Journal: Pharmaceutics

Article Title: Boosting the Activity of Melanoma-Targeting CAR-T Cells in the Presence of Citrate by the Application of Gluconate

doi: 10.3390/pharmaceutics18050551

Figure Lengend Snippet: CAR-T cells maintain robust functionality in the presence of gluconate. ( A ) Schematic outline of the aCEA-28ζ CAR construct. ( B ) CAR-T cells were generated via retroviral transduction of bulk T cells obtained from healthy human donors. CAR expression on aCEA-28ζ CAR-T cells was determined prior to and after MACS. One representative donor out of three is depicted (three biological replicates). ( C ) CAR-triggered secretion of IL-2 and IFNγ from aCEA-28ζ CAR-T cells stimulated with BxPC-3 cells or left unstimulated (w/o) after 48 h, as measured by ELISA. At the beginning of stimulation, either gluconate was added at a concentration of 100 µM or PBS was, serving as a negative control. Data represent means ± SEM of three donors (three biological replicates), p values were calculated by two-way-ANOVA, ns indicates not significant. ( D ) Cytotoxicity of aCEA-28ζ CAR-T cells after a 24 h co-culture with BxPC-3 cells and 293T cells at the depicted effector to target cell ratios. At the beginning of co-culture, either gluconate was added at a concentration of 100 µM or PBS was, serving as a negative control. Data represent means ± SEM of three donors (three biological replicates), p values were calculated by two-way-ANOVA, ns indicates not significant. ( E ) In vitro “stress test”. Anti-CEA-28ζ CAR-T cells (1 × 10 5 CAR-T cells) underwent three rounds (R1-R3) of incubation with GFP-labeled CEA + BxPC-3 cells (1 × 10 5 tumor cells at the start of each round). After each round, CAR-T cells (live CD3 + CAR cells) (left panel) and BxPC-3 cells (right panel) were enumerated by flow cytometry using counting beads. At the beginning of each round, either gluconate was added at a concentration of 100 µM (CART + gluconate) or PBS was (CART + PBS). Further controls comprised PBS alone without CAR-T cells (PBS) and gluconate alone without CAR-T cells (gluconate). Data represent means ± SEM of three donors (three biological replicates), p values were calculated by two-way-ANOVA, ns indicates not significant.

Article Snippet: BxPC-3 cells (ATCC CRL-1420), 293T cells (ATCC CRL-3216 American Type Culture Collection, Manassas, VA, USA), A375 cells, A375M cells, and IGR39 cells were cultured in DMEM, 1% ( w / v ) GlutaMAX (Gibco, ThermoFisher), 100 IU/mL penicillin, 100 μg/mL streptomycin (Pan-Biotech), and 10% ( v / v ) heat-inactivated FCS (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Construct, Generated, Retroviral, Transduction, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Negative Control, Co-Culture Assay, In Vitro, Incubation, Labeling, Flow Cytometry

Journal: ACS Omega

Article Title: Pyrazole–Cyclotriphosphazene Hybrids: Synthesis, Structural Insights, and Cytotoxic Effects against Pancreatic Cancer Cells

doi: 10.1021/acsomega.5c11955

Figure Lengend Snippet: (a) MTT assay (Data represent mean values of four technical and three biological replicates, with error bars indicating standard deviation. Statistical analysis was performed using two-way ANOVA with Dunnett’s post-test (ns p > 0.5, *** p < 0.001, **** p < 0.0001). (b) IC 50 values calculated from MTT assay, and ( c) colony formation assay in PANC-1 and MIA-PaCa-2 cells following 24-h treatment with compound 3a .

Article Snippet: PANC-1 (CRL-1469) and MIA PaCa-2 (CRL-1420) cells were purchased from American Type Culture Collection (ATCC).

Techniques: MTT Assay, Standard Deviation, Colony Assay

Journal: ACS Omega

Article Title: Pyrazole–Cyclotriphosphazene Hybrids: Synthesis, Structural Insights, and Cytotoxic Effects against Pancreatic Cancer Cells

doi: 10.1021/acsomega.5c11955

Figure Lengend Snippet: Fluorescence microscopy images of MIA-PaCa-2 cells after 24-h treatment with 3a , staining of H 2 -DCFDA (green, ROS), MitoTracker (red, mitochondria), and DAPI (blue, nuclei), along with their merged image. Scale bar is 150 μm.

Article Snippet: PANC-1 (CRL-1469) and MIA PaCa-2 (CRL-1420) cells were purchased from American Type Culture Collection (ATCC).

Techniques: Fluorescence, Microscopy, Staining

Journal: ACS Omega

Article Title: Pyrazole–Cyclotriphosphazene Hybrids: Synthesis, Structural Insights, and Cytotoxic Effects against Pancreatic Cancer Cells

doi: 10.1021/acsomega.5c11955

Figure Lengend Snippet: Effects of 4a-I on (a) cell viability and (b) colony formation in PANC-1 and MIA-PaCa-2 cells following 24-h treatment. Data represents the MTT assay mean values ± standard deviation from four technical and three biological replicates. Two-way ANOVA determined statistical significance with Dunnett’s posthoc test (ns: p > 0.05).

Article Snippet: PANC-1 (CRL-1469) and MIA PaCa-2 (CRL-1420) cells were purchased from American Type Culture Collection (ATCC).

Techniques: MTT Assay, Standard Deviation

Journal: ACS Omega

Article Title: Pyrazole–Cyclotriphosphazene Hybrids: Synthesis, Structural Insights, and Cytotoxic Effects against Pancreatic Cancer Cells

doi: 10.1021/acsomega.5c11955

Figure Lengend Snippet: Effects of 5a-I on cell viability in PANC-1 and MIA-PaCa-2 cells following 24-h treatment. Data represents the MTT assay mean values ± standard deviation from four technical and three biological replicates. Two-way ANOVA determined statistical significance with Dunnett’s posthoc test (ns: p > 0.05).

Article Snippet: PANC-1 (CRL-1469) and MIA PaCa-2 (CRL-1420) cells were purchased from American Type Culture Collection (ATCC).

Techniques: MTT Assay, Standard Deviation

Journal: ACS Omega

Article Title: Pyrazole–Cyclotriphosphazene Hybrids: Synthesis, Structural Insights, and Cytotoxic Effects against Pancreatic Cancer Cells

doi: 10.1021/acsomega.5c11955

Figure Lengend Snippet: Effects of 3b on (a) cell viability and (b) colony formation in PANC-1 and MIA-PaCa-2 cells following 24-h treatment. Data represents the MTT assay mean values ± standard deviation from four technical and three biological replicates. Two-way ANOVA determined statistical significance with Dunnett’s posthoc test (ns: p > 0.05).

Article Snippet: PANC-1 (CRL-1469) and MIA PaCa-2 (CRL-1420) cells were purchased from American Type Culture Collection (ATCC).

Techniques: MTT Assay, Standard Deviation

Journal: ACS Omega

Article Title: Pyrazole–Cyclotriphosphazene Hybrids: Synthesis, Structural Insights, and Cytotoxic Effects against Pancreatic Cancer Cells

doi: 10.1021/acsomega.5c11955

Figure Lengend Snippet: Effects of 4b-I on (a) cell viability and (b) colony formation in PANC-1 and MIA-PaCa-2 cells following 24-h treatment. Data represents the MTT assay mean values ± standard deviation from four technical and three biological replicates. Two-way ANOVA determined statistical significance with Dunnett’s posthoc test (ns: p > 0.05).

Article Snippet: PANC-1 (CRL-1469) and MIA PaCa-2 (CRL-1420) cells were purchased from American Type Culture Collection (ATCC).

Techniques: MTT Assay, Standard Deviation

Journal: ACS Omega

Article Title: Pyrazole–Cyclotriphosphazene Hybrids: Synthesis, Structural Insights, and Cytotoxic Effects against Pancreatic Cancer Cells

doi: 10.1021/acsomega.5c11955

Figure Lengend Snippet: Effects of 5b-I on (a) cell viability and (b) colony formation in PANC-1 and MIA-PaCa-2 cells following 24-h treatment. Data represent the MTT assay mean values ± standard deviation from four technical and three biological replicates. Two-way ANOVA determined statistical significance with Dunnett’s posthoc test (ns: p > 0.05).

Article Snippet: PANC-1 (CRL-1469) and MIA PaCa-2 (CRL-1420) cells were purchased from American Type Culture Collection (ATCC).

Techniques: MTT Assay, Standard Deviation