Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD243 , REA495 , 50 , 130-124-440 , PE (APC) , Miltenyi Biotec.

Techniques: Imaging

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Paraquat accumulation in MDR1-transfected HEK293 cells. HEK293 cells were stably transfected with empty vector (EV) or full-length human MDR1 plasmids. (A) Protein expression of MDR1 was detected by Western blot analysis. β-actin was used as a loading control. HEK-EV and MDR1 transfected cells were treated with rhodamine 123 (25 μM) or paraquat (100 μM) for 30 min (uptake period), washed, and then incubated in fresh culture medium for 60 min (efflux period). (B) Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. (C) Intracellular paraquat accumulation was quantified by ELISA and normalized to protein lysate concentrations. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with HEK-EV cells.

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Transfection, Stable Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Expression and function of MDR1 transporter in RPTEC cells. (A) Protein expression of MDR1 in RPTEC cells was detected by Western blot analysis. (B) RPTEC cells were treated with rhodamine 123 (25 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period) and then treated with the culture media with or without PSC833 (2 μM) for 60 min (efflux period). Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. Data are presented as mean ± SE (n = 4). Asterisk (*) represents statistically significant differences (p < 0.05) compared with RPTEC cells treated only with rhodamine 123 (no PSC833).

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Western Blot, Fluorescence

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Paraquat accumulation in RPTEC cells following MDR1 inhibition. RPTEC cells were treated with paraquat (100 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period), washed, and then incubated in fresh culture media with or without PSC833 (2 μM) for 60 min (efflux period). (A) Intracellular paraquat accumulation was quantified by ELISA normalized to protein concentrations of the cellular lysates. (B) Paraquat concentrations in the culture media were quantified by ELISA and compared with levels detected in RPTEC cell lysates. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells treated only with paraquat (no PSC833).

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Paraquat accumulation in RPTEC cells following siRNA knockdown of MDR1. RPTEC cells were transfected with siRNA duplexes targeted against human MDR1. (A) Protein expression of MDR1 at 72 h was assessed by Western blot analysis. (B) RPTEC cells were treated with rhodamine 123 (25 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period), washed, and then incubated in fresh culture media with or without PSC833 (2 μM) for 60 min (efflux period). MDR1 siRNA transfected RPTEC cells were treated with rhodamine (25 μM) for 30 min (uptake period), washed, and then incubated in fresh culture media for 60 min (efflux period). Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. (C) RPTEC cells were treated as described in (B) using paraquat (100 μM) as a substrate. Intracellular paraquat accumulation was quantified by ELISA and normalized to protein concentrations of the cellular lysates. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells without PSC833 or MDR1 siRNA.

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Knockdown, Transfection, Expressing, Western Blot, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Cytotoxicity of paraquat in RPTEC cells following MDR1 inhibition. RPTEC cells were treated with different concentrations of paraquat (0–25 mM) for 3 h in the presence and absence of the MDR1 inhibitor PSC833 (2 μM) (uptake period) and then incubated in fresh culture media with or without PSC833 for 69 h (efflux period). (A) Cytotoxicity was assessed using the LDH assay and expressed as percent of LDH released into the cell culture media relative to total LDH activity. (B) RPTEC cells were pretreated with vehicle or PSC833 (2 μM) for 2 h and then treated with vehicle or paraquat (100 μM) for 24 h. Protein expression of Ho-1 was semi-quantified by Western blot. β-actin was used as a loading control. Data are presented as mean ± SE (n = 3). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells treated by paraquat (no PSC833).

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Inhibition, Incubation, Lactate Dehydrogenase Assay, Cell Culture, Activity Assay, Expressing, Western Blot, Control

Journal: Frontiers in Physiology

Article Title: Leveraging Mathematical Modeling to Quantify Pharmacokinetic and Pharmacodynamic Pathways: Equivalent Dose Metric

doi: 10.3389/fphys.2019.00616

Figure Lengend Snippet: Doxorubicin and TQR combination studies in the MDA-MB-468 MDR1 cell line. Timecourses of the mean intracellular concentration of doxorubicin with corresponding standard deviations are shown for each treatment condition in (A) . Doxorubicin accumulation increases along with TQR concentrations. Equations (1–3) were fit to the data, and the best-fit models are overlaid on the data (smooth lines) in a. Model parameter fits corresponding to the best-fit models are shown in (B–D) . Similar k EF and k FB vales are observed across all TQR concentrations. There is a trend of decreasing k FE values with increasing TQR concentrations (C) , consistent with MDR1 inhibition by TQR. Cell counts of MDA-MB-468 MDR1 following combination treatment with TQR and doxorubicin are show in panels (E-G) . In each plot, a fixed concentration of doxorubicin is applied with variable TQR concentrations. These counts are fit with Equations (4–6) as described in section Model Fits, and the best-fit model is overlaid on the cell counts [smooth lines in panels (E–G) ]. Error bars represent the 95% CI from six experimental replicates for each treatment condition. Model parameters with corresponding 95% CI are shown in (H–J) as a function of TQR concentration. For each doxorubicin concentration, the death rate ( k d,a and k d,b ) increased with TQR concentration (H,I) .

Article Snippet: To specifically modulate doxorubicin pharmacokinetics, the H2BmRFP-expressing MDA-MB-468 cell line (MDA-MB-468 H2B ) was transduced to express a green fluorescent protein (GFP)-tagged MDR1 protein (ABCB1 gene, Origene Technologies, Rockville, MD).

Techniques: Concentration Assay, Inhibition

Journal: Frontiers in Physiology

Article Title: Leveraging Mathematical Modeling to Quantify Pharmacokinetic and Pharmacodynamic Pathways: Equivalent Dose Metric

doi: 10.3389/fphys.2019.00616

Figure Lengend Snippet: Treatment response in MDA-MB-468 MDR1 (left column) and SUM-149PT (right column) cell lines under doxorubicin monotherapy. The top row [panels (A,E) ] shows cell counts over time from treatment response studies for each cell line. For these studies, cells were treated with a fixed concentration of doxorubicin for 24 h. These counts are fit to Equations (4–6) as described in section Model Fits, and the best-fit model is overlaid on the cell counts [smooth lines in (A,E) ]. Error bars represent the 95% CI from six experimental replicates for each treatment condition. Model parameters with corresponding 95% CI are shown in the bottom three rows as a function of doxorubicin concentration. Panels (B–D) show fits from the MDA-MB-468 MDR1 experiments, and panels (F–H) show fits from the SUM-149PT experiments. For each doxorubicin concentration for each cell line, the death rate ( k d,a and k d,b ) increased with increasing doxorubicin concentrations.

Article Snippet: To specifically modulate doxorubicin pharmacokinetics, the H2BmRFP-expressing MDA-MB-468 cell line (MDA-MB-468 H2B ) was transduced to express a green fluorescent protein (GFP)-tagged MDR1 protein (ABCB1 gene, Origene Technologies, Rockville, MD).

Techniques: Concentration Assay

Journal: Frontiers in Physiology

Article Title: Leveraging Mathematical Modeling to Quantify Pharmacokinetic and Pharmacodynamic Pathways: Equivalent Dose Metric

doi: 10.3389/fphys.2019.00616

Figure Lengend Snippet: Leveraging equivalent dose to estimate the effect of TQR in the MDA-MB-468 MDR1 cell line. The equivalent dose for each doxorubicin monotherapy condition was first calculated with the PK model parameters measured in the doxorubicin uptake studies. The equivalent dose statistic was then estimated for each co-treatment condition by matching treatment response parameters from co-treatment conditions to those from doxorubicin monotherapy conditions. Parameter values from all doxorubicin monotherapy and co-treatment conditions are plotted as a function of equivalent dose (A-C) . A subset of responses from doxorubicin monotherapy and co-treatment conditions are color-coded to their estimated equivalent dose (D–F) . Similar dynamics are observed with similarly-colored data, demonstrating the efficacy of the parameter matching in comparing treatment response timecourses. As TQR impairs the function of the MDR1 pump, we hypothesized the effect of TQR is limited to the k FE parameter (G) . With estimates of equivalent dose for all treatment conditions, the k FE value for each TQR concentration was estimated with the optimization routine summarized by Equation (7) (H) . These values, calculated with treatment response data, agree well with direct measurements of k FE reported in . We note the large confidence intervals are a result of the optimization approach, in which the value (1/ k FE ) was optimized.

Article Snippet: To specifically modulate doxorubicin pharmacokinetics, the H2BmRFP-expressing MDA-MB-468 cell line (MDA-MB-468 H2B ) was transduced to express a green fluorescent protein (GFP)-tagged MDR1 protein (ABCB1 gene, Origene Technologies, Rockville, MD).

Techniques: Concentration Assay

Journal: Frontiers in Physiology

Article Title: Leveraging Mathematical Modeling to Quantify Pharmacokinetic and Pharmacodynamic Pathways: Equivalent Dose Metric

doi: 10.3389/fphys.2019.00616

Figure Lengend Snippet: Comparison of MDA-MB-468 H2B and MDA-MB-468 MDR1 cell lines using equivalent dose. The intracellular doxorubicin concentration with 95% CI for each cell line is shown in (A) . The MDA-MB-468 H2B line demonstrates increased intracellular accumulation of doxorubicin relative to the MDA-MB-468 MDR1 line. Equations (1–3) are fit to the doxorubicin uptake data, and the best-fit models are overlaid on the data in a (smooth line). The corresponding parameters with 95% CI are shown in (B–D) . The MDA-MB-468 H2B data are shown in red, and the MDA-MB-468 MDR1 data are shown in blue. Notably, the efflux of drug from the MDA-MB-468 MDR1 ( k FE ) line is significantly greater than the corresponding rate in the MDA-MB-468 H2B line ( p < 0.05). Treatment response is traditionally summarized by cell survival and plotted against applied drug concentration. The cell count relative to control for each cell line is shown as a function of extracellular doxorubicin concentration and equivalent dose in (E,F) , respectively. While a significant difference is observed when comparing these cell lines via EC 50 calculated with the extracellular doxorubicin concentration, no significant difference is observed when comparing the EC 50 statistic derived from the equivalent dose. The equivalent dose can account for the differing pharmacokinetic properties to reveal similar doxorubicin pharmacodynamics in these cell lines.

Article Snippet: To specifically modulate doxorubicin pharmacokinetics, the H2BmRFP-expressing MDA-MB-468 cell line (MDA-MB-468 H2B ) was transduced to express a green fluorescent protein (GFP)-tagged MDR1 protein (ABCB1 gene, Origene Technologies, Rockville, MD).

Techniques: Concentration Assay, Cell Counting, Derivative Assay