positive control snu449 cell line  (ATCC)

Journal: Toxicological Sciences

Article Title: Nonclinical safety assessment of SBT777101, a CAR-engineered regulatory T cell therapy for autoimmune diseases

doi: 10.1093/toxsci/kfag125

Figure Lengend Snippet: SBT777101 demonstrates minimal antigen-specific activation without impact on normal human cell survival. (A) Representative flow cytometry plots showing surface (top) and intracellular (bottom) binding of SBT777101 scFv-Fc (right) relative to isotype control (left) in SNU449 cells. (B) In vitro functional characterization using SNU449 cells ± CV-beads. Left: Mean ± SD CD69 expression (only SBT777101 data shown; no activation observed in UT controls). Data analyzed by two-way ANOVA with Sidak’s multiple comparisons test. Right: Cytotoxicity measured as the number of viable target cells remaining, normalized to no-Treg controls. Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test. (C) Percentage of UT (top) or SBT777101 (bottom) Tregs expressing CD69 after 24-h co-culture with normal human primary cells or CV-beads (positive control). Data represent mean ± SD from three replicates. Independent experiments are separated by dashed vertical lines. Comparisons were made for media alone compared with in the presence of primary cells for each type and analyzed by two-tailed unpaired t -test. (D) Cytotoxicity (mean ± SD) of SBT777101 and UT Tregs against the same primary cell panel, normalized to no-Treg controls. Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test. (E) Fold-change in CD71 expression (mean ± SD; relative to media control) across three donors after 72-h culture with media, CitP − SKNBE2 cells, CitP + SNU449 cells, human brain astrocytes (HBA), or CV-beads. CV beads were prepared in RPMI or AGM media as appropriate. Full lysis controls (B, D) represent target cells treated with Triton X-100. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. All experiments included 3 technical replicates. AGM, astrocyte growth medium; CitP, citrullinated protein; CV, citrullinated vimentin; HMEC, human mammary epithelial cells; NHBE, normal human bronchial epithelial cells; NHDF, normal human dermal fibroblasts; PrEC, prostate epithelial cells; RPTEC, renal proximal tubule epithelial cells; scFv, single-chain variable fragment; UT, untransduced.

Article Snippet: Primary human cells (mammary epithelial cells [HMEC; Lonza Cat#CC-2551], normal bronchial epithelial cells [NHBE; Lonza Cat#CC2451], renal proximal tubule epithelial cells [RPTEC; Lonza Cat#CC2553], prostate epithelial cells [PrEC; Lonza Cat#CC2555], hepatocytes [Lonza Cat#HUCPG], brain astrocytes [HBA; Neuromics Cat#HMP202], iPSC-derived myocytes [Tempo Bioscience Cat#1014], iPSC-derived cortical neurons [Tempo Bioscience Cat#201], and monocyte-derived macrophages [internally derived]) and the positive-control SNU449 cell line (ATCC Cat#CRL-2234) were assessed for binding of the SBT777101 scFv by flow cytometry.

Techniques: Activation Assay, Flow Cytometry, Binding Assay, Control, In Vitro, Functional Assay, Expressing, Co-culture Assay, Positive Control, Two Tailed Test, Lysis

Journal: Hepatology (Baltimore, Md.)

Article Title: The MASTL/YBX1/PAK4 axis regulated by stress-activated STK24 triggers lenvatinib resistance and tumor progression in HCC

doi: 10.1097/HEP.0000000000001392

Figure Lengend Snippet: MASTL promotes lenvatinib resistance and the proliferation of HCC cells in vitro and in vivo. (A) Immunoblot (top panel) and qRT-PCR (bottom panel) results showing the relative MASTL expression in various HCC cell lines. (B) Immunoblot and qRT-PCR results showing the efficiency of MASTL overexpression in MHCC97H cells and MASTL-knockdown in SNU449 cells. (C) IC 50 values of lenvatinib in MASTL-overexpressing, MASTL-knockdown, and control cell lines. (D) Colony formation assay of MASTL-overexpressing, MASTL-knockdown, and control cell lines treated with lenvatinib in 6-well plates for 12 days. (E–H) Subcutaneous tumor models were established using the indicated cells, and the mice were treated with lenvatinib or vehicle (n=6). Tumor volumes were measured at the indicated time points, and tumor weights were measured at the endpoint. (I) Bubble plot displaying the results of the KEGG pathway analysis of MASTL-overexpressing cell lines. (J) Immunoblot analysis of MAPK signaling pathway activation in the indicated cell lines. (K) Bubble plot showing the results of the GO: BP analysis of MASTL-overexpressing cell lines. (L) Differential bidirectional chart showing the top 10 upregulated and downregulated genes in the “regulation of cell growth” gene set from the GO: BP analysis. All the experiments were repeated a minimum of 3 times. p <0.05 was considered to indicate statistical significance. * p <0.05, ** p <0.01, *** p <0.001. Abbreviations: BP, biological process; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; MASTL, microtubule-associated serine/threonine kinase-like; qRT-PCR, quantitative reverse-transcription PCR.

Article Snippet: The PLC/PRF/5 and SNU449 cell lines were obtained from the American Type Culture Collection (ATCC), while the MHCC97H cell line was sourced from the Chinese Academy of Sciences.

Techniques: In Vitro, In Vivo, Western Blot, Quantitative RT-PCR, Expressing, Over Expression, Knockdown, Control, Colony Assay, Activation Assay, Reverse Transcription

Journal: Cancer Gene Therapy

Article Title: Combined downregulation of TGF-β1 and GRP78 is responsible for overcoming acquired sorafenib resistance, which is initiated by rewiring the cell surface CD44-GRP78-IGF-1R signaling circuit

doi: 10.1038/s41417-025-00937-1

Figure Lengend Snippet: A Validation of TGF-β1 and HSP27 mRNA downregulation in SNU449 and sorafenib-resistant SNU449 (SNU449-SR)cells was examined by quantitative real-time PCR after 36 h of infection with adenovirus expressing shRNA targeting TGF-β1, shRNA targeting HSP27 or shRNAs targeting HSP27/TGF-β1 at an MOI of 100. B (top) Cell viability of SNU449 and sorafenib-resistant SNU449 (SNU449-SR) cells was examined after 24 h of infection with adenovirus expressing shRNA targeting TGF-β1, shRNA targeting HSP27 or shRNAs targeting HSP27/TGF-β1 at an MOI of 100, followed by sorafenib (15 μM) treatment for 24 h in HCC and sorafenib-resistant HCC. The cells were infected with adenovirus expressing nonsense shRNA as a negative control at an MOI of 100. Cell viability was tested by Trypan blue. The error bars represent the standard errors from three independent experiments. (bottom) Clonogenic assays of SNU449 and SNU449-SR cells were performed after infection with adenovirus expressing shRNA of TGF-β1 or shRNA of HSP27 or shRNAs of HSP27/TGF-β1 followed by sorafenib (15 μM) treatment in HCC and sorafenib-resistant HCC and incubation for an additional 14 days after appropriate dilution of treated samples. MOI multiplicity of infection, NC negative control. C Transcriptome profiling analysis of SNU449 and SNU449-SR through shRNA of HSP27 or shRNA of TGF-β1 or shRNAs of HSP27/TGF-β1. The expression values of the DEGs are shown in a heatmap. Gene expression levels are visualized as row-standardized z -scores ranging from green (−1) to red (+1) across all samples. The rows are organized by hierarchical clustering analysis with complete linkage and Euclidean distance as a measure of similarity from samples of SNU449 and SNU449-SR (shRNA of HSP27 or shRNA of TGF-β1 or shRNAs of HSP27/TGF-β1). D SNU449 or SNU449-SR cells were infected with adenovirus expressing 100 MOI of shRNA of HSP27 or shRNA of TGF-β1 or shRNAs of HSP27/TGF-β1. Scatter plots of the relative probe set intensities after infection were subsequently generated. Normalized probe set intensities of various infections are plotted. The numbers indicate over 2-fold variation in intensities outside of two diagonal lines. E Cellular levels of GRP78 or vimentin mRNA (left) or protein (right) before and after acquired sorafenib resistance in SNU449 cells were examined by real-time PCR or western blotting, respectively. F SNU449 or SNU449-SR cells were infected with adenovirus expressing shRNAs of HSP27/TGF-β1 at 100 MOI, and the endogenous cellular level of GRP78 or vimentin mRNA was subsequently examined via real-time PCR after RNA extraction. The cells were infected with adenovirus expressing nonsense shRNA as a negative control (NC) at an MOI of 100. G SNU449 or SNU449-SR cells were infected with adenovirus expressing shRNA targeting HSP27, shRNA targeting TGF-β1 or shRNAs targeting HSP27/TGF-β1 at an MOI of 100. The cells were infected with adenovirus expressing nonsense shRNA as a negative control at an MOI of 100. After 48 h, the expression levels of vimentin, GRP78 and β-actin were detected via western blot analysis. The statistical significance was determined by Student’s t tests ( A , E , F ) or two-way analysis of variance (ANOVA)( B ). Data are shown as mean values ± SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns not significant.

Article Snippet: The human cancer cell lines SNU449 (human hepatocellular carcinoma), SNU398 (human hepatocellular carcinoma), and A549 (human lung carcinoma) were purchased from the Korean Cell Line Bank, and 293A, a subclone of the human embryonic kidney 293 cell line, was purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Infection, Expressing, shRNA, Negative Control, Incubation, Gene Expression, Generated, Western Blot, RNA Extraction

Journal: Cancer Gene Therapy

Article Title: Combined downregulation of TGF-β1 and GRP78 is responsible for overcoming acquired sorafenib resistance, which is initiated by rewiring the cell surface CD44-GRP78-IGF-1R signaling circuit

doi: 10.1038/s41417-025-00937-1

Figure Lengend Snippet: A To verify whether GRP78 downregulation is responsible for sorafenib resistance, a siRNA targeting GRP78 was transfected into SNU449 cells or SNU449-SR cells, which were subsequently subjected to sorafenib treatment (15 μM) for 24 h, and viability was examined via MTS assay. B Clonogenic activity of SNU449 or SNU449-SR cells was examined after 24 h of transfection with GRP78 siRNA followed by sorafenib treatment (15 μM) for 24 h, after which the cells were incubated for an additional 14 days after appropriate dilution of the treated samples. C Validation of GRP78 downregulation by screening 5 different newly designed GRP78 shRNAs. Five different synthesized shRNAs were transfected into A549 or SNU449 cells for 48 h, followed by real-time PCR after RNA extraction (top) or western blotting (bottom). D Overall validation of GRP78 downregulation by screening shRNAs via shuttle vector transfection. Shuttle vector plasmid subclones for the expression of 6 different shRNAs were transfected into 293A cells for 48 h, followed by real-time PCR after RNA extraction. E The potency of selected shGRP78 was confirmed after infection of SNU449 with an adenoviral vector expressing the 4th shRNA of GRP78 at various MOIs for 48 h, followed by real-time PCR after RNA extraction (left) or western blotting (right). F Clonogenic assay of SNU449 or SNU449-SR after infection with adenovirus expressing GRP78 2nd or 4th shRNA (MOI of 100) for 48 h and incubation for an additional 14 days after appropriate dilution of the treated samples. The statistical significance was determined by Student’s t tests ( C , D , E ) or two-way analysis of variance (ANOVA)( A ). Data are shown as mean values ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The human cancer cell lines SNU449 (human hepatocellular carcinoma), SNU398 (human hepatocellular carcinoma), and A549 (human lung carcinoma) were purchased from the Korean Cell Line Bank, and 293A, a subclone of the human embryonic kidney 293 cell line, was purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Transfection, MTS Assay, Activity Assay, Incubation, Biomarker Discovery, Synthesized, Real-time Polymerase Chain Reaction, RNA Extraction, Western Blot, Plasmid Preparation, Expressing, Infection, shRNA, Clonogenic Assay

Journal: Cancer Gene Therapy

Article Title: Combined downregulation of TGF-β1 and GRP78 is responsible for overcoming acquired sorafenib resistance, which is initiated by rewiring the cell surface CD44-GRP78-IGF-1R signaling circuit

doi: 10.1038/s41417-025-00937-1

Figure Lengend Snippet: A The viability of SNU449, sorafenib-resistant SNU449 (SNU449-SR), SNU398 and sorafenib-resistant SNU398 (SNU398-SR) cells was examined after 48 h of infection with adenoviruses expressing shHSP27, shTGF-β1, shGRP78 or shvimentin at an MOI of 100. The cells were infected with adenovirus expressing nonsense shRNA as a negative control at an MOI of 100. Cell viability was tested via the MTS assay. B Clonogenic assays of SNU449, sorafenib-resistant SNU449 (SNU449-SR) (left), SNU398, and sorafenib-resistant SNU398 (SNU398-SR) (right) were performed after 48 h of infection with adenoviruses expressing shHSP27, shTGF-β1, shGRP78, or shvimentin and incubated for an additional 14 days after appropriate dilution of the treated samples. C Cell viability of the SNU449, sorafenib-resistant SNU449 (SNU449-SR), SNU398, and sorafenib-resistant SNU398 (SNU398-SR) strains was examined after 48 h of infection with adenoviruses expressing shTGF-β1, shHSP27/shTGF-β1, or shTGF-β1/shGRP78. The cells were infected with adenovirus expressing nonsense shRNA as a negative control at an MOI of 100. Cell viability was tested via both the Trypan blue assay or the MTS assay (supplementary Fig. ), respectively. D Clonogenic assays of SNU449, sorafenib-resistant SNU449 (SNU449-SR) (left), SNU398 and sorafenib-resistant SNU398 (SNU398-SR) (right) were performed after 48 h of infection with adenovirus expressing shHSP27/shTGF-β1, shTGF-β1/shvimentin or shTGF-β1/shGRP78, and the cells were incubated for an additional 14 days after appropriate dilution of the treated samples. E The surface GRP78 of SNU449 cells after acquired sorafenib resistance or LY294002 pretreatment (150 μM, 1 h) was measured via biotinylation, followed by the precipitation of avidin-agarose beads and the release of cell surface proteins for the detection of GRP78 on the cell surface via western blotting. F The levels of GRP78 on the SNU398 cell surface after acquired sorafenib resistance or LY294002 pretreatment (150 μM, 1 h) were measured via biotinylation, followed by the precipitation of avidin-agarose beads and the release of cell surface proteins for the detection of GRP78 on the cell surface via western blotting. G GRP78 mRNA levels in SNU449 cells with sorafenib resistance were measured after adenoviral infection with shRNAs expressing TGF-β1, GRP78 or both TGF-β1 and GRP78. H Cell surface GRP78 levels in SNU449 cells after acquired sorafenib resistance were measured after adenoviral infection with shRNAs expressing TGF-β1, GRP78, or both TGF-β1 and GRP78. I Cell surface GRP78 expression in SNU398 cells after acquired sorafenib resistance was measured after adenoviral infection with shRNAs expressing TGF-β1, GRP78, or both TGF-β1 and GRP78. The statistical significance was determined by Student’s t tests. Data are shown as mean values ± SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The human cancer cell lines SNU449 (human hepatocellular carcinoma), SNU398 (human hepatocellular carcinoma), and A549 (human lung carcinoma) were purchased from the Korean Cell Line Bank, and 293A, a subclone of the human embryonic kidney 293 cell line, was purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Infection, Expressing, shRNA, Negative Control, MTS Assay, Incubation, Avidin-Biotin Assay, Western Blot

Journal: Cancer Gene Therapy

Article Title: Combined downregulation of TGF-β1 and GRP78 is responsible for overcoming acquired sorafenib resistance, which is initiated by rewiring the cell surface CD44-GRP78-IGF-1R signaling circuit

doi: 10.1038/s41417-025-00937-1

Figure Lengend Snippet: A Phosphorylation of Akt, ERK, JNK, p38, and HSP27, including IRE1α, was examined in SNU449 or SNU398 cells after sorafenib treatment (10 μM and 5 μM, respectively) in a time-dependent manner. The numbers indicate the relative band intensity of various phosphorylated proteins corresponding to their own control proteins after sorafenib treatment in a time-dependent manner. B Total cellular and cell surface levels of GRP78 were examined in SNU449 or SNU398 cells after sorafenib treatment (10 μM and 5 μM, respectively) in a time-dependent manner. After sorafenib treatment, GRP78 in the cell lysate was examined via western blotting, whereas the expression of the cell surface GRP78 (csGRP78) protein was measured after biotintylation, followed by avidin pulldown of the cells. The numbers indicate the relative band intensity csGRP78 corresponding to total GRP78 protein after sorafenib treatment in a time-dependent manner. C The surface of GRP78 was examined in SNU449 or SNU398 cells after treatment with NAC (10 μM, 2 h), a p38 inhibitor (SB203580, 20 μM, 1 h), a PI3K inhibitor (LY294002, 20 μM, 1 h), a JNK inhibitor (SP600125, 20 μM, 1 h), a JNK/p38 activator (AEBSF, 10 μM, 1 h), or an IRE1α inhibitor (20 μM, 1 h) before sorafenib treatment (10 μM, 5 μM for 2 h). The numbers indicate the relative band intensity of various phosphorylated proteins or cell surface proteins corresponding to their own control proteins after sorafenib treatment and/or various inhibitor. Each band intensity was measured using a densitometer. D Phosphorylation of IGF1R, CD44, and cell surface CD44 (csCD44) was examined in SNU449 or SNU398 cells after sorafenib treatment (10 μM or 5 μM) in a time-dependent manner. The numbers indicate the relative band intensity of phosphorylated IGF-1R protein or csCD44 corresponding to its own control protein after sorafenib treatment in a time-dependent manner. Each band intensity was measured using a densitometer. E (top) CD44 involvement in the process of acquired sorafenib resistance was demonstrated by CD44 downregulation during sorafenib treatment (2 h). SNU449 cells (left) or SNU398 cells (right) infected with adenovirus expressing shRNA against CD44 were treated with sorafenib for 2 h. Then, the phosphorylation of IGF1R, Akt, ERK, and CD44 at the cell surface membrane and total cell lysate was detected. The numbers indicate the relative band intensity of various phosphorylated proteins or cell surface proteins corresponding to their own control proteins after sorafenib treatment and/or CD44 downregulation. Each band intensity was measured using a densitometer. (bottom) The viability of SNU449 (left) cells was examined after 48 h of infection with adenovirus expressing shCD44 with or without sorafenib (10 μM, 2 h) via the Trypan blue assay. The viability of SNU398 (right) cells was examined after stable retroviral infection with pBabe-puro-CD44. After 2 days of sorafenib treatment (5 μM), cell viability was tested via the Trypan blue assay. F (top) SNU449 cells (left) or SNU398 cells (right) were lysed after sorafenib treatment for 2 h and subjected to immunoprecipitation with anti-GRP78 antibodies to detect the interaction between GRP78 and IGF1R (bottom). GRP78 mRNA expression was examined after 2 days of sorafenib treatment via real-time PCR after RNA extraction from SNU449 cells or SNU398 cells. The statistical significance was determined by Student’s t tests (E, right) or two-way analysis of variance (ANOVA)(E, left). Data are shown as mean values ± SD. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Article Snippet: The human cancer cell lines SNU449 (human hepatocellular carcinoma), SNU398 (human hepatocellular carcinoma), and A549 (human lung carcinoma) were purchased from the Korean Cell Line Bank, and 293A, a subclone of the human embryonic kidney 293 cell line, was purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Phospho-proteomics, Control, Western Blot, Expressing, Avidin-Biotin Assay, Infection, shRNA, Membrane, Retroviral, Immunoprecipitation, Real-time Polymerase Chain Reaction, RNA Extraction

Journal: Cancer Gene Therapy

Article Title: Combined downregulation of TGF-β1 and GRP78 is responsible for overcoming acquired sorafenib resistance, which is initiated by rewiring the cell surface CD44-GRP78-IGF-1R signaling circuit

doi: 10.1038/s41417-025-00937-1

Figure Lengend Snippet: A SNU449 cells (left) or SNU398 cells were pretreated with MG132 (20 μM, 1 h) before sorafenib treatment (10 μM, 5 μM for 2 h, respectively). GRP78 and CD44 at the cell surface membrane, as well as their total cell lysates, were subsequently examined. B Wild-type HSP27, nonphosphorylatable HSP27(3A) (Serine 15,78,82 alterations to alanine) and phosphomimetic HSP27(3D) (Serine 15,78,82 alterations to aspartic acid) mutant plasmids were transfected into the SNU449 (left) or SNU398 (right) strains. The phosphorylation of IGF1R, Akt, ERK, or HSP27 was subsequently examined. CD44 at the cell surface membrane as well as its total cell lysate were also examined. C SNU449 (left) or SNU398 (right) cells infected with adenovirus expressing HSP27 shRNA were treated with sorafenib for 2 h. Then, the phosphorylation of IGF1R, Akt, ERK, or IRE1α was examined. GRP78 and CD44 at the cell surface membrane, as well as their total cell lysates, were also examined. D SNU449 cells infected with adenovirus expressing shRNA targeting GRP78 were treated with sorafenib for 2 h. Then, the phosphorylation of IGF1R, Akt, GRP78, and CD44 at the cell surface membrane and total cell lysates was also examined. The numbers indicate the relative band intensity of various phosphorylated proteins or cell surface proteins corresponding to their own control proteins after sorafenib treatment and/or GRP78 downregulation. Each band intensity was measured using a densitometer. E SNU449 cells were lysed and subjected to immunoprecipitation with anti-HSP27 (top, left) or anti-GRP78 antibodies (top, right) to detect the interaction between HSP27 and CD44 or between GRP78 and CD44 in total cell lysate before sorafenib treatment (left) or after sorafenib treatment (right). F SNU449 cells were lysed and subjected to immunoprecipitation with anti-HSP27 (left) or anti-GRP78 antibodies (right) to detect the interaction between HSP27 and CD44 or between GRP78 and CD44 after sorafenib treatment in a time-dependent manner. G SNU449 and SNU398 were cultured in confocal plates at 1 × 10 5 cells/well and incubated with sorafenib at 10 μM and 5 μM, respectively. Then, the cells were fixed in 4% PFA for 15 min at room temperature and incubated with primary antibodies against CD44, HSP27, CD44 or GRP78 at room temperature for 2 h, followed by incubation with Alexa Fluor 488-conjugated anti-rabbit IgG antibody or Alexa Fluor 647-conjugated anti-mouse IgG antibody at room temperature for 1 h to obtain fluorescence images. Nuclei were stained with DAPI at room temperature for 5 min. H SNU449 and SNU398 were cultured in confocal plates at 1 × 10 5 cells/well and incubated with sorafenib at 10 μM and 5 μM, respectively. Then, the cells were treated with both MemBrite® 543/560 cell surface prestaining solution at 37 °C for 5 min and MemBrite® solution at 4 °C for 30 min to capture Z-stack confocal images.

Article Snippet: The human cancer cell lines SNU449 (human hepatocellular carcinoma), SNU398 (human hepatocellular carcinoma), and A549 (human lung carcinoma) were purchased from the Korean Cell Line Bank, and 293A, a subclone of the human embryonic kidney 293 cell line, was purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Membrane, Mutagenesis, Transfection, Phospho-proteomics, Infection, Expressing, shRNA, Control, Immunoprecipitation, Cell Culture, Incubation, Fluorescence, Staining

Journal: Cancer Gene Therapy

Article Title: Combined downregulation of TGF-β1 and GRP78 is responsible for overcoming acquired sorafenib resistance, which is initiated by rewiring the cell surface CD44-GRP78-IGF-1R signaling circuit

doi: 10.1038/s41417-025-00937-1

Figure Lengend Snippet: A SNU449 (left) or SNU398 (right) cells were pretreated with 2DGal (10 mM, 12 h) before sorafenib treatment (10 μM, 5 μM for 2 h, respectively). GRP78 and CD44 at the cell surface membrane, as well as their total cell lysates, were subsequently examined. Phosphorylated IGF1R, IGF1R, and FUT1 proteins in cell lysates were also examined. B SNU449 (left) or SNU398 (right) cells were pretreated with 2DGal (10 mM, 12 h) before sorafenib treatment (10 μM, 5 μM for 2 h, respectively). Then, the cells were lysed and subjected to immunoprecipitation with an anti-GRP78 antibody to detect the interaction between GRP78 and CD44 in total cell lysates. C After CD44-overexpressing SNU398 cells were selected via retroviral infection with pBabe-puro-CD44, the selected cells were treated with sorafenib (5 μM, 2 h). Then, the cells were lysed and subjected to immunoprecipitation with an anti-CD44 antibody to detect the interaction between CD44 and fucosylation-related factors and H antigen from total cell lysate (right). ( D ) Sorafenib-resistant SNU398 (SNU398-SR) tumors were grown in male BALB/c nude mice. Tumors were established via the subcutaneous injection of 2 × 10 6 cells and were allowed to grow to an average size of 70–120 mm 3 . PBS and adenoviruses were intratumorally injected twice every other day. Oncolytic adenoviruses coexpressing shHSP27-shTGF-β1 or shTGF-β1-shGRP78, shHSP27-shTGF-β1-shHIF1α or shTGF-β1-shGRP78-shHIF1α were intratumorally injected into SNU398-SR tumors. Tumor growth was measured every 2 days for more than 14 days via calipers. The arrows indicate when the oncolytic adenovirus was administered. The statistical significance was determined by two-way analysis of variance (ANOVA). Data are shown as mean values ± SD. ** p < 0.01; **** p < 0.0001; ns not significant.

Article Snippet: The human cancer cell lines SNU449 (human hepatocellular carcinoma), SNU398 (human hepatocellular carcinoma), and A549 (human lung carcinoma) were purchased from the Korean Cell Line Bank, and 293A, a subclone of the human embryonic kidney 293 cell line, was purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Membrane, Immunoprecipitation, Retroviral, Infection, Injection

Journal: Cancer Gene Therapy

Article Title: Combined downregulation of TGF-β1 and GRP78 is responsible for overcoming acquired sorafenib resistance, which is initiated by rewiring the cell surface CD44-GRP78-IGF-1R signaling circuit

doi: 10.1038/s41417-025-00937-1

Figure Lengend Snippet: A SNU449 cells are less sensitive to sorafenib and maintain a high level of CD44. Upon sorafenib treatment of SNU449, rapid p38 inactivation occurs, resulting in decreased Akt/ERK activity due to inactivation of PDGFR, VEGFR, and c-Kit. Independent of the induction of decreased cell viability, sorafenib-induced ER stress activates IRE1α along with apoptosis. The activation of IRE1α is associated with an increase in CD44 fucosylation and JNK phosphorylation, which leads to an increase in the binding of CD44 and GRP78, followed by an increase in these complexes in the cell membrane concomitant with efficient rewiring of the GRP78-IGF1R-PI3K-Akt signaling cascade to provide strong survival potential as well as a continuous positive feedback loop, resulting in sustained strong sorafenib resistance. B SNU398 is associated with increased sensitivity to sorafenib. Compared with SNU449 cells, SNU398 cells presented lower baseline CD44 expression and weaker survival signaling pathways. Upon sorafenib treatment, the inhibition of Akt and ERK phosphorylation is more sustained, leading to a weaker acquisition of resistance. Unlike in SNU449, the CD44‒GRP78 interaction is less prominent because of the reduced expression of CD44, and lower fucosylation activation leads to lower activation of the IGF1R‒PI3K‒Akt signaling cascade, which makes SNU398 less capable of sustaining resistance after sorafenib treatment.

Article Snippet: The human cancer cell lines SNU449 (human hepatocellular carcinoma), SNU398 (human hepatocellular carcinoma), and A549 (human lung carcinoma) were purchased from the Korean Cell Line Bank, and 293A, a subclone of the human embryonic kidney 293 cell line, was purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Binding Assay, Membrane, Expressing, Protein-Protein interactions, Inhibition