cell line  (ATCC)

Journal: bioRxiv

Article Title: Defective Microhomology-Mediated End-joining in SMARCB1-Deficient Tumors

doi: 10.1101/2025.11.20.689563

Figure Lengend Snippet: (A) Volcano plot showing DepMap CRISPR screen results highlighting significant hits in the Fanconi Anemia pathway. Genes in the FA/BRCA pathway are highlighted in red circles, while homologous recombination (HR) genes are highlighted in blue diamonds. (B) Clonogenic survival assays in RPE, G401, and A204 cells following expression of sgRNAs targeting non-targeting control (NT), FANCF , FANCA , FANCG , or BRCA2 . (C) Immunoblot of FANCA, FANCF, FANCG, and BRCA2 in RPE TP53 -/- cells expressing sgNT, sgFANCA, sgFANCF, sgFANCG, and sgBRCA2. Vinculin serves as a loading control. (D) Immunoblot of FANCD2, ubiquitinated FANCD2 (Ub-FANCD2), and SMARCB1 in RPE TP53 -/- WT and SMARCB1 -KO cells treated with or without MMC (30 ng/mL, 24 hours). α-tubulin serves as a loading control. Quantification of Ub-FANCD2/FANCD2 ratio is shown on the right. (E) Quantification (left) and representative immunofluorescence images (right) of FANCD2 nuclear foci formation in RPE TP53 -/- WT and SMARCB1 -KO cells with or without MMC treatment (30 ng/mL, 24 hours). Nuclei are stained with DAPI (scale bars = 2 μm). Data are shown as individual data points representing the number of FANCD2 foci per nucleus; the mean is indicated by a horizontal bar. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (*, P < 0.05; ****, P < 0.0001).

Article Snippet: This study utilized a panel of human cell lines, including HEK293T (ATCC CRL-3216), immortalized retinal pigment epithelial cells (RPE WT and RPE TP53 −/− ), K562 (ATCC CCL-243), U2OS (ATCC HTB-96), and rhabdoid tumor cell lines G401 (ATCC CRL-1441), A204 (ATCC HTB-82), and VA-ES-BJ (ATCC CRL-2138).

Techniques: CRISPR, Homologous Recombination, Expressing, Control, Western Blot, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Defective Microhomology-Mediated End-joining in SMARCB1-Deficient Tumors

doi: 10.1101/2025.11.20.689563

Figure Lengend Snippet: (A) MMEJ reporter assay in K562 cells expressing the indicated sgRNAs. Data are presented as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (****, P < 0.0001). (B) Immunoblot of endogenous POLQ, SMARCB1, and GAPDH in WT RPE cells with empty vector, SMARCB1 -KO RPE cells with empty vector, and SMARCB1 -KO RPE cells reconstituted with WT SMARCB1. (C) MMEJ reporter assay in HEK293T WT and SMARCB1 -KO cells with or without 3xFLAG POLQ transfection. (D) Immunoblot of Flag-tagged POLQ expression in HEK293T WT and SMARCB1 -KO cells with or without 3xFLAG POLQ transfection. (E) Immunoblot of endogenous POLQ, SMARCB1, and GAPDH in WT RPE cells, SMARCB1 -KO RPE cells, and rhabdoid tumor cell lines (G401, A204). (F) Representative immunofluorescence images showing nuclear foci of HA–tagged endogenous POLQ in U2OS cells expressing sg NT or sg SMARCB1 . Quantification of HA– POLQ foci per cell is shown below. Data are shown as individual data points representing the number of HA-POLQ foci per nucleus; the mean is indicated by a horizontal bar. Statistical significance was calculated using Mann-Whitney U test (****, P < 0.0001). (G) Percentage somatic SNVs attributed to COSMIC single base substitution signatures (v3.3) for Rhabdoid Tumors (n = 56), Wilms Tumor (n = 81), and Neuroblastoma (n = 135). Data are shown as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (****, P < 0.0001).

Article Snippet: This study utilized a panel of human cell lines, including HEK293T (ATCC CRL-3216), immortalized retinal pigment epithelial cells (RPE WT and RPE TP53 −/− ), K562 (ATCC CCL-243), U2OS (ATCC HTB-96), and rhabdoid tumor cell lines G401 (ATCC CRL-1441), A204 (ATCC HTB-82), and VA-ES-BJ (ATCC CRL-2138).

Techniques: Reporter Assay, Expressing, Western Blot, Plasmid Preparation, Transfection, Immunofluorescence, MANN-WHITNEY, Wilms Tumor Assay

Journal: bioRxiv

Article Title: Defective Microhomology-Mediated End-joining in SMARCB1-Deficient Tumors

doi: 10.1101/2025.11.20.689563

Figure Lengend Snippet: (A) Volcano plot showing proteins identified in WT-SMARCB1 versus MT5-SMARCB1 coimmunoprecipitates (n = 2). The x-axis represents the log₂ fold change (WT/MT5), and the y-axis represents the –log₁₀ p-value. Proteins enriched in WT-SMARCB1 (Up, red) or MT5-SMARCB1 (Down, blue) are highlighted, whereas non-significant proteins are shown in grey. Nucleoporins are marked as black-filled diamonds with white outlines and labeled with gene symbols. (B) Gene Ontology enrichment analysis of proteins identified by mass spectrometry as enriched in WT-SMARCB1 versus MT5-SMARCB1 co-immunoprecipitates, showing the top biological processes. (C) Co-immunoprecipitation analysis. HEK293T HA-FKBP12 F36V -SMARCB1 knock-in cells were treated with dTAG-1 to induce endogenous SMARCB1 degradation and transfected with EV, V5-tagged WT-SMARCB1, or V5-tagged MT5-SMARCB1 mutant. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting for NUP214, NUP188, NUP 153, NUP133, NUP93, SMARCB1, and V5. (D) Co-immunoprecipitation analysis. Cell lysates of WT HEK293T and HA-FKBP12 F36V -SMARCB1 knock-in HEK293T cells were immunoprecipitated with anti-HA antibody, followed by immunoblotting for NUP214, NUP188, NUP 153, NUP93, SMARCB1, and HA. (E) Quantification of cytoplasmic-to-nuclear mRNA ratios of POLQ in RPE TP53 −/− SMARCB1 -KO cells reconstituted with EV, WT-SMARCB1, or MT5-SMARCB1, measured by qRT-PCR (n = 3). Data are shown as the mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (****, P < 0.0001). (F) Quantification of cytoplasmic-to-nuclear mRNA ratios of POLQ and in TP53 −/− RPE, G401 and A204 cells, measured by qRT-PCR (n = 3). Data are shown as the mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (****, P < 0.0001). (G) Quantification of cytoplasmic-to-nuclear mRNA ratios of POLQ and in TP53 −/− RPE expressing sgNT, sgNUP153-1, sgNUP214-1, sgNUP93-2, measured by qRT-PCR (n = 3). Data are shown as the mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (****, P < 0.0001). (H) Immunoblot of POLQ, NUP153, NUP214, NUP93 and GAPDH in RPE TP53 -/- 3xFLAG-POLQ knock-in cells expressing sgNT, sgPOLQ, sgNUP153, sgNUP214, and sgNUP93.

Article Snippet: This study utilized a panel of human cell lines, including HEK293T (ATCC CRL-3216), immortalized retinal pigment epithelial cells (RPE WT and RPE TP53 −/− ), K562 (ATCC CCL-243), U2OS (ATCC HTB-96), and rhabdoid tumor cell lines G401 (ATCC CRL-1441), A204 (ATCC HTB-82), and VA-ES-BJ (ATCC CRL-2138).

Techniques: Labeling, Mass Spectrometry, Immunoprecipitation, Knock-In, Transfection, Mutagenesis, Western Blot, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Defective Microhomology-Mediated End-joining in SMARCB1-Deficient Tumors

doi: 10.1101/2025.11.20.689563

Figure Lengend Snippet: (A) Survival curves of RPE TP53 -/- WT, rhabdoid tumor cell lines (A204, G401), and epithelioid sarcoma cell line (VA-ES-BJ) treated with indicated concentrations of E7820. (B) Survival curves of U2OS WT and U2OS SMARCB1 -KO clone 4 and 7 cells treated with indicated concentrations of E7820. (C) Survival curves of U2OS SMARCB1 -KO cells overexpressing empty vector, WT-SMARCB1, or MT5-SMARCB1 and treated with indicated concentrations of E7820. (D) Survival curves of RPE TP53 -/- WT, rhabdoid tumor cell lines (A204, G401), and epithelioid sarcoma cell line (VA-ES-BJ) treated with indicated concentrations of Indisulam. (E) Survival curves of U2OS WT and U2OS SMARCB1 -KO clone 4 and 7 cells treated with indicated concentrations of Indisulam. (F) Survival curves of U2OS SMARCB1 -KO cells overexpressing empty vector, WT-SMARCB1, or MT5-SMARCB1 and treated with indicated concentrations of Indisulam. (G) Immunoblot of RBM39, FANCA, FANCD2, and γ-H2AX in A204 and G401 cells treated with DMSO, 250 nM E7820 or Indisulam. α-Tubulin serves as a loading control. (H-I) Survival curves of (H) RPE TP53 -/- WT, rhabdoid tumor cell lines (A204, G401), and epithelioid sarcoma cell line (VA-ES-BJ) and (I) RPE TP53 -/- WT and SMARCB1 -KO cells reconstituted with EV, WT-SMARCB1, or MT5-SMARCB1 treated with indicated concentrations THZ531. (J-K) Survival curves of (J) RPE TP53 -/- WT, rhabdoid tumor cell lines (A204, G401), and epithelioid sarcoma cell line (VA-ES-BJ) and (K) RPE TP53 -/- WT and SMARCB1 -KO cells reconstituted with EV, WT-SMARCB1, or MT5-SMARCB1 treated with indicated concentrations BSJ-5-63. (L) Immunoblot of BRCA2 and BRCA1 in A204 and G401 cells treated with DMSO, 100nM THZ531, or 50nM BSJ-5-63. GAPDH serves as a loading control. (M) Survival curves of RPE TP53 -/- WT and SMARCB1 -KO cells and rhabdoid tumor cell lines (A204, G401) treated with indicated concentrations of 5-Nitro-2-furaldehyde.

Article Snippet: This study utilized a panel of human cell lines, including HEK293T (ATCC CRL-3216), immortalized retinal pigment epithelial cells (RPE WT and RPE TP53 −/− ), K562 (ATCC CCL-243), U2OS (ATCC HTB-96), and rhabdoid tumor cell lines G401 (ATCC CRL-1441), A204 (ATCC HTB-82), and VA-ES-BJ (ATCC CRL-2138).

Techniques: Plasmid Preparation, Western Blot, Control

Journal: Cancer immunology research

Article Title: Glypican-3-specific CAR T cells co-expressing IL15 and IL21 have superior expansion and antitumor activity against hepatocellular carcinoma

doi: 10.1158/2326-6066.CIR-19-0293

Figure Lengend Snippet: A) GPC3-CAR T cells were co-cultured with 51Cr-labeled GPC3-positive (Huh-7, Hep3B, G401, A549-GPC3) and negative (A549) target cells at the indicated effector-to-target (E:T) ratios. 51Cr-release was detected after four hours as a measure of GPC3-CAR T cytotoxicity (mean ± SEM, pooled results from two independent experiments evaluating four donors). B) Indicated T cell groups were cultured with Huh-7 tumor cells at a 1:1 ratio and concentration of indicated effector cytokines released into the supernatant (+24 hrs) was measured by Luminex assay (combined data from three independent experiments evaluating six donors). Comparison with two-way ANOVA. C) Surface expression of CD4 (top) and CD8 (bottom) populations within CAR-positive cells at baseline (left) and after two consecutive stimulations with Huh-7 cells (right, E:T = 1:1) as measured by FACS (mean ± SD, combined data from three independent experiments evaluating six donors), one-way ANOVA. *p<0.05, ** p<0.01, ***p<0.001.

Article Snippet: Cell lines The HCC cell line Hep3B, rhabdoid tumor cell line G401, lung carcinoma cell line A549, and human embryonic kidney cell line 293T were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in Dulbecco’s Modified Eagle Media with 10% fetal bovine serum (FBS) except for 293T cells which were cultured in Iscove Modified Dulbecco Media with 10% FBS.

Techniques: Cell Culture, Labeling, Concentration Assay, Luminex, Comparison, Expressing