Fig 1: CRISPR library screening identified ELF2 as a driver of topotecan resistance.A The schematic diagram illustrates the workflow of genome-wide CRISPR-Cas9 knockout library screening (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats). B The scatter plot depicts the results for topotecan positively selected hits in the CRISPR-Cas9 screening, with the top 20 hits shown in red. C KEGG analysis of the top 50 topotecan positively selected hits identified through genome-wide CRISPR-Cas9 knockout screening. D Relative cell viability of WERI-Rb1 and Y79 cells following treatment with topotecan or vehicle for 96 hours (n = 3). E, F ELF2 protein expression in WERI-Rb1 and Y79 cells under topotecan treatment (n = 3). Data are presented as means ± SD. Statistical analysis was performed using two-tailed Student’s t test (D) or one-way ANOVA and Tukey’s multiple comparison test (F); ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.
Fig 2: Gene disruption of ELF2 enhances resistance to topotecan in Y79 retinoblastoma cells.A A reduction of ELF2 protein expression in ELF2 knockout (KO; sgELF2) cells compared to non-targeting control (NTC) cells. B Western blot analysis of total caspase-3 and cleaved caspase-3 in topotecan (TPT)-treated ELF2 KO and NTC cells. C, D Quantitative analysis of apoptotic cells by TUNEL assay in topotecan-treated ELF2 KO and NTC cells (n = 3; scale bar: 50 µm). The relative TUNEL-positive rate was normalized to the mean value of the NT + NaCl group (set as 1.0). E Seven days after the transplantation of NTC or ELF2 KO (sgELF2) cells (denoted as day 0), mice were subjected to an intraperitoneal injection of saline or 0.05 mg/kg topotecan once daily, Monday through Friday, for three consecutive weeks (total 15 doses). Following the initiation of dosing, each mouse was monitored with callipers, and tumor volumes were plotted graphically (n = 5). F The macroscopic appearance of xenotransplanted tumors 21 days following topotecan treatment. G and H Representative western blot image and quantitative analysis of ELF2 and Caspase-3 proteins in topotecan-treated ELF2 KO (sgELF2) and NTC tumor tissues (n = 5). I Quantitative analysis of apoptotic cells by TUNEL assay in topotecan-treated ELF2 KO (sgELF2) and NTC tumor tissues (n = 5). Data are presented as means ± SD. Statistical analysis was performed using two-tailed Student’s t test (A) or one-way ANOVA and Tukey’s multiple comparison test (B, D, E, H and I); ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.
Fig 3: Gene disruption of ELF2 promotes topotecan resistance in WERI-Rb1 cells.A A reduction of ELF2 protein expression in ELF2 knockout (KO; sgELF2) cells. B Western blot analysis of total caspase-3 and cleaved caspase-3 in topotecan (TPT)-treated ELF2 KO (sgELF2) and non-targeting control (NTC) cells. C, D Quantitative analysis of apoptotic cells by TUNEL assay in topotecan-treated ELF2 KO (sgELF2) and NTC cells (n = 3; scale bar: 50 µm). The relative TUNEL-positive rate was normalized to the mean value of the NT + NaCl group (set as 1.0). E Representative western blot image of ELF2 and Caspase-3 proteins in the ELF2-overexpressing (pCMV3-ELF2-t3) and control (pCMV3-untagged) WERI-Rb1 cells. F, G Quantitative analysis of ELF2 protein, cleaved caspase-3 proteins and total caspase-3 (n = 3). Bar groups in E–G represent: 1 = pCMV3-untagged, 2 = pCMV3-untagged + TPT, 3 = pCMV3-ELF2-t3, 4 = pCMV3-ELF2-t3 + TPT. H Relative cell viability of control and ELF2 overexpressing cells following treatment with topotecan for 72 h (n = 3). In H, the bars represent fold-change comparisons: 2/1 = (pCMV3-untagged + TPT)/(pCMV3-untagged); 4/3 = (pCMV3-ELF2-t3 + TPT)/(pCMV3-ELF2-t3). I and J Quantitative analysis of apoptotic cells by TUNEL assay in topotecan-treated ELF2 overexpressing and control cells (n = 3; scale bar: 50 µm). Data are presented as means ± SD. Statistical analysis was performed using two-tailed Student’s t test (A, H) or one-way ANOVA and Tukey’s multiple comparison test (B, D, F, G and J); ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.
Fig 4: Gene disruption of ELF2 enhances resistance to topotecan through metabolic pathways.A Principal component analysis (PCA) of RNA-seq data from the experimental groups (non-targeting control (NTC), ELF2 knockout (KO; sgELF2), topotecan(TPT)-treated NTC, topotecan-treated ELF2 KO WERI-Rb1 cells; n = 3). B Volcano plots illustrate the genes that were significantly altered in both NTC and ELF2 KO (sgELF2) WERI-Rb1 cells upon stimulation with topotecan. Red and blue dots indicate genes exhibiting a log2|fold change | > 1 and FDR < 0.05. C Venn diagram of differentially expressed genes (DEGs) between the two data sets. D Gene Ontology and E KEGG enrichment analysis of the identified genes from (C).
Fig 5: Gene disruption of ELF2 impairs the antitumor effects of topotecan in vivo.A Workflow of in vivo mouse xenotransplantation study. Seven days after the transplantation of non-targeting control (NTC) or ELF2 knockout (sgELF2) WERI-Rb1 cells (denoted as day 0), mice were subjected to an intraperitoneal injection of saline or 0.1 mg/kg topotecan (TPT) once daily, Monday through Friday, for 3 consecutive weeks (total 15 doses). B Following the initiation of dosing, each mouse was monitored with callipers, and tumor volumes were plotted graphically (n = 6). C The macroscopic appearance of xenotransplanted tumors 21 days following topotecan treatment. D and E Representative western blot image and quantitative analysis of ELF2 and Caspase-3 proteins in topotecan-treated ELF2 KO (sgELF2) and NTC tumor tissues (n = 6). F, G Representative image and quantitative analysis of apoptotic cells by TUNEL assay in topotecan-treated ELF2 KO (sgELF2) and NTC tumor tissues (n = 6; scale bar: 50 µm). Data are presented as means ± SD. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test (B, E and G); ****p < 0.0001, **p < 0.01, *p < 0.05.
Supplier Page from Sino Biological, Inc. for Human ELF2 Gene ORF cDNA clone expression plasmid