negative control vector  (Sino Biological)

Journal: Cell Death & Disease

Article Title: Loss of ELF2 drives topotecan resistance in retinoblastoma revealed by genome-wide CRISPR-Cas9 screening

doi: 10.1038/s41419-025-08335-z

Figure Lengend Snippet: 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.

Article Snippet: The human ELF2 ORF clone expression plasmid (HG12537-UT) and pCMV3-untagged negative control vector (CV011; Sino Biological, Beijing, China) were transfected into WERI-Rb1 and Y79 cells separately using LipofectamineTM 3000 transfection reagent (L3000075; Thermo Fisher Scientific, US).

Techniques: Expressing, Knock-Out, Western Blot, Control, TUNEL Assay, Two Tailed Test, Comparison

Journal: Cell Death & Disease

Article Title: Loss of ELF2 drives topotecan resistance in retinoblastoma revealed by genome-wide CRISPR-Cas9 screening

doi: 10.1038/s41419-025-08335-z

Figure Lengend Snippet: A Heatmap illustrating the expression of 17 genes involved in the metabolic pathways identified in Fig. for each sample across various groups. B The mRNA levels of MT-CYB in WERI-Rb1 cells across different groups. C The mRNA levels of MT-CYB in Y79 cells across different groups. D Heatmap illustrating the expression of 12 mtDNA-encoded genes across groups. E GSEA enrichment plots of OXIDATIVE_PHOSPHORYLATION pathways comparing sgELF2 + topotecan(TPT) vs. NTC + TPT. F , G Representative Western blot and quantitative analysis of MT-CYB expression in control (NTC + pCMV3-untagged), ELF2 knockout (sgELF2 + pCMV3-untagged), and ELF2 rescue (sgELF2 + pCMV3-ELF2-t3) cells. H , I Quantitative analysis of mitochondrial DNA copy number and cellular ATP levels in WERI-Rb1 cells across various groups. J The relative contribution of mitochondrial and glycolytic ATP across different groups. Data are presented as means ± SD. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test ( B , C , G , H , I and J ); **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The human ELF2 ORF clone expression plasmid (HG12537-UT) and pCMV3-untagged negative control vector (CV011; Sino Biological, Beijing, China) were transfected into WERI-Rb1 and Y79 cells separately using LipofectamineTM 3000 transfection reagent (L3000075; Thermo Fisher Scientific, US).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Knock-Out, Comparison

Journal: Cell Death Discovery

Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression

doi: 10.1038/s41420-025-02717-0

Figure Lengend Snippet: A WT or G2E3-KO AsPC-1 cells were transfected with control plasmid or plasmids encoding G2E3-FLAG. The cells were then analyzed by SDS-PAGE and immunoblotting with antibodies to the indicate proteins. B Quantification of ratio of LC3B-II, GABARAP and GABARAPL1 to Vinculin. The indicated p-values were calculated using means of three independent experiments ±SEM. n.s., not significant, * p < 0.05, ** p < 0.01. Significance was calculated with Student’s t test. C HEK293TN WT and AsPC-1 WT were transfected with plasmids encoding G2E3-FLAG or control plasmid FLAG. After 48 h, cell lysates and immunoprecipitates were analyzed using SDS-PAGE and immunoblotting with corresponding antibodies. D Immunofluorescent staining of endogenous GABARAP, GABARAPL1, LC3B, and exogenous G2E3 in G2E3 KO AsPC-1cells. G2E3KO AsPC-1 cells were transfected with plasmids encoding G2E3-FLAG. After 48 h, cells were fixed. G2E3-FLAG was stained with anti-FLAG antibody and the co-localization of the endogenous GABARAP, LC3B and GABARAPL1 were visualized under confocal microscope. Scale bar: 10 μm.

Article Snippet: pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological.

Techniques: Transfection, Control, Plasmid Preparation, SDS Page, Western Blot, Staining, Microscopy

Journal: Cell Death Discovery

Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression

doi: 10.1038/s41420-025-02717-0

Figure Lengend Snippet: A WT or G2E3-KO AsPC-1 cells were fixed and stained with Lamp1 (red), LC3 (green), and DAPI (blue). Scale bar 10 μm. B The Pearson’s coefficients of LC3B overlap with Lamp1 in ( A ) were quantified using Fiji software. Mean ± SEM, n = 10. **** p < 0.0001. Significance was calculated with Student’s t test. C Co-localization analysis of endogenous Lamp1 and exogenously expressed G2E3-FLAG. Line plots exemplify degree of co-localization of G2E3 and Lamp1 signals.

Article Snippet: pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological.

Techniques: Staining, Software