Article Figures & Data
Figures
- FIGURE 1.
Decreased liver retention of 64Cu-ATSM or -PTSM in ArKO and ArKO liver tumor–bearing mice. Male wild-type (n = 5), ArKO (n = 5), and ArKO mice with liver tumors (n = 3) were injected with 3.7 MBq of 64Cu-PTSM (A) or 3.7 MBq of 64Cu-ATSM (B). After 1-h uptake, livers were harvested and weighed, and activity was determined. Results are expressed as mean ± SE.
- FIGURE 2.
PET/CT shows decreased retention of 64Cu-ATSM in ArKO tumor–bearing mice. Whole-body PET/CT images were acquired for wild-type (A) and ArKO tumor–bearing (B) mice 1 h after the intravenous injection of 3.7 MBq (100 μCi) of 64Cu-ATSM. Images show protuberant abdomen in ArKO tumor–bearing mouse and clearly decreased activity in right side of enlarged liver (circles).
- FIGURE 3.
Mdr1a and Mdr1b expression is elevated in liver of ArKO mice and ArKO mice with liver tumor (A and B). Mdr1a and Mdr1b mRNA levels were quantified by qRT-PCR using glyceraldehyde-3-phosphate dehydrogenase as a reference gene. Wild-type liver had lowest levels of proteins, correlating with higher 64Cu-ATSM retention. (C) Western blot analysis of Mdr1 protein expression. Anti-MDR1 antibody does not distinguish between Mdr1a and Mdr1b. Approximately 170-kD MDR1 band is highest in ArKO liver tumor. (D) Quantitative analysis of Western blot.
- FIGURE 4.
PET/CT shows decreased retention of 64Cu-ATSM in MES-SA/Dx5 xenografts. One hour after intravenous injection of 3.7 MBq (100 μCi) of 64Cu-ATSM, whole-body PET/CT images were acquired of mice bearing MES-SA xenografts about right shoulder and MES-SA/Dx5 xenografts on left. Axial sections are shown. MES-SA/Dx5 xenograft clearly shows less activity. Results are representative of studies on 3 different mice.
- FIGURE 5.
Knockdown of MDR1 by siRNA decreases protein levels. Protein extracts were prepared from control transfected–MES-SA/Dx5 cells or MDR1 siRNA–transfected cells 24 h after start of transfection. Relative levels of MDR1 protein were assessed by Western blotting. Data are representative of 3 independent experiments.
- FIGURE 6.
MES-SA/Dx5 cells retain lower levels of 64Cu-ATSM/PTSM and show more rapid efflux; knockdown of MDR1 protein leads to increased retention and slower efflux. After transfection with control or MDR1 siRNA duplexes, cells were incubated with radiotracers for up to 1 h (A and C), and medium was removed. In efflux experiments, cells were loaded with radiotracer for 15 min. Medium was then replaced with cold medium, and incubation continued for times up to 2 h (B and D). Cells were quickly washed and lysed in buffered detergent solution. Activity in cell lysates was determined and expressed as counts per milligram of protein. (A and B) Uptake and efflux of 64Cu-ATSM. (C and D) Uptake and efflux of 64Cu-PTSM. Results are expressed as mean ± SEM. Data represent average of 3 independent experiments with triplicates in each group. Lines in graph were hand-drawn.
Tables
Gene Primer sequence siRNA duplex sequence MDR1(ABCB1) forward 5′-TCGTAGGAGTGTCCGTGGAT-3′ MDR1 (ABCB1) reverse 5′-CATTGGCGAGCCTGGTAG-3′ Mdr1a (Abcb1a) forward 5′-TACGCCTACTATTACACCG-3′ Mdr1a (Abcb1a) reverse 5′-CCAGCCTATCTCCTGATT-3′ Mdr1b (Abcb1b) forward 5′-TCGCTTGTCTACAGTTCG-3′ Mdr1b (Abcb1b) reverse 5′-GCATTATTTCCTGGTTCA-3′ 18S forward 5′-GGAATTGACGGAAGGGCACCACC-3′ 18S reverse 5′-GTGCAGCCCCGGACATCTAAGG-3′ GAPDH forward 5′-CTGCCATTTGCAGTGGCAAAGTGG-3′ GAPDH reverse 5′-TTGTCATGGATGACCTTGGCCAGG-3′ MDR1 siRNA 5′-GAGUGGGCACAAACCAGAUAAUAUU-3′ 5′-AAUAUUAUCUGGUUUGUGCCCACUC-3′ 5′-CCAUAAAUGUAAGGUUUCUACGGGA-3′ 5′-UCCCGUAGAAACCUUACAUUUAUGG-3′ 5′-GCUCGCCAAUGAUGCUGCUCAAGUU-3′ 5′-AACUUGAGCAGCAUCAUUGGCGAGC-3′ Lamin A/C control siRNA 5′-CUGGACUUCCAGAAGAACAdTdT-3′ 5′-UGUUCUUCUGGAAGUCCAGdTdT-3′ GADPH = glyceraldehyde-3-phosphate dehydrogenase.
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