Journal: Journal of Ovarian Research

Article Title: 5-methylcytosine regulated CCNL2 promotes tumorigenesis and cisplatin resistance of ovarian cancer with therapeutic implications

doi: 10.1186/s13048-025-01753-9

Figure Lengend Snippet: CCNL2 is upregulated in OC and associated with poor prognosis. A ) CCNL2 expression level in OC tissues ( n = 10) and fallopian tube tissues ( n = 10) from the GEO dataset, GSE69428 . B ) Western blot analysis of CCNL2 protein expression levels in serous ovarian cancer tissues (SOCs) ( n = 7) and fallopian tube tissues (FTs) ( n = 6). C ) CCNL2 protein expression was assessed in OC ( n = 100) and normal tissues ( n = 25) in the CPTAC database. D ) CCNL2 protein expression level was analyzed across different OC grades in the CPTAC database. E ) Pan-cancer analysis of CCNL2 at the protein level based on the CPTAC database. F ) Statistical analysis was conducted on CCNL2 expression levels assessed via IHC staining of tissue microarrays. The proportion of tissues with high CCNL2 expression was higher in SOCs ( n = 130) compared to FTs ( n = 50). G , H ) Representative images with high and low CCNL2 expression levels from tissue microarrays via IHC staining. I , J ) Kaplan-Meier analyses of the Qilu cohort demonstrated the effect of CCNL2 on the PFS ( I ) and OS ( J ) in OC patients. K , L ) Kaplan-Meier analyses demonstrated how CCNL2 affects the PFS ( L ) and OS (K) of OC patients based on the Kaplan-Meier cohort. The p-value was obtained using two-tailed unpaired Student’s t-test (for A , C , D ), Chi-square test (for F), and log-rank test (for I, J, K, L ). Data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant

Article Snippet: Tissues were then blocked and incubated overnight at 4°C with primary antibody against Ki-67 (1:100, Origene, TA500265) and CCNL2 (1:150, Elabscience, E-AB-40247).

Techniques: Expressing, Western Blot, Immunohistochemistry, Two Tailed Test

Journal: Journal of Ovarian Research

Article Title: 5-methylcytosine regulated CCNL2 promotes tumorigenesis and cisplatin resistance of ovarian cancer with therapeutic implications

doi: 10.1186/s13048-025-01753-9

Figure Lengend Snippet: CCNL2 promotes the proliferation of ovarian cancer. A ) Western blot analysis of CCNL2 in stably CCNL2-depleted and CCNL2-overexpressed OC cell lines. B ) The effect of CCNL2 on cell proliferation was assessed by colony formation assay in HEY and OV90 cells ( n = 3). Quantified data are shown on the right. C ) CCK8 assay was performed to assess the proliferation ability after CCNL2 knockdown or overexpression in HEY cells ( n = 3). D ) The EdU assay was performed in OV90 cells with CCNL2 knockdown ( n = 3). Scale bar, 100 μm. Quantified data are shown on the right. E , F ) Transwell assays were performed in HEY and OV90 cells with CCNL2 knockdown ( n = 3). Quantified data are shown on the right. G - I ) The image of subcutaneous tumors ( G ), tumor mass ( H ), and IHC images of Ki-67 and CCNL2 ( I ) in control and CCNL2 knockdown groups ( n = 5 per group). Scale bar, 100 μm. J - L ) The image of subcutaneous tumors ( J ), tumor mass ( K ), and IHC images of Ki-67 and CCNL2 ( L ) in control and CCNL2 overexpression groups ( n = 5 per group). Scale bar, 100 μm. All quantification analyses were based on three independent experiments. Data are mean ± SD. The p-value was obtained using two-tailed unpaired Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant

Article Snippet: Tissues were then blocked and incubated overnight at 4°C with primary antibody against Ki-67 (1:100, Origene, TA500265) and CCNL2 (1:150, Elabscience, E-AB-40247).

Techniques: Western Blot, Stable Transfection, Colony Assay, CCK-8 Assay, Knockdown, Over Expression, EdU Assay, Control, Two Tailed Test

Journal: Journal of Ovarian Research

Article Title: 5-methylcytosine regulated CCNL2 promotes tumorigenesis and cisplatin resistance of ovarian cancer with therapeutic implications

doi: 10.1186/s13048-025-01753-9

Figure Lengend Snippet: Knockdown of CCNL2 enhances the sensitivity of OC cells to cisplatin. A ) CCK8 assay was conducted to assess the effect of the CCNL2 depletion on chemosensitivity. OC cells were treated with various concentrations of cisplatin for 48 h ( n = 3). B ) Apoptotic cells were analyzed using Annexin V/PI staining in HEY and OV90 cells with cisplatin treatment (5 µg/ml, 48 h) ( n = 3). The bar graphs on the right display the percentage of apoptotic cells. C ) Clonogenic assay was performed with CCNL2 depletion under cisplatin treatment in OV90 cells. D - G ) The image of subcutaneous tumors ( D ), tumor mass ( E ), IHC images of Ki-67 ( F ), and tumor growth curves ( G ) in CCNL2 knockdown and control groups with cisplatin treatment (4 mg/kg every three days) ( n = 5 per group). Scale bar, 100 μm. All quantification analyses were based on three independent experiments. Data are mean ± SD. Student’s unpaired t-test was used to calculate the p value.* P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant

Article Snippet: Tissues were then blocked and incubated overnight at 4°C with primary antibody against Ki-67 (1:100, Origene, TA500265) and CCNL2 (1:150, Elabscience, E-AB-40247).

Techniques: Knockdown, CCK-8 Assay, Staining, Clonogenic Assay, Control

Journal: Journal of Ovarian Research

Article Title: 5-methylcytosine regulated CCNL2 promotes tumorigenesis and cisplatin resistance of ovarian cancer with therapeutic implications

doi: 10.1186/s13048-025-01753-9

Figure Lengend Snippet: YBX1 enhances CCNL2 mRNA stability via an m 5 C-dependent manner. A ) Hub proteins were screened by Cytoscape from proteins pulled by biotinylated-CCNL2 probe. B ) Relative CCNL2 mRNA expression after YBX1 knockdown in HEY and OV90 cells ( n = 3). C ) Relative CCNL2 protein expression was analyzed with YBX1 knockdown in HEY and OV90 cells. D ) Colony formation assay was conducted to assess the rescue effect of YBX1 on CCNL2 overexpression ( n = 3). E ) Correlation analysis of CCNL2 and m 5 C signatures in ovarian cancer tissues based on TCGA database. F ) The half-life of CCNL2 mRNA was analyzed by RT-qPCR after YBX1 knockdown with Actinomycin D treatment in HEY and OV90 cells. G ) Immunoblotting of YBX1 after RNA pull-down assays with full-length biotinylated-CCNL2 probe and negative RNA probe. H ) RIP assays showing the interaction between YBX1 and CCNL2 mRNA ( n = 3). The agarose gel electrophoresis results above showed the results of RT-PCR. The NC lane meant negative control without the template. The IgG and Anti-YBX1 lanes used products pulled by IgG or anti-YBX1 antibodies separately. The input lane used input as the template. I ) MeRIP assays were conducted after YBX1 knockdown. Primers used in the Negative Control group were designed for non-m 5 C modification sites, while primers used in the other two groups were designed for the m 5 C modification sites ( n = 3). J ) Relative luciferase activity of CCNL2-WT or CCNL2-mut in YBX1 overexpression or control cells ( n = 3). K ) Relative luciferase activity of CCNL2-WT was detected after YBX1 overexpression or W65A mutation ( n = 3). All quantification analyses were based on three independent experiments. Data are mean ± SD. Student’s unpaired t-test was used to calculate the p value. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. The relative enrichment of CCNL2 in the RIP assay was normalized by input. The fold enrichment of CCNL2 in the MeRIP assay was normalized by input and IgG

Article Snippet: Tissues were then blocked and incubated overnight at 4°C with primary antibody against Ki-67 (1:100, Origene, TA500265) and CCNL2 (1:150, Elabscience, E-AB-40247).

Techniques: Expressing, Knockdown, Colony Assay, Over Expression, Quantitative RT-PCR, Western Blot, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Negative Control, Modification, Luciferase, Activity Assay, Control, Mutagenesis

Journal: Journal of Ovarian Research

Article Title: 5-methylcytosine regulated CCNL2 promotes tumorigenesis and cisplatin resistance of ovarian cancer with therapeutic implications

doi: 10.1186/s13048-025-01753-9

Figure Lengend Snippet: YBX1 cooperates with MATR3 to maintain the stability of CCNL2 mRNA. A ) CO-IP assay demonstrated the interaction between YBX1 and MATR3. B ) CCNL2 protein expression was measured with MATR3 depletion by western blot in HEY and OV90 cells. C ) The half-life of CCNL2 mRNA was analyzed by RT-qPCR with MATR3 knockdown in OV90 cells with actinomycin D treatment ( n = 3). D ) CCNL2 mRNA expression was analyzed in indicated HEY cells ( n = 3). E ) The half-life of CCNL2 mRNA was analyzed by RT-qPCR in indicate OV90 cells with actinomycin D treatment ( n = 3). F , G ) CCNL2 protein expression was measured in indicated HEY and OV90 cells. H ) Colony formation assay was conducted to investigate the regulation mechanism of YBX1-MATR3-CCNL2 axis. I ) CCK8 assay confirmed the regulation mechanism of YBX1-MATR3-CCNL2 axis in HEY cells. All quantification analyses were based on three independent experiments. Data are mean ± SD. Student’s unpaired t-test was used to calculate the p value. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant

Article Snippet: Tissues were then blocked and incubated overnight at 4°C with primary antibody against Ki-67 (1:100, Origene, TA500265) and CCNL2 (1:150, Elabscience, E-AB-40247).

Techniques: Co-Immunoprecipitation Assay, Expressing, Western Blot, Quantitative RT-PCR, Knockdown, Colony Assay, CCK-8 Assay

Journal: Journal of Ovarian Research

Article Title: 5-methylcytosine regulated CCNL2 promotes tumorigenesis and cisplatin resistance of ovarian cancer with therapeutic implications

doi: 10.1186/s13048-025-01753-9

Figure Lengend Snippet: Identification of a small molecule YB-B1 which enhances chemotherapy sensitivity. A ) Schematic diagram of virtual screening for YBX1 inhibitors. B ) Chemical structure of YB-B1 and its docking with the YBX1 protein. C , D ) SPR assay was performed to evaluate the affinity between YB-B1 and YBX1 human protein. RU, resonance unit. E ) Western blot analyses were performed to evaluate the effects of YB-B1 treatment over the indicated time in HEY and OV90 cells. F ) The line chart illustrating the time-dependent trends of CCNL2 and YBX1 protein expression during YB-B1 treatment. G ) Dose-response curve for YB-B1 in HEY and OV90 cells after 48 h of treatment ( n = 3). H ) Clonogenic assay with YB-B1 treatment in HEY and OV90 cells ( n = 3). I ) Cell viability was assessed in OV90 cells after 48 h with cisplatin and YB-B1 treatment ( n = 3). J , K ) Apoptotic cells were analyzed using Annexin V/PI staining in OV90 cells with cisplatin and YB-B1 treatment for 48 h ( n = 3). L - O ) The PDX model was constructed using fresh ovarian cancer tissues and divided into Cisplatin and Cisplatin + YB-B1 groups for corresponding treatments. The image of subcutaneous tumors ( L ), tumor mass ( M ), and IHC images of Ki-67 ( N ) in Cisplatin and Cisplatin + YB-B1 groups ( n = 5 per group). Scale bar, 50 μm. Quantified data are shown on the right. All quantification analyses were based on three independent experiments. Data are mean ± SD. Student’s unpaired t-test was used to calculate the p value. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant

Article Snippet: Tissues were then blocked and incubated overnight at 4°C with primary antibody against Ki-67 (1:100, Origene, TA500265) and CCNL2 (1:150, Elabscience, E-AB-40247).

Techniques: SPR Assay, Western Blot, Expressing, Clonogenic Assay, Staining, Construct

Journal: Journal of Ovarian Research

Article Title: 5-methylcytosine regulated CCNL2 promotes tumorigenesis and cisplatin resistance of ovarian cancer with therapeutic implications

doi: 10.1186/s13048-025-01753-9

Figure Lengend Snippet: Graphic abstract of the role of CCNL2 in ferroptosis and its regulatory mechanism. YBX1 cooperates with MATR3 to regulate CCNL2, driving OC cells into the ferrotosis resistance state. LIP referes to labile iron pool

Article Snippet: Tissues were then blocked and incubated overnight at 4°C with primary antibody against Ki-67 (1:100, Origene, TA500265) and CCNL2 (1:150, Elabscience, E-AB-40247).

Techniques: