human psg5l rb1  (Addgene inc)

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 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.

Article Snippet: The human WERI-Rb1 and Y79 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Genome Wide, CRISPR, Knock-Out, Library Screening, Expressing, 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 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 WERI-Rb1 and Y79 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

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 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.

Article Snippet: The human WERI-Rb1 and Y79 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vivo, Transplantation Assay, Control, Knock-Out, Injection, Saline, Western Blot, TUNEL Assay, 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 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 ).

Article Snippet: The human WERI-Rb1 and Y79 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: RNA Sequencing, Control, Knock-Out

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 WERI-Rb1 and Y79 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

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

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Cytotoxic effect of L12 treatment and its dependency on the RB1 pathway functionality. (A) Depiction of the structure of L12. (B) Western blotting (WB) analysis of RB1 and phosphorylated Ser 780 RB1 proteins using an anti‐RB1 (RB1) and anti‐phospho‐Ser 780 RB1 (pRB1) antibodies. Total lysates from various cell lines were probed, including the RB1‐deficient human retinoblastoma Y79, the osteosarcoma U2OS with a constitutively phosphorylated RB1 at Ser 780 (pRB1), the mammary gland epithelial cell line MCF10A, and the adenocarcinoma alveolar basal epithelial A549, both with functional RB1 pathways, as well as the RB1‐deficient small cell lung carcinoma U1690. α‐tubulin (αTubulin) served as a loading control. Cells treated with different L12 concentrations for 24 h were assessed for cell accumulation in the sub‐G1 fraction (indicating dead cells). The histograms depict mean ± SD of the percentages of cells in the sub‐G1 fractions ( N = 3; * p < .05). (C) Examination of L12's impact on the downstream gene target of TUBG, RB1 , was conducted by WB analysis using an anti‐RB1 antibody on MCF10A and A549 cell lysates. α‐tubulin and actin were utilized as loading controls. Numbers on the western blot indicate variations in RB1 expression compared to the vehicle control. To account for protein loading discrepancies, RB1's protein concentration was normalized to its ratio with the respective loading control for each treatment. The graph displays relative RB1 protein expression across MCF10A ( N = 4) and A549 ( N = 3) cell lines when treated with varying L12 concentrations, with data presented as mean ± SD. Please note that the intensity of the RB1 bands may vary between figures as a result of adjusted exposure times for western blot analysis. These adjustments were made to prevent overexposure and to accurately capture differences in RB1 expression levels across experimental conditions.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Western Blot, Functional Assay, Control, Expressing, Protein Concentration

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Influence of TUBG1, E2F1, and RB1 protein levels on the cytotoxic effect of L12 treatment. (A and B) The MCF10A cell lines used include: Control (MCF10A, non‐modified parental cells), MCF10A sh TUBG (stably expressing TUBG shRNA) and MCF10A sh TUBG TUBG1 (stably co‐expressing TUBG shRNA and a sh‐resistant TUBG1 gene). (A) MCF10A cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. DNA content was measured by nuclear counter to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction (indicative of dead cells). Histograms display the results, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (B) Western blotting (WB) was performed to analyze TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs illustrate relative protein expression (Student's t test, N = 3, * p < .05, ** p < .01). (C and D) The U2OS cell lines used include: Control (U2OS, non‐modified parental cells), U2OS sh E2F1 (transiently expressing E2F1 shRNA) and U2OS E2F1 sh E2F1 (transiently co‐expressing E2F1 sgRNA and a E2F1 gene). U2OS cells were treated with DMSO (vehicle) or 50 nM L12 for 24 h. (C) DNA content was measured to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction. Histograms show representative data, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 4; Student's t test, * p < .01, ** p < .01). (D) WB was used to analyze E2F1 and procaspase 3 protein levels in total lysates using anti‐E2F1 and anti‐procaspase 3 antibodies. GAPDH served as the loading control. Graphs display relative protein expression (Student's t test, N = 4, * p < .05, ** p < .01). The numbers above the blots (WB) represent the normalized intensity of the protein bands. (E and F) The A549 cell lines used include: Control (A549, non‐modified parental cells), A549 sg RB1 (stably expressing RB1 sgRNA) and A549 RB1 sgRNA RB1 (stably co‐expressing RB1 sgRNA and a sg‐resistant RB1 gene). (E) A549 cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. The cell cycle profile and percentage of sub‐G1 cells were determined. Histograms represent the results, and graphs show mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (F) WB analyzed TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs depict relative protein expression (Student's t test, N = 3, * p < .05, **** p < .0001). To ensure accurate comparisons of RB1 protein levels under different conditions, Western blot exposure times were optimized for each experiment to balance signal detection and prevent overexposure, enabling the detection of subtle differences in RB1 expression.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Control, Modification, Stable Transfection, Expressing, shRNA, Western Blot

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Variable cytotoxic effects of L12 in cells expressing TUBG1 vs. TUBG2. (A) U2OS cells, U2OS cells stably expressing Flag‐tagged TUBG2 (TUBG2‐Flag), or TUBG1 single guide (sg) RNA (sg TUBG1 , resulting in TUBG1 knockout) co‐expressing either sg‐resistant TUBG1 or a sg‐resistant TUBG2 were treated with DMSO (vehicle) or varying concentrations of L12 for 24 h. Total lysates were analyzed by western blotting (WB; N = 3). Antibodies targeting the C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) of TUBG were used to detect endogenous and recombinant TUBG proteins, with actin serving as a loading control. DNA content was measured using a nuclear counter, and histograms display cell cycle profile changes, specifically in the sub‐G1 fraction. Graphs present normalized TUBG1 and TUBG2 levels (from WB) relative to actin and mean ± SD percentages of cells in the sub‐G1 fraction ( N = 3). A schematic highlights the GTPase domain (residues 5–255) and DNA‐binding domain (DBD; residues 334–451) of the human TUBG1 (h‐ TUBG1 ) gene. Amino acid differences between TUBG1 and TUBG2 are shown, with gray and blue indicating TUBG1‐specific residues and magenta denoting TUBG2‐specific residues. (B) WB analysis of cytosolic (Cytosol) and chromatin fractions from the indicated U2OS cells. Antibodies targeting TUBG's C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) were used to detect endogenous and recombinant TUBG proteins. T3320 preferentially detects TUBG1, while T6557 preferentially detects TUBG2, especially in fractionated samples, where T6557's specificity for TUBG1 improves. Densitometric analysis of TUBG1 and TUBG2 levels are shown, normalized to α‐tubulin (cytosolic marker) or histone (chromatin marker). An anti‐flag antibody was used to detect TUBG2–Flag. (C) WB analysis of total lysates shows RB1, TUBG1, and TUBG2 protein levels using anti‐RB, T3320, and T6557 antibodies. An α‐tubulin (αTubulin) served as a loading control. The graphs depict relative RB1, TUBG1, and TUBG2 expression across the indicated cell lines (RB1: TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 8; U2OS‐TUBG2 Flag, N = 3; TUBG1: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG1, N = 3; TUBG2: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 3). (D) Confocal microscopy images of U2OS‐TUBG2‐Flag cells stained with an anti‐TUBG and anti‐Flag antibodies. Images highlight the location of TUBG2‐Flag at γ‐tubules and centrosomes. Colocalization pixel maps (CPM) of magenta/red and green channels are included. White regions signify colocalization, with arrowheads and arrows indicating γ‐tubules and centrosomes, respectively. Scale bars: 10 μm. Graphs show the fluorescence intensity of T3320 (TUBG1) or anti‐Flag (TUBG2) found at γ‐tubules and centrosomes (Student's t test, **** p < .0001; γ‐tubules: N = 127; centrosomes: N = 108). Note that the issue of antibody specificity encountered in WB analysis does not affect immunofluorescence assays, as the proteins maintain a different conformation that is not influenced by SDS treatment. (E) Confocal microscopy images of TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2 cells stained with anti‐TUBG antibodies. Graphs display the mean percentages of cells with γ‐tubules ( N = 5; Student's t test, ** p < .01) Hoechst was used for nuclear staining. Scale bars: 10 μm.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Expressing, Stable Transfection, Knock-Out, Western Blot, Recombinant, Control, Binding Assay, Marker, Confocal Microscopy, Staining, Fluorescence, Immunofluorescence

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Cytotoxic effect of L12 treatment and its dependency on the RB1 pathway functionality. (A) Depiction of the structure of L12. (B) Western blotting (WB) analysis of RB1 and phosphorylated Ser 780 RB1 proteins using an anti‐RB1 (RB1) and anti‐phospho‐Ser 780 RB1 (pRB1) antibodies. Total lysates from various cell lines were probed, including the RB1‐deficient human retinoblastoma Y79, the osteosarcoma U2OS with a constitutively phosphorylated RB1 at Ser 780 (pRB1), the mammary gland epithelial cell line MCF10A, and the adenocarcinoma alveolar basal epithelial A549, both with functional RB1 pathways, as well as the RB1‐deficient small cell lung carcinoma U1690. α‐tubulin (αTubulin) served as a loading control. Cells treated with different L12 concentrations for 24 h were assessed for cell accumulation in the sub‐G1 fraction (indicating dead cells). The histograms depict mean ± SD of the percentages of cells in the sub‐G1 fractions ( N = 3; * p < .05). (C) Examination of L12's impact on the downstream gene target of TUBG, RB1 , was conducted by WB analysis using an anti‐RB1 antibody on MCF10A and A549 cell lysates. α‐tubulin and actin were utilized as loading controls. Numbers on the western blot indicate variations in RB1 expression compared to the vehicle control. To account for protein loading discrepancies, RB1's protein concentration was normalized to its ratio with the respective loading control for each treatment. The graph displays relative RB1 protein expression across MCF10A ( N = 4) and A549 ( N = 3) cell lines when treated with varying L12 concentrations, with data presented as mean ± SD. Please note that the intensity of the RB1 bands may vary between figures as a result of adjusted exposure times for western blot analysis. These adjustments were made to prevent overexposure and to accurately capture differences in RB1 expression levels across experimental conditions.

Article Snippet: Human RB1 (RRID:Addgene_10720) was prepared using a Quikchange Mutagenesis Kit (Stratagene) and the following oligos (modified bases underlined): 5′C AAAACCCCCCGAAA G AC C GC G GCCACCGCCGCC3′ and 5′GGCGGCGGT GGC C GC G GT C TTTCGGGGGGTTTTG3′ (RRID:Addgene_229856).

Techniques: Western Blot, Functional Assay, Control, Expressing, Protein Concentration

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Influence of TUBG1, E2F1, and RB1 protein levels on the cytotoxic effect of L12 treatment. (A and B) The MCF10A cell lines used include: Control (MCF10A, non‐modified parental cells), MCF10A sh TUBG (stably expressing TUBG shRNA) and MCF10A sh TUBG TUBG1 (stably co‐expressing TUBG shRNA and a sh‐resistant TUBG1 gene). (A) MCF10A cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. DNA content was measured by nuclear counter to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction (indicative of dead cells). Histograms display the results, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (B) Western blotting (WB) was performed to analyze TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs illustrate relative protein expression (Student's t test, N = 3, * p < .05, ** p < .01). (C and D) The U2OS cell lines used include: Control (U2OS, non‐modified parental cells), U2OS sh E2F1 (transiently expressing E2F1 shRNA) and U2OS E2F1 sh E2F1 (transiently co‐expressing E2F1 sgRNA and a E2F1 gene). U2OS cells were treated with DMSO (vehicle) or 50 nM L12 for 24 h. (C) DNA content was measured to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction. Histograms show representative data, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 4; Student's t test, * p < .01, ** p < .01). (D) WB was used to analyze E2F1 and procaspase 3 protein levels in total lysates using anti‐E2F1 and anti‐procaspase 3 antibodies. GAPDH served as the loading control. Graphs display relative protein expression (Student's t test, N = 4, * p < .05, ** p < .01). The numbers above the blots (WB) represent the normalized intensity of the protein bands. (E and F) The A549 cell lines used include: Control (A549, non‐modified parental cells), A549 sg RB1 (stably expressing RB1 sgRNA) and A549 RB1 sgRNA RB1 (stably co‐expressing RB1 sgRNA and a sg‐resistant RB1 gene). (E) A549 cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. The cell cycle profile and percentage of sub‐G1 cells were determined. Histograms represent the results, and graphs show mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (F) WB analyzed TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs depict relative protein expression (Student's t test, N = 3, * p < .05, **** p < .0001). To ensure accurate comparisons of RB1 protein levels under different conditions, Western blot exposure times were optimized for each experiment to balance signal detection and prevent overexposure, enabling the detection of subtle differences in RB1 expression.

Article Snippet: Human RB1 (RRID:Addgene_10720) was prepared using a Quikchange Mutagenesis Kit (Stratagene) and the following oligos (modified bases underlined): 5′C AAAACCCCCCGAAA G AC C GC G GCCACCGCCGCC3′ and 5′GGCGGCGGT GGC C GC G GT C TTTCGGGGGGTTTTG3′ (RRID:Addgene_229856).

Techniques: Control, Modification, Stable Transfection, Expressing, shRNA, Western Blot

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Variable cytotoxic effects of L12 in cells expressing TUBG1 vs. TUBG2. (A) U2OS cells, U2OS cells stably expressing Flag‐tagged TUBG2 (TUBG2‐Flag), or TUBG1 single guide (sg) RNA (sg TUBG1 , resulting in TUBG1 knockout) co‐expressing either sg‐resistant TUBG1 or a sg‐resistant TUBG2 were treated with DMSO (vehicle) or varying concentrations of L12 for 24 h. Total lysates were analyzed by western blotting (WB; N = 3). Antibodies targeting the C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) of TUBG were used to detect endogenous and recombinant TUBG proteins, with actin serving as a loading control. DNA content was measured using a nuclear counter, and histograms display cell cycle profile changes, specifically in the sub‐G1 fraction. Graphs present normalized TUBG1 and TUBG2 levels (from WB) relative to actin and mean ± SD percentages of cells in the sub‐G1 fraction ( N = 3). A schematic highlights the GTPase domain (residues 5–255) and DNA‐binding domain (DBD; residues 334–451) of the human TUBG1 (h‐ TUBG1 ) gene. Amino acid differences between TUBG1 and TUBG2 are shown, with gray and blue indicating TUBG1‐specific residues and magenta denoting TUBG2‐specific residues. (B) WB analysis of cytosolic (Cytosol) and chromatin fractions from the indicated U2OS cells. Antibodies targeting TUBG's C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) were used to detect endogenous and recombinant TUBG proteins. T3320 preferentially detects TUBG1, while T6557 preferentially detects TUBG2, especially in fractionated samples, where T6557's specificity for TUBG1 improves. Densitometric analysis of TUBG1 and TUBG2 levels are shown, normalized to α‐tubulin (cytosolic marker) or histone (chromatin marker). An anti‐flag antibody was used to detect TUBG2–Flag. (C) WB analysis of total lysates shows RB1, TUBG1, and TUBG2 protein levels using anti‐RB, T3320, and T6557 antibodies. An α‐tubulin (αTubulin) served as a loading control. The graphs depict relative RB1, TUBG1, and TUBG2 expression across the indicated cell lines (RB1: TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 8; U2OS‐TUBG2 Flag, N = 3; TUBG1: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG1, N = 3; TUBG2: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 3). (D) Confocal microscopy images of U2OS‐TUBG2‐Flag cells stained with an anti‐TUBG and anti‐Flag antibodies. Images highlight the location of TUBG2‐Flag at γ‐tubules and centrosomes. Colocalization pixel maps (CPM) of magenta/red and green channels are included. White regions signify colocalization, with arrowheads and arrows indicating γ‐tubules and centrosomes, respectively. Scale bars: 10 μm. Graphs show the fluorescence intensity of T3320 (TUBG1) or anti‐Flag (TUBG2) found at γ‐tubules and centrosomes (Student's t test, **** p < .0001; γ‐tubules: N = 127; centrosomes: N = 108). Note that the issue of antibody specificity encountered in WB analysis does not affect immunofluorescence assays, as the proteins maintain a different conformation that is not influenced by SDS treatment. (E) Confocal microscopy images of TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2 cells stained with anti‐TUBG antibodies. Graphs display the mean percentages of cells with γ‐tubules ( N = 5; Student's t test, ** p < .01) Hoechst was used for nuclear staining. Scale bars: 10 μm.

Article Snippet: Human RB1 (RRID:Addgene_10720) was prepared using a Quikchange Mutagenesis Kit (Stratagene) and the following oligos (modified bases underlined): 5′C AAAACCCCCCGAAA G AC C GC G GCCACCGCCGCC3′ and 5′GGCGGCGGT GGC C GC G GT C TTTCGGGGGGTTTTG3′ (RRID:Addgene_229856).

Techniques: Expressing, Stable Transfection, Knock-Out, Western Blot, Recombinant, Control, Binding Assay, Marker, Confocal Microscopy, Staining, Fluorescence, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: KRAS G12D -driven pentose phosphate pathway remodeling imparts a targetable vulnerability synergizing with MRTX1133 for durable remissions in PDAC

doi: 10.1016/j.xcrm.2025.101966

Figure Lengend Snippet:

Article Snippet: In addition, the cDNA sequences of E2F1 and RING1 gene were cloned into the pRK5-HA vector. pCMV3-His-Rb (#HG10137-NH) was purchased from Sino Biological Inc. (Beijing, China). pRK5-HA-Ubi, pRK5- Ring1 , pRK5- UBE2T , deletions-mutant p53 plasmids were constructed in our previous article.

Techniques: Mutagenesis, Recombinant, Virus, CCK-8 Assay, Membrane, Concentration Assay, Modification, Transfection, Agarose Gel Electrophoresis, Plasmid Preparation, Extraction, Bicinchoninic Acid Protein Assay, Staining, RNA Extraction, H&E Stain, Viability Assay, Reporter Assay, Labeling, Western Blot, Sequencing, Control, Software

Journal: Cell Reports Medicine

Article Title: KRAS G12D -driven pentose phosphate pathway remodeling imparts a targetable vulnerability synergizing with MRTX1133 for durable remissions in PDAC

doi: 10.1016/j.xcrm.2025.101966

Figure Lengend Snippet:

Article Snippet: In addition, the cDNA sequences of E2F1 and RING1 gene were cloned into the pRK5-HA vector. pCMV3-His-Rb (#HG10137-NH) was purchased from Sino Biological Inc. (Beijing, China). pRK5-HA-Ubi, pRK5- Ring1 , pRK5- UBE2T , deletions-mutant p53 plasmids were constructed in our previous article.

Techniques: Mutagenesis, Recombinant, Virus, CCK-8 Assay, Membrane, Concentration Assay, Modification, Transfection, Agarose Gel Electrophoresis, Plasmid Preparation, Extraction, Bicinchoninic Acid Protein Assay, Staining, RNA Extraction, H&E Stain, Viability Assay, Reporter Assay, Labeling, Western Blot, Sequencing, Control, Software