Review



aspc 1  (ATCC)


Bioz Verified Symbol ATCC is a verified supplier
Bioz Manufacturer Symbol ATCC manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 99
      Buy from Supplier

    Structured Review

    ATCC aspc 1
    BMH-21 preferentially inhibits cell viability and induces apoptosis in pancreatic cancer cells. A, Effect of BMH-21 on cell viability of HPDEC and pancreatic cancer cells (Capan-2, <t>AsPC-1,</t> BxPC-3, PANC-1, HPAF-II, and MIA PaCa-2). Cells were treated with indicated concentrations of BMH-21 for 24 hours, and cell viability was analyzed via MTT assay. B–E, Effect of BMH-21 on apoptosis induction in HPDEC and pancreatic cancer cells. B, Effect of BMH-21 on PARP in HPDEC and pancreatic cancer cells as assessed using WB. Briefly, cells were treated with indicated concentrations of BMH-21 for 12 hours. Total PARP and cleaved PARP (cl-PARP) were detected by WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin antibody. C and D, Flow cytometric analysis to detect apoptotic cells in control and BMH-21 (2 μmol/L)–treated HPDEC and MIA PaCa-2 cells using Alexa Fluor 488–labeled Annexin V. Representative flow cytometric images showing percent Alexa Fluor 488–tagged Annexin V–positive cells. E, The bar graph represents the percentage of live and apoptotic HPDEC and MIA PaCa-2 cells after BMH-21 treatment. The data represent the mean ± SEM of three samples. The P values were expressed as follows: **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. PI, propidium iodide.
    Aspc 1, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 3408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aspc 1/product/ATCC
    Average 99 stars, based on 3408 article reviews
    aspc 1 - by Bioz Stars, 2026-03
    99/100 stars

    Images

    1) Product Images from "Ubiquitination of Oncogenic Mutant p53 via Attenuation of Ribosome Biogenesis Machinery Effectively Inhibits Pancreatic Tumor Growth"

    Article Title: Ubiquitination of Oncogenic Mutant p53 via Attenuation of Ribosome Biogenesis Machinery Effectively Inhibits Pancreatic Tumor Growth

    Journal: Molecular Cancer Therapeutics

    doi: 10.1158/1535-7163.MCT-25-0097

    BMH-21 preferentially inhibits cell viability and induces apoptosis in pancreatic cancer cells. A, Effect of BMH-21 on cell viability of HPDEC and pancreatic cancer cells (Capan-2, AsPC-1, BxPC-3, PANC-1, HPAF-II, and MIA PaCa-2). Cells were treated with indicated concentrations of BMH-21 for 24 hours, and cell viability was analyzed via MTT assay. B–E, Effect of BMH-21 on apoptosis induction in HPDEC and pancreatic cancer cells. B, Effect of BMH-21 on PARP in HPDEC and pancreatic cancer cells as assessed using WB. Briefly, cells were treated with indicated concentrations of BMH-21 for 12 hours. Total PARP and cleaved PARP (cl-PARP) were detected by WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin antibody. C and D, Flow cytometric analysis to detect apoptotic cells in control and BMH-21 (2 μmol/L)–treated HPDEC and MIA PaCa-2 cells using Alexa Fluor 488–labeled Annexin V. Representative flow cytometric images showing percent Alexa Fluor 488–tagged Annexin V–positive cells. E, The bar graph represents the percentage of live and apoptotic HPDEC and MIA PaCa-2 cells after BMH-21 treatment. The data represent the mean ± SEM of three samples. The P values were expressed as follows: **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. PI, propidium iodide.
    Figure Legend Snippet: BMH-21 preferentially inhibits cell viability and induces apoptosis in pancreatic cancer cells. A, Effect of BMH-21 on cell viability of HPDEC and pancreatic cancer cells (Capan-2, AsPC-1, BxPC-3, PANC-1, HPAF-II, and MIA PaCa-2). Cells were treated with indicated concentrations of BMH-21 for 24 hours, and cell viability was analyzed via MTT assay. B–E, Effect of BMH-21 on apoptosis induction in HPDEC and pancreatic cancer cells. B, Effect of BMH-21 on PARP in HPDEC and pancreatic cancer cells as assessed using WB. Briefly, cells were treated with indicated concentrations of BMH-21 for 12 hours. Total PARP and cleaved PARP (cl-PARP) were detected by WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin antibody. C and D, Flow cytometric analysis to detect apoptotic cells in control and BMH-21 (2 μmol/L)–treated HPDEC and MIA PaCa-2 cells using Alexa Fluor 488–labeled Annexin V. Representative flow cytometric images showing percent Alexa Fluor 488–tagged Annexin V–positive cells. E, The bar graph represents the percentage of live and apoptotic HPDEC and MIA PaCa-2 cells after BMH-21 treatment. The data represent the mean ± SEM of three samples. The P values were expressed as follows: **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. PI, propidium iodide.

    Techniques Used: MTT Assay, Stripping Membranes, Control, Labeling

    Expression pattern of RPA194 and targeting RiBi in pancreatic cancer. A, In silico analysis of RPA194 expression in human normal and pancreatic tumor tissues. The box plot shows significant ( P = 0.01) overexpression of RPA194 in pancreatic tumor tissues compared with normal pancreatic tissues. B, Differential expression of RPA194 in various pancreatic cancer cell lines by WB analysis. C, Effect of BMH-21 on the RPA194 protein level in normal (HPDEC) and pancreatic cancer cells (Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells). Briefly, cells were treated with indicated concentrations of BMH-21 for 3, 6, and 12 hours, and the RPA194 protein level was determined by WB analysis. D, Effect of BMH-21 (2 μmol/L) on the localization of RPA194 (red) and UBTF (green) in Capan-2, MIA PaCa-2, and HPAF-II cells as determined by immunofluorescence. Scale bar, 5 μm. The quantification of immunofluorescence images is provided in Supplementary Fig. S3. E, Effect of BMH-21 on pre-rRNA synthesis in HPDEC, Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Cells were treated with BMH-21 (2 μmol/L) for 6 hours. qPCR was performed using primers for 5′ ETS expression at 851 regions. The bar graph indicates fold expression of 5′ ETS in control and BMH-21–treated cells. F–I, Effect of BMH-21 on the recruitment of RNA Pol I catalytic subunits (RPA194 and RPA135) on rDNA by ChIP assay. The cells were grown in 150-mm dishes to 90% confluency and treated with 2 μmol/L BMH-21 for 24 hours. IP was performed using RPA194 or RPA135 antibodies, and purified chromatin was amplified by qPCR. F and G, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in Capan-2 cells. H and I, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in MIA PaCa-2 cells. Values in bar graphs represent the mean ± SEM of three replicates. ns, P ≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
    Figure Legend Snippet: Expression pattern of RPA194 and targeting RiBi in pancreatic cancer. A, In silico analysis of RPA194 expression in human normal and pancreatic tumor tissues. The box plot shows significant ( P = 0.01) overexpression of RPA194 in pancreatic tumor tissues compared with normal pancreatic tissues. B, Differential expression of RPA194 in various pancreatic cancer cell lines by WB analysis. C, Effect of BMH-21 on the RPA194 protein level in normal (HPDEC) and pancreatic cancer cells (Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells). Briefly, cells were treated with indicated concentrations of BMH-21 for 3, 6, and 12 hours, and the RPA194 protein level was determined by WB analysis. D, Effect of BMH-21 (2 μmol/L) on the localization of RPA194 (red) and UBTF (green) in Capan-2, MIA PaCa-2, and HPAF-II cells as determined by immunofluorescence. Scale bar, 5 μm. The quantification of immunofluorescence images is provided in Supplementary Fig. S3. E, Effect of BMH-21 on pre-rRNA synthesis in HPDEC, Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Cells were treated with BMH-21 (2 μmol/L) for 6 hours. qPCR was performed using primers for 5′ ETS expression at 851 regions. The bar graph indicates fold expression of 5′ ETS in control and BMH-21–treated cells. F–I, Effect of BMH-21 on the recruitment of RNA Pol I catalytic subunits (RPA194 and RPA135) on rDNA by ChIP assay. The cells were grown in 150-mm dishes to 90% confluency and treated with 2 μmol/L BMH-21 for 24 hours. IP was performed using RPA194 or RPA135 antibodies, and purified chromatin was amplified by qPCR. F and G, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in Capan-2 cells. H and I, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in MIA PaCa-2 cells. Values in bar graphs represent the mean ± SEM of three replicates. ns, P ≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Techniques Used: Expressing, In Silico, Over Expression, Quantitative Proteomics, Immunofluorescence, Control, Purification, Amplification

    BMH-21 induces WT p53 protein expression by several folds but not the mutant p53 expression in pancreatic cancer cells. A, Effect of BMH-21 on the protein level of WT and mutant p53 in Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Briefly, cells were treated with 2 μmol/L BMH-21 for 24 hours, and 40–100 μg cell lysate was subjected to WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin or cyclophilin B antibodies. B, The bar graph represents the quantification of blots represented in A . The blots were quantified using ImageJ software and normalized with respective loading controls. C and D, The effect of BMH-21 on the localization of WT and mutant p53 levels in Capan-2 and MIA PaCa-2 cells was evaluated by immunofluorescence. Scale bar, 5 μm. E, The bar graphs represent the quantification of immunofluorescence of p53 shown in C and D . The quantification was done using ImageJ software. ns, P ≥ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
    Figure Legend Snippet: BMH-21 induces WT p53 protein expression by several folds but not the mutant p53 expression in pancreatic cancer cells. A, Effect of BMH-21 on the protein level of WT and mutant p53 in Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Briefly, cells were treated with 2 μmol/L BMH-21 for 24 hours, and 40–100 μg cell lysate was subjected to WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin or cyclophilin B antibodies. B, The bar graph represents the quantification of blots represented in A . The blots were quantified using ImageJ software and normalized with respective loading controls. C and D, The effect of BMH-21 on the localization of WT and mutant p53 levels in Capan-2 and MIA PaCa-2 cells was evaluated by immunofluorescence. Scale bar, 5 μm. E, The bar graphs represent the quantification of immunofluorescence of p53 shown in C and D . The quantification was done using ImageJ software. ns, P ≥ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Techniques Used: Expressing, Mutagenesis, Stripping Membranes, Software, Immunofluorescence

    RPA194 interacts with both WT and mutant p53, and BMH-21–induced RPA194 degradation exposes mutant p53 for its ubiquitination. A and B, RPA194 interacts with WT p53 in Capan-2 cells, truncated p53 (Δp53) in AsPC-1, and mutant p53 R248W in MIA PaCa-2 cells. Cells were treated with BMH-21 (2 μmol/L) or BMH-21 + MG132 (10 μmol/L) for 24 hours. A, RPA194 immunoprecipitated samples were subjected to WB analysis using p53 (HRP-conjugated) antibody (top). IgG antibody was used as the isotype control for IP. Eighty micrograms of protein for Capan-2 and 40 μg of protein for AsPC-1 and MIA PaCa-2 cells were used as inputs. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). B, Effect of BMH-21 on ubiquitination of RPA194 and RPA194 binding partners in pancreatic cancer cells (bottom). RPA194 was immunoprecipitated using the RPA194 antibody, and WB analysis was performed using the anti-ubiquitin antibody. The results indicate that RPA194 or its binding partners are ubiquitinated by BMH-21. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). C, Effect of BMH-21 on the ubiquitination level of RPA194 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. Briefly, untreated, BMH-21, and BMH-21 + MG132–treated samples were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB analysis using the RPA194 antibody. D, Effect of BMH-21 on the ubiquitination level of p53 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. The cells were treated as mentioned in C , and protein lysates were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB using HRP-conjugated p53 antibody. Hundred micrograms of protein for Capan-2 and 40 μg protein for MIA PaCa-2 cells were used as inputs.
    Figure Legend Snippet: RPA194 interacts with both WT and mutant p53, and BMH-21–induced RPA194 degradation exposes mutant p53 for its ubiquitination. A and B, RPA194 interacts with WT p53 in Capan-2 cells, truncated p53 (Δp53) in AsPC-1, and mutant p53 R248W in MIA PaCa-2 cells. Cells were treated with BMH-21 (2 μmol/L) or BMH-21 + MG132 (10 μmol/L) for 24 hours. A, RPA194 immunoprecipitated samples were subjected to WB analysis using p53 (HRP-conjugated) antibody (top). IgG antibody was used as the isotype control for IP. Eighty micrograms of protein for Capan-2 and 40 μg of protein for AsPC-1 and MIA PaCa-2 cells were used as inputs. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). B, Effect of BMH-21 on ubiquitination of RPA194 and RPA194 binding partners in pancreatic cancer cells (bottom). RPA194 was immunoprecipitated using the RPA194 antibody, and WB analysis was performed using the anti-ubiquitin antibody. The results indicate that RPA194 or its binding partners are ubiquitinated by BMH-21. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). C, Effect of BMH-21 on the ubiquitination level of RPA194 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. Briefly, untreated, BMH-21, and BMH-21 + MG132–treated samples were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB analysis using the RPA194 antibody. D, Effect of BMH-21 on the ubiquitination level of p53 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. The cells were treated as mentioned in C , and protein lysates were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB using HRP-conjugated p53 antibody. Hundred micrograms of protein for Capan-2 and 40 μg protein for MIA PaCa-2 cells were used as inputs.

    Techniques Used: Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation, Control, Binding Assay, Incubation

    Effect of BMH-21 on pancreatic tumor growth in the orthotopic xenograft mouse models. A and B, Schematic diagram depicting the experimental plan of the mouse xenograft study to investigate the therapeutic efficacy of BMH-21 against pancreatic cancer. Luciferase-labeled MIA PaCa-2 and AsPC-1 cells (1 × 10 6 ) were implanted in the pancreas of athymic nude mice ( n = 8/group). The vehicle and BMH-21 (25 mg/kg body weight) treatments started at 4–6 days after implantation of pancreatic cancer cells. Bioluminescence imaging of all the mice was performed at indicated days. C, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the MIA PaCa-2 luciferase xenograft at various time points. D and E, The average radiance (photons/second/cm 2 /sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the MIA PaCa-2 luciferase xenograft model. F, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the AsPC-1 luciferase xenograft at various time points. G and H, The average radiance (photons/second/cm 2 /sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the AsPC-1 luciferase xenograft model. I, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the MIA PaCa-2 luciferase xenograft mouse model. J, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the AsPC-1 luciferase xenograft mouse model. K, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated groups in MIA PaCa-2 luciferase xenograft mice. L, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated AsPC-1 luciferase xenograft mice. Tumor volume was calculated using the ellipsoid formula [0.5 × (W 2 × L)], where W and L represent the shortest and longest tumor diameters, respectively. M, Representative IHC staining images showing the expression of RPA194, Ki-67, PCNA, and mutant p53 in excised MIA PaCa-2 luciferase cell–derived xenograft tumors of vehicle-treated and BMH-21–treated mice. Magnification: 20×; scale bar, 50 μm. N, The bar graphs represent the quantification of IHC staining of RPA194, PCNA, Ki-67, and mutant p53 in pancreatic tumors excised from mice orthotopically xenografted with MIA PaCa-2 luciferase cells represented in M . The quantification of IHC staining was done using ImageJ software. Values in bar graphs represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P = 0.0003; ****, P ≤ 0.0001.
    Figure Legend Snippet: Effect of BMH-21 on pancreatic tumor growth in the orthotopic xenograft mouse models. A and B, Schematic diagram depicting the experimental plan of the mouse xenograft study to investigate the therapeutic efficacy of BMH-21 against pancreatic cancer. Luciferase-labeled MIA PaCa-2 and AsPC-1 cells (1 × 10 6 ) were implanted in the pancreas of athymic nude mice ( n = 8/group). The vehicle and BMH-21 (25 mg/kg body weight) treatments started at 4–6 days after implantation of pancreatic cancer cells. Bioluminescence imaging of all the mice was performed at indicated days. C, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the MIA PaCa-2 luciferase xenograft at various time points. D and E, The average radiance (photons/second/cm 2 /sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the MIA PaCa-2 luciferase xenograft model. F, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the AsPC-1 luciferase xenograft at various time points. G and H, The average radiance (photons/second/cm 2 /sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the AsPC-1 luciferase xenograft model. I, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the MIA PaCa-2 luciferase xenograft mouse model. J, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the AsPC-1 luciferase xenograft mouse model. K, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated groups in MIA PaCa-2 luciferase xenograft mice. L, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated AsPC-1 luciferase xenograft mice. Tumor volume was calculated using the ellipsoid formula [0.5 × (W 2 × L)], where W and L represent the shortest and longest tumor diameters, respectively. M, Representative IHC staining images showing the expression of RPA194, Ki-67, PCNA, and mutant p53 in excised MIA PaCa-2 luciferase cell–derived xenograft tumors of vehicle-treated and BMH-21–treated mice. Magnification: 20×; scale bar, 50 μm. N, The bar graphs represent the quantification of IHC staining of RPA194, PCNA, Ki-67, and mutant p53 in pancreatic tumors excised from mice orthotopically xenografted with MIA PaCa-2 luciferase cells represented in M . The quantification of IHC staining was done using ImageJ software. Values in bar graphs represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P = 0.0003; ****, P ≤ 0.0001.

    Techniques Used: Drug discovery, Luciferase, Labeling, Imaging, Immunohistochemistry, Expressing, Mutagenesis, Derivative Assay, Software



    Similar Products

    aspc 1  (ATCC)
    99
    ATCC aspc 1
    BMH-21 preferentially inhibits cell viability and induces apoptosis in pancreatic cancer cells. A, Effect of BMH-21 on cell viability of HPDEC and pancreatic cancer cells (Capan-2, <t>AsPC-1,</t> BxPC-3, PANC-1, HPAF-II, and MIA PaCa-2). Cells were treated with indicated concentrations of BMH-21 for 24 hours, and cell viability was analyzed via MTT assay. B–E, Effect of BMH-21 on apoptosis induction in HPDEC and pancreatic cancer cells. B, Effect of BMH-21 on PARP in HPDEC and pancreatic cancer cells as assessed using WB. Briefly, cells were treated with indicated concentrations of BMH-21 for 12 hours. Total PARP and cleaved PARP (cl-PARP) were detected by WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin antibody. C and D, Flow cytometric analysis to detect apoptotic cells in control and BMH-21 (2 μmol/L)–treated HPDEC and MIA PaCa-2 cells using Alexa Fluor 488–labeled Annexin V. Representative flow cytometric images showing percent Alexa Fluor 488–tagged Annexin V–positive cells. E, The bar graph represents the percentage of live and apoptotic HPDEC and MIA PaCa-2 cells after BMH-21 treatment. The data represent the mean ± SEM of three samples. The P values were expressed as follows: **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. PI, propidium iodide.
    Aspc 1, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aspc 1/product/ATCC
    Average 99 stars, based on 1 article reviews
    aspc 1 - by Bioz Stars, 2026-03
    99/100 stars
    		  Buy from Supplier
    aspc  (CLS Cell Lines Service GmbH)
    90
    CLS Cell Lines Service GmbH aspc
    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression <t>in</t> <t>AsPC-1</t> cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.
    Aspc, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aspc/product/CLS Cell Lines Service GmbH
    Average 90 stars, based on 1 article reviews
    aspc - by Bioz Stars, 2026-03
    90/100 stars
    		  Buy from Supplier
    aspc  (ATCC)
    99
    ATCC aspc
    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression <t>in</t> <t>AsPC-1</t> cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.
    Aspc, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aspc/product/ATCC
    Average 99 stars, based on 1 article reviews
    aspc - by Bioz Stars, 2026-03
    99/100 stars
    		  Buy from Supplier
    crl  (ATCC)
    99
    ATCC crl
    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression <t>in</t> <t>AsPC-1</t> cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.
    Crl, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/crl/product/ATCC
    Average 99 stars, based on 1 article reviews
    crl - by Bioz Stars, 2026-03
    99/100 stars
    		  Buy from Supplier
    aspc 1 cells  (ATCC)
    99
    ATCC aspc 1 cells
    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression <t>in</t> <t>AsPC-1</t> cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.
    Aspc 1 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aspc 1 cells/product/ATCC
    Average 99 stars, based on 1 article reviews
    aspc 1 cells - by Bioz Stars, 2026-03
    99/100 stars
    		  Buy from Supplier
    aspc 1 cell lines  (ATCC)
    99
    ATCC aspc 1 cell lines
    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression <t>in</t> <t>AsPC-1</t> cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.
    Aspc 1 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aspc 1 cell lines/product/ATCC
    Average 99 stars, based on 1 article reviews
    aspc 1 cell lines - by Bioz Stars, 2026-03
    99/100 stars
    		  Buy from Supplier
    human 141 aspc1  (ATCC)
    99
    ATCC human 141 aspc1
    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression <t>in</t> <t>AsPC-1</t> cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.
    Human 141 Aspc1, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human 141 aspc1/product/ATCC
    Average 99 stars, based on 1 article reviews
    human 141 aspc1 - by Bioz Stars, 2026-03
    99/100 stars
    		  Buy from Supplier
    adenocarcinoma cells  (ATCC)
    99
    ATCC adenocarcinoma cells
    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression <t>in</t> <t>AsPC-1</t> cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.
    Adenocarcinoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adenocarcinoma cells/product/ATCC
    Average 99 stars, based on 1 article reviews
    adenocarcinoma cells - by Bioz Stars, 2026-03
    99/100 stars
    		  Buy from Supplier
    saturation binding analysis  (ATCC)
    99
    ATCC saturation binding analysis
    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression <t>in</t> <t>AsPC-1</t> cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.
    Saturation Binding Analysis, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/saturation binding analysis/product/ATCC
    Average 99 stars, based on 1 article reviews
    saturation binding analysis - by Bioz Stars, 2026-03
    99/100 stars
    		  Buy from Supplier

    Image Search Results


    BMH-21 preferentially inhibits cell viability and induces apoptosis in pancreatic cancer cells. A, Effect of BMH-21 on cell viability of HPDEC and pancreatic cancer cells (Capan-2, AsPC-1, BxPC-3, PANC-1, HPAF-II, and MIA PaCa-2). Cells were treated with indicated concentrations of BMH-21 for 24 hours, and cell viability was analyzed via MTT assay. B–E, Effect of BMH-21 on apoptosis induction in HPDEC and pancreatic cancer cells. B, Effect of BMH-21 on PARP in HPDEC and pancreatic cancer cells as assessed using WB. Briefly, cells were treated with indicated concentrations of BMH-21 for 12 hours. Total PARP and cleaved PARP (cl-PARP) were detected by WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin antibody. C and D, Flow cytometric analysis to detect apoptotic cells in control and BMH-21 (2 μmol/L)–treated HPDEC and MIA PaCa-2 cells using Alexa Fluor 488–labeled Annexin V. Representative flow cytometric images showing percent Alexa Fluor 488–tagged Annexin V–positive cells. E, The bar graph represents the percentage of live and apoptotic HPDEC and MIA PaCa-2 cells after BMH-21 treatment. The data represent the mean ± SEM of three samples. The P values were expressed as follows: **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. PI, propidium iodide.

    Journal: Molecular Cancer Therapeutics

    Article Title: Ubiquitination of Oncogenic Mutant p53 via Attenuation of Ribosome Biogenesis Machinery Effectively Inhibits Pancreatic Tumor Growth

    doi: 10.1158/1535-7163.MCT-25-0097

    Figure Lengend Snippet: BMH-21 preferentially inhibits cell viability and induces apoptosis in pancreatic cancer cells. A, Effect of BMH-21 on cell viability of HPDEC and pancreatic cancer cells (Capan-2, AsPC-1, BxPC-3, PANC-1, HPAF-II, and MIA PaCa-2). Cells were treated with indicated concentrations of BMH-21 for 24 hours, and cell viability was analyzed via MTT assay. B–E, Effect of BMH-21 on apoptosis induction in HPDEC and pancreatic cancer cells. B, Effect of BMH-21 on PARP in HPDEC and pancreatic cancer cells as assessed using WB. Briefly, cells were treated with indicated concentrations of BMH-21 for 12 hours. Total PARP and cleaved PARP (cl-PARP) were detected by WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin antibody. C and D, Flow cytometric analysis to detect apoptotic cells in control and BMH-21 (2 μmol/L)–treated HPDEC and MIA PaCa-2 cells using Alexa Fluor 488–labeled Annexin V. Representative flow cytometric images showing percent Alexa Fluor 488–tagged Annexin V–positive cells. E, The bar graph represents the percentage of live and apoptotic HPDEC and MIA PaCa-2 cells after BMH-21 treatment. The data represent the mean ± SEM of three samples. The P values were expressed as follows: **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. PI, propidium iodide.

    Article Snippet: Capan-2, AsPC-1, MIA PaCa-2, HPAF-II, BxPC-3, PANC-1, and HPNE were obtained from ATCC.

    Techniques: MTT Assay, Stripping Membranes, Control, Labeling

    Expression pattern of RPA194 and targeting RiBi in pancreatic cancer. A, In silico analysis of RPA194 expression in human normal and pancreatic tumor tissues. The box plot shows significant ( P = 0.01) overexpression of RPA194 in pancreatic tumor tissues compared with normal pancreatic tissues. B, Differential expression of RPA194 in various pancreatic cancer cell lines by WB analysis. C, Effect of BMH-21 on the RPA194 protein level in normal (HPDEC) and pancreatic cancer cells (Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells). Briefly, cells were treated with indicated concentrations of BMH-21 for 3, 6, and 12 hours, and the RPA194 protein level was determined by WB analysis. D, Effect of BMH-21 (2 μmol/L) on the localization of RPA194 (red) and UBTF (green) in Capan-2, MIA PaCa-2, and HPAF-II cells as determined by immunofluorescence. Scale bar, 5 μm. The quantification of immunofluorescence images is provided in Supplementary Fig. S3. E, Effect of BMH-21 on pre-rRNA synthesis in HPDEC, Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Cells were treated with BMH-21 (2 μmol/L) for 6 hours. qPCR was performed using primers for 5′ ETS expression at 851 regions. The bar graph indicates fold expression of 5′ ETS in control and BMH-21–treated cells. F–I, Effect of BMH-21 on the recruitment of RNA Pol I catalytic subunits (RPA194 and RPA135) on rDNA by ChIP assay. The cells were grown in 150-mm dishes to 90% confluency and treated with 2 μmol/L BMH-21 for 24 hours. IP was performed using RPA194 or RPA135 antibodies, and purified chromatin was amplified by qPCR. F and G, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in Capan-2 cells. H and I, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in MIA PaCa-2 cells. Values in bar graphs represent the mean ± SEM of three replicates. ns, P ≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Journal: Molecular Cancer Therapeutics

    Article Title: Ubiquitination of Oncogenic Mutant p53 via Attenuation of Ribosome Biogenesis Machinery Effectively Inhibits Pancreatic Tumor Growth

    doi: 10.1158/1535-7163.MCT-25-0097

    Figure Lengend Snippet: Expression pattern of RPA194 and targeting RiBi in pancreatic cancer. A, In silico analysis of RPA194 expression in human normal and pancreatic tumor tissues. The box plot shows significant ( P = 0.01) overexpression of RPA194 in pancreatic tumor tissues compared with normal pancreatic tissues. B, Differential expression of RPA194 in various pancreatic cancer cell lines by WB analysis. C, Effect of BMH-21 on the RPA194 protein level in normal (HPDEC) and pancreatic cancer cells (Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells). Briefly, cells were treated with indicated concentrations of BMH-21 for 3, 6, and 12 hours, and the RPA194 protein level was determined by WB analysis. D, Effect of BMH-21 (2 μmol/L) on the localization of RPA194 (red) and UBTF (green) in Capan-2, MIA PaCa-2, and HPAF-II cells as determined by immunofluorescence. Scale bar, 5 μm. The quantification of immunofluorescence images is provided in Supplementary Fig. S3. E, Effect of BMH-21 on pre-rRNA synthesis in HPDEC, Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Cells were treated with BMH-21 (2 μmol/L) for 6 hours. qPCR was performed using primers for 5′ ETS expression at 851 regions. The bar graph indicates fold expression of 5′ ETS in control and BMH-21–treated cells. F–I, Effect of BMH-21 on the recruitment of RNA Pol I catalytic subunits (RPA194 and RPA135) on rDNA by ChIP assay. The cells were grown in 150-mm dishes to 90% confluency and treated with 2 μmol/L BMH-21 for 24 hours. IP was performed using RPA194 or RPA135 antibodies, and purified chromatin was amplified by qPCR. F and G, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in Capan-2 cells. H and I, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in MIA PaCa-2 cells. Values in bar graphs represent the mean ± SEM of three replicates. ns, P ≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Article Snippet: Capan-2, AsPC-1, MIA PaCa-2, HPAF-II, BxPC-3, PANC-1, and HPNE were obtained from ATCC.

    Techniques: Expressing, In Silico, Over Expression, Quantitative Proteomics, Immunofluorescence, Control, Purification, Amplification

    BMH-21 induces WT p53 protein expression by several folds but not the mutant p53 expression in pancreatic cancer cells. A, Effect of BMH-21 on the protein level of WT and mutant p53 in Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Briefly, cells were treated with 2 μmol/L BMH-21 for 24 hours, and 40–100 μg cell lysate was subjected to WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin or cyclophilin B antibodies. B, The bar graph represents the quantification of blots represented in A . The blots were quantified using ImageJ software and normalized with respective loading controls. C and D, The effect of BMH-21 on the localization of WT and mutant p53 levels in Capan-2 and MIA PaCa-2 cells was evaluated by immunofluorescence. Scale bar, 5 μm. E, The bar graphs represent the quantification of immunofluorescence of p53 shown in C and D . The quantification was done using ImageJ software. ns, P ≥ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Journal: Molecular Cancer Therapeutics

    Article Title: Ubiquitination of Oncogenic Mutant p53 via Attenuation of Ribosome Biogenesis Machinery Effectively Inhibits Pancreatic Tumor Growth

    doi: 10.1158/1535-7163.MCT-25-0097

    Figure Lengend Snippet: BMH-21 induces WT p53 protein expression by several folds but not the mutant p53 expression in pancreatic cancer cells. A, Effect of BMH-21 on the protein level of WT and mutant p53 in Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Briefly, cells were treated with 2 μmol/L BMH-21 for 24 hours, and 40–100 μg cell lysate was subjected to WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin or cyclophilin B antibodies. B, The bar graph represents the quantification of blots represented in A . The blots were quantified using ImageJ software and normalized with respective loading controls. C and D, The effect of BMH-21 on the localization of WT and mutant p53 levels in Capan-2 and MIA PaCa-2 cells was evaluated by immunofluorescence. Scale bar, 5 μm. E, The bar graphs represent the quantification of immunofluorescence of p53 shown in C and D . The quantification was done using ImageJ software. ns, P ≥ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Article Snippet: Capan-2, AsPC-1, MIA PaCa-2, HPAF-II, BxPC-3, PANC-1, and HPNE were obtained from ATCC.

    Techniques: Expressing, Mutagenesis, Stripping Membranes, Software, Immunofluorescence

    RPA194 interacts with both WT and mutant p53, and BMH-21–induced RPA194 degradation exposes mutant p53 for its ubiquitination. A and B, RPA194 interacts with WT p53 in Capan-2 cells, truncated p53 (Δp53) in AsPC-1, and mutant p53 R248W in MIA PaCa-2 cells. Cells were treated with BMH-21 (2 μmol/L) or BMH-21 + MG132 (10 μmol/L) for 24 hours. A, RPA194 immunoprecipitated samples were subjected to WB analysis using p53 (HRP-conjugated) antibody (top). IgG antibody was used as the isotype control for IP. Eighty micrograms of protein for Capan-2 and 40 μg of protein for AsPC-1 and MIA PaCa-2 cells were used as inputs. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). B, Effect of BMH-21 on ubiquitination of RPA194 and RPA194 binding partners in pancreatic cancer cells (bottom). RPA194 was immunoprecipitated using the RPA194 antibody, and WB analysis was performed using the anti-ubiquitin antibody. The results indicate that RPA194 or its binding partners are ubiquitinated by BMH-21. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). C, Effect of BMH-21 on the ubiquitination level of RPA194 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. Briefly, untreated, BMH-21, and BMH-21 + MG132–treated samples were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB analysis using the RPA194 antibody. D, Effect of BMH-21 on the ubiquitination level of p53 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. The cells were treated as mentioned in C , and protein lysates were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB using HRP-conjugated p53 antibody. Hundred micrograms of protein for Capan-2 and 40 μg protein for MIA PaCa-2 cells were used as inputs.

    Journal: Molecular Cancer Therapeutics

    Article Title: Ubiquitination of Oncogenic Mutant p53 via Attenuation of Ribosome Biogenesis Machinery Effectively Inhibits Pancreatic Tumor Growth

    doi: 10.1158/1535-7163.MCT-25-0097

    Figure Lengend Snippet: RPA194 interacts with both WT and mutant p53, and BMH-21–induced RPA194 degradation exposes mutant p53 for its ubiquitination. A and B, RPA194 interacts with WT p53 in Capan-2 cells, truncated p53 (Δp53) in AsPC-1, and mutant p53 R248W in MIA PaCa-2 cells. Cells were treated with BMH-21 (2 μmol/L) or BMH-21 + MG132 (10 μmol/L) for 24 hours. A, RPA194 immunoprecipitated samples were subjected to WB analysis using p53 (HRP-conjugated) antibody (top). IgG antibody was used as the isotype control for IP. Eighty micrograms of protein for Capan-2 and 40 μg of protein for AsPC-1 and MIA PaCa-2 cells were used as inputs. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). B, Effect of BMH-21 on ubiquitination of RPA194 and RPA194 binding partners in pancreatic cancer cells (bottom). RPA194 was immunoprecipitated using the RPA194 antibody, and WB analysis was performed using the anti-ubiquitin antibody. The results indicate that RPA194 or its binding partners are ubiquitinated by BMH-21. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). C, Effect of BMH-21 on the ubiquitination level of RPA194 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. Briefly, untreated, BMH-21, and BMH-21 + MG132–treated samples were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB analysis using the RPA194 antibody. D, Effect of BMH-21 on the ubiquitination level of p53 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. The cells were treated as mentioned in C , and protein lysates were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB using HRP-conjugated p53 antibody. Hundred micrograms of protein for Capan-2 and 40 μg protein for MIA PaCa-2 cells were used as inputs.

    Article Snippet: Capan-2, AsPC-1, MIA PaCa-2, HPAF-II, BxPC-3, PANC-1, and HPNE were obtained from ATCC.

    Techniques: Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation, Control, Binding Assay, Incubation

    Effect of BMH-21 on pancreatic tumor growth in the orthotopic xenograft mouse models. A and B, Schematic diagram depicting the experimental plan of the mouse xenograft study to investigate the therapeutic efficacy of BMH-21 against pancreatic cancer. Luciferase-labeled MIA PaCa-2 and AsPC-1 cells (1 × 10 6 ) were implanted in the pancreas of athymic nude mice ( n = 8/group). The vehicle and BMH-21 (25 mg/kg body weight) treatments started at 4–6 days after implantation of pancreatic cancer cells. Bioluminescence imaging of all the mice was performed at indicated days. C, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the MIA PaCa-2 luciferase xenograft at various time points. D and E, The average radiance (photons/second/cm 2 /sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the MIA PaCa-2 luciferase xenograft model. F, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the AsPC-1 luciferase xenograft at various time points. G and H, The average radiance (photons/second/cm 2 /sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the AsPC-1 luciferase xenograft model. I, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the MIA PaCa-2 luciferase xenograft mouse model. J, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the AsPC-1 luciferase xenograft mouse model. K, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated groups in MIA PaCa-2 luciferase xenograft mice. L, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated AsPC-1 luciferase xenograft mice. Tumor volume was calculated using the ellipsoid formula [0.5 × (W 2 × L)], where W and L represent the shortest and longest tumor diameters, respectively. M, Representative IHC staining images showing the expression of RPA194, Ki-67, PCNA, and mutant p53 in excised MIA PaCa-2 luciferase cell–derived xenograft tumors of vehicle-treated and BMH-21–treated mice. Magnification: 20×; scale bar, 50 μm. N, The bar graphs represent the quantification of IHC staining of RPA194, PCNA, Ki-67, and mutant p53 in pancreatic tumors excised from mice orthotopically xenografted with MIA PaCa-2 luciferase cells represented in M . The quantification of IHC staining was done using ImageJ software. Values in bar graphs represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P = 0.0003; ****, P ≤ 0.0001.

    Journal: Molecular Cancer Therapeutics

    Article Title: Ubiquitination of Oncogenic Mutant p53 via Attenuation of Ribosome Biogenesis Machinery Effectively Inhibits Pancreatic Tumor Growth

    doi: 10.1158/1535-7163.MCT-25-0097

    Figure Lengend Snippet: Effect of BMH-21 on pancreatic tumor growth in the orthotopic xenograft mouse models. A and B, Schematic diagram depicting the experimental plan of the mouse xenograft study to investigate the therapeutic efficacy of BMH-21 against pancreatic cancer. Luciferase-labeled MIA PaCa-2 and AsPC-1 cells (1 × 10 6 ) were implanted in the pancreas of athymic nude mice ( n = 8/group). The vehicle and BMH-21 (25 mg/kg body weight) treatments started at 4–6 days after implantation of pancreatic cancer cells. Bioluminescence imaging of all the mice was performed at indicated days. C, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the MIA PaCa-2 luciferase xenograft at various time points. D and E, The average radiance (photons/second/cm 2 /sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the MIA PaCa-2 luciferase xenograft model. F, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the AsPC-1 luciferase xenograft at various time points. G and H, The average radiance (photons/second/cm 2 /sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the AsPC-1 luciferase xenograft model. I, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the MIA PaCa-2 luciferase xenograft mouse model. J, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the AsPC-1 luciferase xenograft mouse model. K, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated groups in MIA PaCa-2 luciferase xenograft mice. L, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated AsPC-1 luciferase xenograft mice. Tumor volume was calculated using the ellipsoid formula [0.5 × (W 2 × L)], where W and L represent the shortest and longest tumor diameters, respectively. M, Representative IHC staining images showing the expression of RPA194, Ki-67, PCNA, and mutant p53 in excised MIA PaCa-2 luciferase cell–derived xenograft tumors of vehicle-treated and BMH-21–treated mice. Magnification: 20×; scale bar, 50 μm. N, The bar graphs represent the quantification of IHC staining of RPA194, PCNA, Ki-67, and mutant p53 in pancreatic tumors excised from mice orthotopically xenografted with MIA PaCa-2 luciferase cells represented in M . The quantification of IHC staining was done using ImageJ software. Values in bar graphs represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P = 0.0003; ****, P ≤ 0.0001.

    Article Snippet: Capan-2, AsPC-1, MIA PaCa-2, HPAF-II, BxPC-3, PANC-1, and HPNE were obtained from ATCC.

    Techniques: Drug discovery, Luciferase, Labeling, Imaging, Immunohistochemistry, Expressing, Mutagenesis, Derivative Assay, Software

    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression in AsPC-1 cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.

    Journal: Research

    Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

    doi: 10.34133/research.1105

    Figure Lengend Snippet: Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression in AsPC-1 cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.

    Article Snippet: Pan02 mouse pancreatic cancer cells (Cytion, 300501), AsPC-1 human pancreatic cancer cells (American Type Culture Collection [ATCC], CRL-1682), and Jurkat immortalized human T lymphocytes (ATCC, TIB-152) were cultured in RPMI 1640 medium (Thermo Fisher, 11875093) supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific, FB12999102) and 1% (v/v) 10,000 U/ml penicillin–streptomycin (Gibco, 16140122).

    Techniques: Binding Assay, Electroporation, Membrane, Flow Cytometry, Control, Expressing, Imaging, Cell Characterization

    In vitro assay for longitudinal combinatorial treatment evaluation. (A) In vitro multicellular tumor spheroid (MCTS) assay to assess the treatment response to electroporation and CAR T-cell therapy. (1) MCTSs were formed within a low-adherent U-bottom 96-well plate and (2) then moved to a 4-well rectangular plate with low-conductivity buffer to (3) deliver electroporation via parallel-plate electrodes. (4) The MCTSs were immediately moved back into the original U-bottom well, where (5) adjuvant CAR T-cell therapy or sham was delivered. (B) Live (green) and dead (red) imaging of AsPC-1 MCTSs at 3 and 72 h after treatment at different electric field magnitudes; the scale bar is 1 mm. Normalized absorbance for the XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay at (C) 3 and (D) 72 h post-treatment at different electric fields; one-way ANOVA with Tukey’s post hoc and correction; mean ± SD; n = 3. (E) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs over time and across different electric field intensities. (F) Normalized green fluorescent protein (GFP) intensity and (G) MCTS area over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); multiple one-sample Wilcoxon signed-ranked tests between that timepoint and the initial zero timepoint ( # P < 0.05); n = 3.

    Journal: Research

    Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

    doi: 10.34133/research.1105

    Figure Lengend Snippet: In vitro assay for longitudinal combinatorial treatment evaluation. (A) In vitro multicellular tumor spheroid (MCTS) assay to assess the treatment response to electroporation and CAR T-cell therapy. (1) MCTSs were formed within a low-adherent U-bottom 96-well plate and (2) then moved to a 4-well rectangular plate with low-conductivity buffer to (3) deliver electroporation via parallel-plate electrodes. (4) The MCTSs were immediately moved back into the original U-bottom well, where (5) adjuvant CAR T-cell therapy or sham was delivered. (B) Live (green) and dead (red) imaging of AsPC-1 MCTSs at 3 and 72 h after treatment at different electric field magnitudes; the scale bar is 1 mm. Normalized absorbance for the XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay at (C) 3 and (D) 72 h post-treatment at different electric fields; one-way ANOVA with Tukey’s post hoc and correction; mean ± SD; n = 3. (E) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs over time and across different electric field intensities. (F) Normalized green fluorescent protein (GFP) intensity and (G) MCTS area over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); multiple one-sample Wilcoxon signed-ranked tests between that timepoint and the initial zero timepoint ( # P < 0.05); n = 3.

    Article Snippet: Pan02 mouse pancreatic cancer cells (Cytion, 300501), AsPC-1 human pancreatic cancer cells (American Type Culture Collection [ATCC], CRL-1682), and Jurkat immortalized human T lymphocytes (ATCC, TIB-152) were cultured in RPMI 1640 medium (Thermo Fisher, 11875093) supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific, FB12999102) and 1% (v/v) 10,000 U/ml penicillin–streptomycin (Gibco, 16140122).

    Techniques: In Vitro, Electroporation, Adjuvant, Imaging

    In vitro evaluation of anti-tumor efficacy and infiltration following IRE and CAR T-cell therapy. (A) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs, (B) deep-red intensity of CellTracker-stained CAR T cells, and (C) merged images over time for the CAR-T-cell-only and combinatorial treatments (both). (D) Measured MCTS area and (E) normalized eGFP intensity over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); n ≥ 3. (F) Comparison of deep-red intensity within the tumor spheroid over time; 2-tailed t tests between groups at each timepoint ( # P < 0.05; ## P < 0.01); n ≥ 3.

    Journal: Research

    Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

    doi: 10.34133/research.1105

    Figure Lengend Snippet: In vitro evaluation of anti-tumor efficacy and infiltration following IRE and CAR T-cell therapy. (A) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs, (B) deep-red intensity of CellTracker-stained CAR T cells, and (C) merged images over time for the CAR-T-cell-only and combinatorial treatments (both). (D) Measured MCTS area and (E) normalized eGFP intensity over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); n ≥ 3. (F) Comparison of deep-red intensity within the tumor spheroid over time; 2-tailed t tests between groups at each timepoint ( # P < 0.05; ## P < 0.01); n ≥ 3.

    Article Snippet: Pan02 mouse pancreatic cancer cells (Cytion, 300501), AsPC-1 human pancreatic cancer cells (American Type Culture Collection [ATCC], CRL-1682), and Jurkat immortalized human T lymphocytes (ATCC, TIB-152) were cultured in RPMI 1640 medium (Thermo Fisher, 11875093) supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific, FB12999102) and 1% (v/v) 10,000 U/ml penicillin–streptomycin (Gibco, 16140122).

    Techniques: In Vitro, Staining, Comparison

    Subcutaneous mouse model of human pancreatic cancer for comparing tumor response and survival following combinatorial IRE and CAR T-cell treatment. (A) Schematic of the treatment timeline: NSG mice were inoculated with MSLN + AsPC-1 cells on day 0. Mice received IRE followed by peritumoral CAR T-cell injection on day 25 and were tracked for 35 d until day 60. (B) Pre- and post-treatment IVIS imaging verifies successful peritumoral FLuc + αMLSN CAR T-cell injection. (C) Relative rodent weight over time post-treatment; mean ± standard error of the mean (SEM); n = 24. (D) Representative images of rodents over the post-treatment tracking period (blanks for mice sacrificed). (E) Tumor measurements from inoculation for all mice. (F) The average measured tumor volume from inoculation; mean ± SEM; n = 6 (the sample size drops as mice reach the tumor size endpoint). (G) Progression-free survival and (H) overall survival for each group; Kaplan–Meier with Bonferroni multiple comparisons; n = 6 (* P < 0.05; ** P < 0.01).

    Journal: Research

    Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

    doi: 10.34133/research.1105

    Figure Lengend Snippet: Subcutaneous mouse model of human pancreatic cancer for comparing tumor response and survival following combinatorial IRE and CAR T-cell treatment. (A) Schematic of the treatment timeline: NSG mice were inoculated with MSLN + AsPC-1 cells on day 0. Mice received IRE followed by peritumoral CAR T-cell injection on day 25 and were tracked for 35 d until day 60. (B) Pre- and post-treatment IVIS imaging verifies successful peritumoral FLuc + αMLSN CAR T-cell injection. (C) Relative rodent weight over time post-treatment; mean ± standard error of the mean (SEM); n = 24. (D) Representative images of rodents over the post-treatment tracking period (blanks for mice sacrificed). (E) Tumor measurements from inoculation for all mice. (F) The average measured tumor volume from inoculation; mean ± SEM; n = 6 (the sample size drops as mice reach the tumor size endpoint). (G) Progression-free survival and (H) overall survival for each group; Kaplan–Meier with Bonferroni multiple comparisons; n = 6 (* P < 0.05; ** P < 0.01).

    Article Snippet: Pan02 mouse pancreatic cancer cells (Cytion, 300501), AsPC-1 human pancreatic cancer cells (American Type Culture Collection [ATCC], CRL-1682), and Jurkat immortalized human T lymphocytes (ATCC, TIB-152) were cultured in RPMI 1640 medium (Thermo Fisher, 11875093) supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific, FB12999102) and 1% (v/v) 10,000 U/ml penicillin–streptomycin (Gibco, 16140122).

    Techniques: Injection, Imaging

    Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression in AsPC-1 cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.

    Journal: Research

    Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

    doi: 10.34133/research.1105

    Figure Lengend Snippet: Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression in AsPC-1 cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.

    Article Snippet: Pan02 mouse pancreatic cancer cells (Cytion, 300501), AsPC-1 human pancreatic cancer cells (American Type Culture Collection [ATCC], CRL-1682), and Jurkat immortalized human T lymphocytes (ATCC, TIB-152) were cultured in RPMI 1640 medium (Thermo Fisher, 11875093) supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific, FB12999102) and 1% (v/v) 10,000 U/ml penicillin–streptomycin (Gibco, 16140122).

    Techniques: Binding Assay, Electroporation, Membrane, Flow Cytometry, Control, Expressing, Imaging, Cell Characterization

    In vitro assay for longitudinal combinatorial treatment evaluation. (A) In vitro multicellular tumor spheroid (MCTS) assay to assess the treatment response to electroporation and CAR T-cell therapy. (1) MCTSs were formed within a low-adherent U-bottom 96-well plate and (2) then moved to a 4-well rectangular plate with low-conductivity buffer to (3) deliver electroporation via parallel-plate electrodes. (4) The MCTSs were immediately moved back into the original U-bottom well, where (5) adjuvant CAR T-cell therapy or sham was delivered. (B) Live (green) and dead (red) imaging of AsPC-1 MCTSs at 3 and 72 h after treatment at different electric field magnitudes; the scale bar is 1 mm. Normalized absorbance for the XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay at (C) 3 and (D) 72 h post-treatment at different electric fields; one-way ANOVA with Tukey’s post hoc and correction; mean ± SD; n = 3. (E) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs over time and across different electric field intensities. (F) Normalized green fluorescent protein (GFP) intensity and (G) MCTS area over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); multiple one-sample Wilcoxon signed-ranked tests between that timepoint and the initial zero timepoint ( # P < 0.05); n = 3.

    Journal: Research

    Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

    doi: 10.34133/research.1105

    Figure Lengend Snippet: In vitro assay for longitudinal combinatorial treatment evaluation. (A) In vitro multicellular tumor spheroid (MCTS) assay to assess the treatment response to electroporation and CAR T-cell therapy. (1) MCTSs were formed within a low-adherent U-bottom 96-well plate and (2) then moved to a 4-well rectangular plate with low-conductivity buffer to (3) deliver electroporation via parallel-plate electrodes. (4) The MCTSs were immediately moved back into the original U-bottom well, where (5) adjuvant CAR T-cell therapy or sham was delivered. (B) Live (green) and dead (red) imaging of AsPC-1 MCTSs at 3 and 72 h after treatment at different electric field magnitudes; the scale bar is 1 mm. Normalized absorbance for the XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay at (C) 3 and (D) 72 h post-treatment at different electric fields; one-way ANOVA with Tukey’s post hoc and correction; mean ± SD; n = 3. (E) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs over time and across different electric field intensities. (F) Normalized green fluorescent protein (GFP) intensity and (G) MCTS area over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); multiple one-sample Wilcoxon signed-ranked tests between that timepoint and the initial zero timepoint ( # P < 0.05); n = 3.

    Article Snippet: Pan02 mouse pancreatic cancer cells (Cytion, 300501), AsPC-1 human pancreatic cancer cells (American Type Culture Collection [ATCC], CRL-1682), and Jurkat immortalized human T lymphocytes (ATCC, TIB-152) were cultured in RPMI 1640 medium (Thermo Fisher, 11875093) supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific, FB12999102) and 1% (v/v) 10,000 U/ml penicillin–streptomycin (Gibco, 16140122).

    Techniques: In Vitro, Electroporation, Adjuvant, Imaging

    In vitro evaluation of anti-tumor efficacy and infiltration following IRE and CAR T-cell therapy. (A) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs, (B) deep-red intensity of CellTracker-stained CAR T cells, and (C) merged images over time for the CAR-T-cell-only and combinatorial treatments (both). (D) Measured MCTS area and (E) normalized eGFP intensity over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); n ≥ 3. (F) Comparison of deep-red intensity within the tumor spheroid over time; 2-tailed t tests between groups at each timepoint ( # P < 0.05; ## P < 0.01); n ≥ 3.

    Journal: Research

    Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

    doi: 10.34133/research.1105

    Figure Lengend Snippet: In vitro evaluation of anti-tumor efficacy and infiltration following IRE and CAR T-cell therapy. (A) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs, (B) deep-red intensity of CellTracker-stained CAR T cells, and (C) merged images over time for the CAR-T-cell-only and combinatorial treatments (both). (D) Measured MCTS area and (E) normalized eGFP intensity over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); n ≥ 3. (F) Comparison of deep-red intensity within the tumor spheroid over time; 2-tailed t tests between groups at each timepoint ( # P < 0.05; ## P < 0.01); n ≥ 3.

    Article Snippet: Pan02 mouse pancreatic cancer cells (Cytion, 300501), AsPC-1 human pancreatic cancer cells (American Type Culture Collection [ATCC], CRL-1682), and Jurkat immortalized human T lymphocytes (ATCC, TIB-152) were cultured in RPMI 1640 medium (Thermo Fisher, 11875093) supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific, FB12999102) and 1% (v/v) 10,000 U/ml penicillin–streptomycin (Gibco, 16140122).

    Techniques: In Vitro, Staining, Comparison

    Subcutaneous mouse model of human pancreatic cancer for comparing tumor response and survival following combinatorial IRE and CAR T-cell treatment. (A) Schematic of the treatment timeline: NSG mice were inoculated with MSLN + AsPC-1 cells on day 0. Mice received IRE followed by peritumoral CAR T-cell injection on day 25 and were tracked for 35 d until day 60. (B) Pre- and post-treatment IVIS imaging verifies successful peritumoral FLuc + αMLSN CAR T-cell injection. (C) Relative rodent weight over time post-treatment; mean ± standard error of the mean (SEM); n = 24. (D) Representative images of rodents over the post-treatment tracking period (blanks for mice sacrificed). (E) Tumor measurements from inoculation for all mice. (F) The average measured tumor volume from inoculation; mean ± SEM; n = 6 (the sample size drops as mice reach the tumor size endpoint). (G) Progression-free survival and (H) overall survival for each group; Kaplan–Meier with Bonferroni multiple comparisons; n = 6 (* P < 0.05; ** P < 0.01).

    Journal: Research

    Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

    doi: 10.34133/research.1105

    Figure Lengend Snippet: Subcutaneous mouse model of human pancreatic cancer for comparing tumor response and survival following combinatorial IRE and CAR T-cell treatment. (A) Schematic of the treatment timeline: NSG mice were inoculated with MSLN + AsPC-1 cells on day 0. Mice received IRE followed by peritumoral CAR T-cell injection on day 25 and were tracked for 35 d until day 60. (B) Pre- and post-treatment IVIS imaging verifies successful peritumoral FLuc + αMLSN CAR T-cell injection. (C) Relative rodent weight over time post-treatment; mean ± standard error of the mean (SEM); n = 24. (D) Representative images of rodents over the post-treatment tracking period (blanks for mice sacrificed). (E) Tumor measurements from inoculation for all mice. (F) The average measured tumor volume from inoculation; mean ± SEM; n = 6 (the sample size drops as mice reach the tumor size endpoint). (G) Progression-free survival and (H) overall survival for each group; Kaplan–Meier with Bonferroni multiple comparisons; n = 6 (* P < 0.05; ** P < 0.01).

    Article Snippet: Pan02 mouse pancreatic cancer cells (Cytion, 300501), AsPC-1 human pancreatic cancer cells (American Type Culture Collection [ATCC], CRL-1682), and Jurkat immortalized human T lymphocytes (ATCC, TIB-152) were cultured in RPMI 1640 medium (Thermo Fisher, 11875093) supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific, FB12999102) and 1% (v/v) 10,000 U/ml penicillin–streptomycin (Gibco, 16140122).

    Techniques: Injection, Imaging