Journal: Investigative Ophthalmology & Visual Science

Article Title: Abemaciclib Inhibits Retinoblastoma Tumor Growth by Targeting CDK1/2

doi: 10.1167/iovs.67.5.72

Figure Lengend Snippet: Abemaciclib reduces retinoblastoma cell viability and CDK1/2 phosphorylation with minimal cytotoxicity to healthy retinal cells. ( A, B ) CCK-8 assays showing dose-dependent inhibition of cell viability by abemaciclib in Y79 and WERI-Rb1 retinoblastoma cells, respectively, with IC₅₀ values indicated. ( C ) CCK-8 assay showing that abemaciclib has no obvious cytotoxic effect on healthy retinal pigment epithelial cells. ( D, E ) Western blot analysis of total and phosphorylated CDK1 and CDK2 in Y79 and WERI-Rb1 cells treated with abemaciclib for 48 hours. ( F, G ) Densitometric quantification of total and phosphorylated CDK1 and CDK2 protein levels normalized to β-actin in Y79 and WERI-Rb1 cells, showing reduced CDK1/2 phosphorylation after abemaciclib treatment. ( H, I ) Quantitative analysis of the phospho-CDK1 (Thr161)/CDK1 and phospho-CDK2 (Thr160)/CDK2 ratios in Y79 and WERI-Rb1 cells treated with increasing doses of abemaciclib. Data are presented as mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). CDK1, cyclin-dependent kinase 1; CDK2, cyclin-dependent kinase 2; CCK-8, Cell Counting Kit-8.

Article Snippet: Abemaciclib (HY-16297A; MedChemExpress, China) was dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) to prepare a 5 mM stock solution.

Techniques: Phospho-proteomics, CCK-8 Assay, Inhibition, Western Blot, Cell Counting

Journal: Investigative Ophthalmology & Visual Science

Article Title: Abemaciclib Inhibits Retinoblastoma Tumor Growth by Targeting CDK1/2

doi: 10.1167/iovs.67.5.72

Figure Lengend Snippet: Transcriptome sequencing analysis of Y79 cells treated with 5 µM abemaciclib for 48 hours. ( A ) Volcano plot showing differential transcript expression between DMSO- and abemaciclib-treated Y79 cells. ( B ) Heatmap of the top 40 DEGs ranked by adjusted P value. ( C, D ) KEGG pathway and GO biological process enrichment analyses of the DEGs. ( E ) GSEA enrichment plots of the p53 and apoptosis signaling pathways. False discovery rate (FDR) ≤0.25 was considered statistically significant (normalized enrichment score [NES]). DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis.

Article Snippet: Abemaciclib (HY-16297A; MedChemExpress, China) was dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) to prepare a 5 mM stock solution.

Techniques: Sequencing, Expressing, Protein-Protein interactions

Journal: Investigative Ophthalmology & Visual Science

Article Title: Abemaciclib Inhibits Retinoblastoma Tumor Growth by Targeting CDK1/2

doi: 10.1167/iovs.67.5.72

Figure Lengend Snippet: Abemaciclib inhibits retinoblastoma cell proliferation by inducing G2/M phase arrest and apoptosis. ( A ) Representative flow cytometry histograms of cell cycle distribution in Y79 and WERI-Rb1 cells treated with DMSO or abemaciclib for 48 hours. ( B ) Statistical analysis of cell cycle phase distribution in Y79 and WERI-Rb1 cells. ( C ) Representative flow cytometry plots of apoptosis in Y79 and WERI-Rb1 cells treated with DMSO or abemaciclib for 48 hours (necrotic cells, Q1; late apoptotic cells, Q2; early apoptotic cells, Q3; and viable cells, Q4). ( D ) Statistical analysis of apoptosis rates in Y79 and WERI-Rb1 cells. ( E ) Western blot analysis of apoptosis-related protein expression (PARP, Cleaved PARP, BAX, and BCL-2) in retinoblastoma cells treated with DMSO or abemaciclib for 48 hours. ( F ) Densitometric quantification of protein levels in E , normalized to β-actin. ( G ) Flow cytometric analysis of EdU incorporation in Y79 and WERI-Rb1 cells treated with DMSO or the indicated concentrations of abemaciclib for 48 hours. ( H ) Percentage of EdU positive cells in Y79 and WERI-Rb1 cells. ( I, J ) Colony formation in soft agar of Y79 and WERI-Rb1 cells treated with DMSO or abemaciclib for 2 weeks, shown as microscopic and macroscopic images. ( K ) Relative colony formation in Y79 and WERI-Rb1cells in the soft agar assay. Data are presented as mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Abemaciclib (HY-16297A; MedChemExpress, China) was dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) to prepare a 5 mM stock solution.

Techniques: Flow Cytometry, Western Blot, Expressing, Soft Agar Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Abemaciclib Inhibits Retinoblastoma Tumor Growth by Targeting CDK1/2

doi: 10.1167/iovs.67.5.72

Figure Lengend Snippet: Abemaciclib induces DNA damage and activates the p53/p21 pathway in retinoblastoma cells. ( A ) Immunofluorescence detection of γ-H2AX in Y79 and WERI-Rb1 cells treated with DMSO or abemaciclib for 48 hours ( scale bar = 50 µm). ( B ) Quantification of mean γ-H2AX fluorescence intensity in Y79 and WERI-Rb1 cells. ( C ) Representative fluorescence images of intracellular ROS in Y79 and WERI-Rb1 cells treated with DMSO or abemaciclib for 48 hours ( scale bar = 100 µm). ( D ) Quantitative analysis of intracellular ROS levels in Y79 and WERI-Rb1 cells. ( E ) Western blot analysis of DNA damage repair-related proteins ATR, CHK1, and Rad51, along with the DNA damage marker γ-H2AX, in retinoblastoma cells treated with abemaciclib for 48 hours. ( F, G ) Densitometric quantification of protein levels in E , normalized to β-actin. ( H ) The mRNA levels of p53 and p21 in Y79 cells treated with abemaciclib for 48 hours, as determined by qRT-PCR. ( I ) Western blot analysis of p53 and p21 in retinoblastoma cells treated with abemaciclib for 48 hours. ( J ) Densitometric quantification of p53 and p21 protein levels normalized to β-actin. Data are presented as mean ± SD of three independent experiments (** P < 0.01, *** P < 0.001, **** P < 0.0001). ROS, reactive oxygen species; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: Abemaciclib (HY-16297A; MedChemExpress, China) was dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) to prepare a 5 mM stock solution.

Techniques: Immunofluorescence, Fluorescence, Western Blot, Marker, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Investigative Ophthalmology & Visual Science

Article Title: Abemaciclib Inhibits Retinoblastoma Tumor Growth by Targeting CDK1/2

doi: 10.1167/iovs.67.5.72

Figure Lengend Snippet: Abemaciclib suppress tumor growth in the retinoblastoma PDX model. ( A ) Gross images of mice bearing retinoblastoma PDX tumors after treatment with vehicle or abemaciclib. ( B ) Photographs of excised PDX tumors. ( C ) Weights of excised PDX tumors. ( D ) Tumor volume curves of PDX tumors in mice treated with vehicle or abemaciclib, measured at the indicated time points. ( E ) Body weight curves of mice during treatment with vehicle or abemaciclib. ( F ) Immunohistochemical staining of BAX and Ki-67 in PDX tumor sections from vehicle- and abemaciclib-treated mice. (** P < 0.01, **** P < 0.0001). PDX, patient-derived xenograft.

Article Snippet: Abemaciclib (HY-16297A; MedChemExpress, China) was dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) to prepare a 5 mM stock solution.

Techniques: Immunohistochemical staining, Staining, Derivative Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Abemaciclib Inhibits Retinoblastoma Tumor Growth by Targeting CDK1/2

doi: 10.1167/iovs.67.5.72

Figure Lengend Snippet: Abemaciclib suppresses intraocular tumor growth in the orthotopic xenograft model. ( A ) Experimental timeline of the orthotopic CDX model. ( B ) Slit-lamp photographs showing intraocular tumor formation in eyes injected with Y79 cells compared with healthy eyes. ( C ) Slit-lamp photographs of representative eyes from vehicle- and abemaciclib-treated mice on days 4 and 18, illustrating progressive intraocular tumor growth in the vehicle group and tumor suppression in the abemaciclib group. ( D ) Body weight curves of mice during treatment with vehicle or abemaciclib. ( E ) H&E staining of tumor-bearing eyes from vehicle- and abemaciclib-treated mice at the experimental endpoint. ( F ) Quantification of intraocular tumor area based on H&E sections from vehicle- and abemaciclib-treated mice. Each point denotes the mean tumor area per mouse. ( G, H ) Immunohistochemical staining of Ki-67 and BAX in orthotopic CDX tumor sections from vehicle- and abemaciclib-treated mice. (*** P < 0.001). CDX, cell line-derived xenograft; H&E, hematoxylin and eosin.

Article Snippet: Abemaciclib (HY-16297A; MedChemExpress, China) was dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) to prepare a 5 mM stock solution.

Techniques: Injection, Staining, Immunohistochemical staining, Derivative Assay