sk co 1  (ATCC)

Journal: STAR Protocols

Article Title: Protocols to evaluate mutant specificity of an oncogene-targeting siRNA using orthogonal in vitro and in vivo approaches

doi: 10.1016/j.xpro.2025.104323

Figure Lengend Snippet: Validation of mutant selectivity of KRAS G12V siRNA in vitro (A) UV melting curves and sequences of 23mer duplexes between the fully modified KRAS guide RNA and the targeted G12V mutant, WT, and G12D mutant RNA. (B) Western blot analysis in A431 cells stably expressing KRAS WT or G12V transiently transfected with siRNAs. Cells were analyzed at 48 hours and 72 hours. Blots were done separately, and densitometry quantification below is based on vinculin control for each individual blot. (C) RT-qPCR analysis in A431 cells transiently transfected with siRNAs at 20 nM. Cells were analyzed at 48 hours. Data shown as mean +/- SEM, experiments performed in duplicate. (D) Luciferase dose-response curve in A431-KRAS-WT or A431-KRAS-G12V cells stably expressing a luciferase reporter. Cells were analyzed at 72 hours. Data shown as mean +/- SEM, experiments performed in triplicate. (E) Volcano plots from RNA-sequencing in SKCO1 cells transiently transfected with siRNAs at 20 nM. Cells were analyzed at 24 hours. This figure includes data published in Stanland et al and has received permission to be shown in this figure.

Article Snippet: SKCO1 , ATCC , Cat #HTB-39; RRID: CVCL_0626.

Techniques: Biomarker Discovery, Mutagenesis, In Vitro, Modification, Western Blot, Stable Transfection, Expressing, Transfection, Control, Quantitative RT-PCR, Luciferase, RNA Sequencing

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: GucyCART has limited efficacy in low antigen models. (A, B) GUCY2C RNA (A) and protein (B) levels in various CRC models relative to the T84 cell line. ** p < 0.01, *** p < 0.001, **** p < 0.0001, One-way ANOVA comparing each cell line to T84 cells. Each data point in (A, B) represents the average from a biological replicate. (C) Tumor burden of LS174T (N = 7) or LoVo (N = 5) cells injected i.p. in NSG mice before i.v. treatment with 5x10 6 control or GucyCART cells. Areas under the curve (AUCs) were quantified, and an unpaired T-test was used for comparisons (ns = p > 0.05). Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Injection, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: CART-conditioned media induce GUCY2C loss in low antigen CRC cells. (A–F ) GucyCART cells were activated by 48 hour co-culture with T84 CRC cells (A–C) or untransduced T cells were activated with anti-CD3/CD2/CD28 beads (D–F) . Conditioned media (CM) were then collected, and fresh LS174T, SKCO1, or LoVo cells were exposed to CM for 48 hours before quantification of GUCY2C protein (B, E) and mRNA (C, F) . Controls include control CART cells (A–C) or beads lacking anti-CD3/CD2/CD28 antibodies (D-F) . Each data point represents the average of biological replicates in separate experiments (N = 3–5 experiments). A paired T-test was used to compare the conditions; ns = p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Co-Culture Assay, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the GAPDH control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: RNA sequencing, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: GucyCART has limited efficacy in low antigen models. (A, B) GUCY2C RNA (A) and protein (B) levels in various CRC models relative to the T84 cell line. ** p < 0.01, *** p < 0.001, **** p < 0.0001, One-way ANOVA comparing each cell line to T84 cells. Each data point in (A, B) represents the average from a biological replicate. (C) Tumor burden of LS174T (N = 7) or LoVo (N = 5) cells injected i.p. in NSG mice before i.v. treatment with 5x10 6 control or GucyCART cells. Areas under the curve (AUCs) were quantified, and an unpaired T-test was used for comparisons (ns = p > 0.05). Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Injection, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: GucyCART induces GUCY2C loss in multiple low antigen models. (A) LS174T, SKCO1, and LoVo CRC cells were exposed to GucyCART for 48 hours. (B) Cytolysis kinetics were quantified over the 48 hour co-culture. AUCs were calculated for each condition, and one-way ANOVA was used to compare GucyCART and control CART at each E:T ratio. Each data point in (B) represents the mean ± SD from n ≥ 3 technical replicates in a single experiment that is representative of 3–5 experiments; **** p < 0.0001. (C, D) Following 48 hours of cytolysis, remaining cells were collected, and GUCY2C mRNA (C) and protein (D) were quantified relative to the epithelia-specific housekeeping control EPCAM. Each data point represents the average of biological replicates in separate experiments (N = 3–5 experiments). One-way ANOVA was used to compare each E:T of GucyCART to control CART; ** p < 0.01, *** p < 0.0001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Co-Culture Assay, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: CART-conditioned media induce GUCY2C loss in low antigen CRC cells. (A–F ) GucyCART cells were activated by 48 hour co-culture with T84 CRC cells (A–C) or untransduced T cells were activated with anti-CD3/CD2/CD28 beads (D–F) . Conditioned media (CM) were then collected, and fresh LS174T, SKCO1, or LoVo cells were exposed to CM for 48 hours before quantification of GUCY2C protein (B, E) and mRNA (C, F) . Controls include control CART cells (A–C) or beads lacking anti-CD3/CD2/CD28 antibodies (D-F) . Each data point represents the average of biological replicates in separate experiments (N = 3–5 experiments). A paired T-test was used to compare the conditions; ns = p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Co-Culture Assay, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: 4PBA rescues IFNγ-induced GUCY2C loss and permits complete cytolysis. (A) LS174T cells were treated with control or 15 ng/mL IFNγ ± 2.5 or 5 mM 4PBA for 48 hours before quantification of CHOP, GUCY2C, and pSTAT1 proteins. Each data point represents the average of biological replicates in separate experiments (N = 3–5 experiments). In each experiment, analytes were examined on the same blot and normalized to the β-tubulin control (shown only below pSTAT1). One-way ANOVA was used to compare conditions to the control, and bars indicate other comparisons; ns = p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001. (B) LS174T or LoVo cells were treated with control or GucyCART at E:T of 1:1 ± 2.5 mM 4PBA. Each data point represents the mean ± SD with n ≥ 3 technical replicates; representative of two experiments. AUCs were calculated for each condition, and one-way ANOVA was used for comparisons; **** p < 0.0001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: GucyCART has limited efficacy in low antigen models. (A, B) GUCY2C RNA (A) and protein (B) levels in various CRC models relative to the T84 cell line. ** p < 0.01, *** p < 0.001, **** p < 0.0001, One-way ANOVA comparing each cell line to T84 cells. Each data point in (A, B) represents the average from a biological replicate. (C) Tumor burden of LS174T (N = 7) or LoVo (N = 5) cells injected i.p. in NSG mice before i.v. treatment with 5x10 6 control or GucyCART cells. Areas under the curve (AUCs) were quantified, and an unpaired T-test was used for comparisons (ns = p > 0.05). Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Injection, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: GucyCART induces GUCY2C loss in multiple low antigen models. (A) LS174T, SKCO1, and LoVo CRC cells were exposed to GucyCART for 48 hours. (B) Cytolysis kinetics were quantified over the 48 hour co-culture. AUCs were calculated for each condition, and one-way ANOVA was used to compare GucyCART and control CART at each E:T ratio. Each data point in (B) represents the mean ± SD from n ≥ 3 technical replicates in a single experiment that is representative of 3–5 experiments; **** p < 0.0001. (C, D) Following 48 hours of cytolysis, remaining cells were collected, and GUCY2C mRNA (C) and protein (D) were quantified relative to the epithelia-specific housekeeping control EPCAM. Each data point represents the average of biological replicates in separate experiments (N = 3–5 experiments). One-way ANOVA was used to compare each E:T of GucyCART to control CART; ** p < 0.01, *** p < 0.0001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Co-Culture Assay, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: CART-conditioned media induce GUCY2C loss in low antigen CRC cells. (A–F ) GucyCART cells were activated by 48 hour co-culture with T84 CRC cells (A–C) or untransduced T cells were activated with anti-CD3/CD2/CD28 beads (D–F) . Conditioned media (CM) were then collected, and fresh LS174T, SKCO1, or LoVo cells were exposed to CM for 48 hours before quantification of GUCY2C protein (B, E) and mRNA (C, F) . Controls include control CART cells (A–C) or beads lacking anti-CD3/CD2/CD28 antibodies (D-F) . Each data point represents the average of biological replicates in separate experiments (N = 3–5 experiments). A paired T-test was used to compare the conditions; ns = p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Co-Culture Assay, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: CART-induced GUCY2C loss is mediated by the IFNγ-JAK-STAT1 signaling axis. (A) LS174T cells and orthogonal approaches of cytokine screening (B) and confirmation (C) , IFNγ neutralizing antibody (D) , pharmacologic JAK1/2 blockade with ruxolitinib (E) , and JAK1/2 genetic knockout (F) were used to define the role of the IFNγ-JAK-STAT signaling axis in GUCY2C loss. (B) LS174T cells were treated with GM-CSF (20 ng/mL), TNFα (1 ng/mL), IL-8 (2 ng/mL), MIP-1α (2 ng/mL), MIP-1β (2 ng/mL), or IFNγ (15 ng/mL) for 48 hours, and GUCY2C protein levels were quantified. (C) LS174T cells were treated with 150 ng/mL IFNγ for 48 hours, and pSTAT1 and GUCY2C protein levels were quantified. (D–F) LS174T cells were treated with conditioned media (CM) for 48 hours from control or anti-CD3/CD2/CD28 bead-activated T cells, and pSTAT1 and GUCY2C protein levels were quantified; (D) 13 μg/mL anti-IFNγ neutralizing antibody was included in some conditions; (E) 2.5 μM ruxolitinib was included in some conditions; (F) LS174T cell pools previously treated with control CRISPR/Cas9 or JAK1 + 2 CRISPR/Cas9 were used. Each data point in (B–F) represents average of biological replicates from separate experiments (N = 3–4 experiments). In (C–F) , pSTAT, GUCY2C, and housekeeping control were examined on the same blot. The housekeeping protein is shown only below GUCY2C. A paired t-test was used to compare the two conditions in (C) ; in (B) and (D–F) , one-way ANOVA was used to compare each condition to the control treatment, and additional comparisons are indicated by bars; ns = p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Knock-Out, Control, CRISPR, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the GAPDH control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: RNA sequencing, Control, Generated

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: 4PBA rescues IFNγ-induced GUCY2C loss and permits complete cytolysis. (A) LS174T cells were treated with control or 15 ng/mL IFNγ ± 2.5 or 5 mM 4PBA for 48 hours before quantification of CHOP, GUCY2C, and pSTAT1 proteins. Each data point represents the average of biological replicates in separate experiments (N = 3–5 experiments). In each experiment, analytes were examined on the same blot and normalized to the β-tubulin control (shown only below pSTAT1). One-way ANOVA was used to compare conditions to the control, and bars indicate other comparisons; ns = p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001. (B) LS174T or LoVo cells were treated with control or GucyCART at E:T of 1:1 ± 2.5 mM 4PBA. Each data point represents the mean ± SD with n ≥ 3 technical replicates; representative of two experiments. AUCs were calculated for each condition, and one-way ANOVA was used for comparisons; **** p < 0.0001. Figure schematics were generated using BioRender.com .

Article Snippet: LS174T (CL-188, ATCC), SKCO1 (HTB-39), LoVo (CCL229, ATCC), and T84 (CCL-248, ATCC) were obtained ATCC.

Techniques: Control, Generated