in HeLa and C33A cells treated with siRNAs against YTHDF3. Data in A , B , C , D , E , F , and G are mean value ± SEM of three biological replicates, student t test, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Cell lysates were incubated with beads conjugated to YTHDF3 or eIF4A3 antibodies (Proteintech).

Techniques: Migration, Quantitative RT-PCR, Expressing, Over Expression, Control, Western Blot, Cell Counting, CCK-8 Assay, EdU Assay, Immunofluorescence, Transwell Assay

Journal: Molecular Cancer

Article Title: CircZFR/YTHDF3 axis drives lymph node metastasis in cervical cancer via FASN translation

doi: 10.1186/s12943-025-02424-5

Figure Lengend Snippet: CircZFR Facilitates FASN Protein Translation via the YTHDF3-Dependent Mechanism. A Western blot analysis of FASN protein expression in HeLa and C33A cells treated with siYTHDF3#1 or siYTHDF3#2. B After treatment with MG132 for indicated times, protein levels of FASN were tested by Western blot analyses of HeLa cells treated with siCtrl, siYTHDF3#1, or siYTHDF3#2. C Coimmunoprecipitation by anti-eIF4A3 verified that the combination between YTHDF3 and eIF4A3 increased after overexpression circZFR and decreased after knocking down circZFR. D RIP assay using eIF4A3 antibody to test the combination between eIF4A3 protein and FASN mRNA in HeLa cells with or without circZFR overexpression. E MeRIP-qPCR assay validated the three m 6 A sites in FASN mRNA. F Schematic image of YTHDF3-WT plasmids, and YTHDF3-MUT plasmids containing m 6 A motif mutations in the CDS region. G Western blot assay tested the FASN protein expression in HeLa cells transfected with empty vector, YTHDF3-WT, or YTHDF3-MUT plasmids. WT, wild type; MUT, mutant. Data in A, B, C, D, E, and G are mean value ± SEM of three biological replicates, student t test, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Cell lysates were incubated with beads conjugated to YTHDF3 or eIF4A3 antibodies (Proteintech).

Techniques: Western Blot, Expressing, Over Expression, Transfection, Plasmid Preparation, Mutagenesis

Journal: Molecular Cancer

Article Title: CircZFR/YTHDF3 axis drives lymph node metastasis in cervical cancer via FASN translation

doi: 10.1186/s12943-025-02424-5

Figure Lengend Snippet: CircZFR Accelerates Lymph Node Metastasis in Vivo in Preclinical Models. A HeLa cells stably expressing either shCtrl or sh-circZFR (1 × 10⁶ cells) were injected into the footpads of mice and harvested popliteal lymph nodes (upper panel). Moreover, HeLa cells stably expressing either empty control or circZFR-OE (1 × 10⁶ cells) were injected into footpads of mice and harvested popliteal lymph nodes (lower panel). B Tumor volume analysis: Left panel shows comparison between empty vector and circZFR-OE groups; right panel shows comparison between shCtrl and sh-circZFR groups. C Representative images of harvested popliteal lymph nodes from the shCtrl, sh-circZFR, empty vector, and circZFR-OE groups. D Western blot analysis of FASN protein levels in popliteal lymph nodes from all four groups. Left: Representative Western blot images. Right: Quantification of FASN protein expression. E Schematic illustration of circZFR molecular mechanism. Overexpression circZFR binds to the m 6 A reader protein YTHDF3, facilitating m 6 A recognition on FASN mRNA and recruiting the translation initiator eIF4A3, boosting FASN translation, thereby facilitating EMT of cervical cancer cells. Statistical analyses of data in B and D were conducted using Student’s t-test. Data are presented as mean ± SEM from independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Cell lysates were incubated with beads conjugated to YTHDF3 or eIF4A3 antibodies (Proteintech).

Techniques: In Vivo, Stable Transfection, Expressing, Injection, Control, Comparison, Plasmid Preparation, Western Blot, Over Expression

Journal: Molecular Cancer

Article Title: CircZFR/YTHDF3 axis drives lymph node metastasis in cervical cancer via FASN translation

doi: 10.1186/s12943-025-02424-5

Figure Lengend Snippet: CircZFR Facilitates FASN Protein Translation via the YTHDF3-Dependent Mechanism. A Western blot analysis of FASN protein expression in HeLa and C33A cells treated with siYTHDF3#1 or siYTHDF3#2. B After treatment with MG132 for indicated times, protein levels of FASN were tested by Western blot analyses of HeLa cells treated with siCtrl, siYTHDF3#1, or siYTHDF3#2. C Coimmunoprecipitation by anti-eIF4A3 verified that the combination between YTHDF3 and eIF4A3 increased after overexpression circZFR and decreased after knocking down circZFR. D RIP assay using eIF4A3 antibody to test the combination between eIF4A3 protein and FASN mRNA in HeLa cells with or without circZFR overexpression. E MeRIP-qPCR assay validated the three m 6 A sites in FASN mRNA. F Schematic image of YTHDF3-WT plasmids, and YTHDF3-MUT plasmids containing m 6 A motif mutations in the CDS region. G Western blot assay tested the FASN protein expression in HeLa cells transfected with empty vector, YTHDF3-WT, or YTHDF3-MUT plasmids. WT, wild type; MUT, mutant. Data in A, B, C, D, E, and G are mean value ± SEM of three biological replicates, student t test, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Cell lysates were incubated with beads conjugated to YTHDF3 or eIF4A3 antibodies (Proteintech).

Techniques: Western Blot, Expressing, Over Expression, Transfection, Plasmid Preparation, Mutagenesis

Journal: Molecular Cancer

Article Title: CircZFR/YTHDF3 axis drives lymph node metastasis in cervical cancer via FASN translation

doi: 10.1186/s12943-025-02424-5

Figure Lengend Snippet: CircZFR Accelerates Lymph Node Metastasis in Vivo in Preclinical Models. A HeLa cells stably expressing either shCtrl or sh-circZFR (1 × 10⁶ cells) were injected into the footpads of mice and harvested popliteal lymph nodes (upper panel). Moreover, HeLa cells stably expressing either empty control or circZFR-OE (1 × 10⁶ cells) were injected into footpads of mice and harvested popliteal lymph nodes (lower panel). B Tumor volume analysis: Left panel shows comparison between empty vector and circZFR-OE groups; right panel shows comparison between shCtrl and sh-circZFR groups. C Representative images of harvested popliteal lymph nodes from the shCtrl, sh-circZFR, empty vector, and circZFR-OE groups. D Western blot analysis of FASN protein levels in popliteal lymph nodes from all four groups. Left: Representative Western blot images. Right: Quantification of FASN protein expression. E Schematic illustration of circZFR molecular mechanism. Overexpression circZFR binds to the m 6 A reader protein YTHDF3, facilitating m 6 A recognition on FASN mRNA and recruiting the translation initiator eIF4A3, boosting FASN translation, thereby facilitating EMT of cervical cancer cells. Statistical analyses of data in B and D were conducted using Student’s t-test. Data are presented as mean ± SEM from independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Cell lysates were incubated with beads conjugated to YTHDF3 or eIF4A3 antibodies (Proteintech).

Techniques: In Vivo, Stable Transfection, Expressing, Injection, Control, Comparison, Plasmid Preparation, Western Blot, Over Expression