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. 2012 Jul 11;103(9):1730–1736. doi: 10.1111/j.1349-7006.2012.02352.x

In vivo imaging of proteasome inhibition using a proteasome‐sensitive fluorescent reporter

Isao Momose 1,✉, Daisuke Tatsuda 1, Shun‐ichi Ohba 1, Tohru Masuda 1, Daishiro Ikeda 1, Akio Nomoto 1
PMCID: PMC7659265  PMID: 22676179

Abstract

A proteasome degrades numerous regulatory proteins that are critical for tumor growth and is therefore recognized as a promising anticancer target. Determining proteasome activity in the tumors of mice bearing xenografts is essential for the development of novel proteasome inhibitors. We developed a system for in vivo imaging of proteasome inhibition in the tumors of living mice, using a proteasome‐sensitive fluorescent reporter, ZsProSensor‐1. This reporter consists of a green fluorescent protein, ZsGreen, fused to mouse ornithine decarboxylase, which is degraded by the proteasome without being ubiquitinated. In stably transfected cells expressing ZsProSensor‐1, the fluorescent reporter was rapidly degraded under steady‐state conditions, whereas it was stabilized in the presence of proteasome inhibitors. Subcutaneous inoculation of the transfected cells into nude mice resulted in tumor formation. When the proteasome inhibitor bortezomib was intravenously administered to mice bearing these tumors, the ZsProSensor‐1 protein accumulated in the tumors and emitted a fluorescent signal in a dose‐dependent manner. Robust fluorescence was sustained for 3 days and then gradually decreased to baseline levels within 15 days. Intravenous administration of bortezomib also showed potent antitumor activity. In contrast, oral administration of bortezomib did not result in fluorescent protein accumulation in tumors or exhibit any antitumor activity. These results indicate that in vivo imaging using the ZsProSensor‐1 fluorescent protein can be used as an indicator of antitumor activity and will be a powerful tool for the development of novel proteasome inhibitors.

A proteasome is an unusually large multi‐enzyme complex that serves as the main pathway for the degradation of intracellular proteins in eukaryotic cells.1, 2 It degrades numerous regulatory proteins, including cyclins, cyclin‐dependent kinase inhibitors (e.g. p21 and p27), tumor suppressors (e.g. p53) and nuclear factor (NF)‐κB inhibitors (e.g. IκB‐α), all of which are critical for tumor growth.3, 4, 5, 6 Proteasome inhibitors can stabilize these regulatory proteins and induce cell cycle arrest, endoplasmic reticulum stress and apoptosis, and, as a result, can limit tumor development.7, 8 Proteasome inhibitors have been recognized as promising candidate anticancer agents.9, 10 Indeed, the proteasome inhibitor bortezomib (also referred to as PS‐341, Velcade11, 12) has been approved for the treatment of multiple myeloma. However, bortezomib has side‐effects, including painful peripheral neuropathy, orthostatic hypotension, pyrexia, cardiac and pulmonary disorders, adverse gastrointestinal events, myelosuppression and thrombocytopenia asthenia.13, 14, 15, 16 These issues emphasize the continued need for the development of novel proteasome inhibitors.

Animal experiments are essential for the development of new proteasome inhibitors. In most cases, the effects of candidate inhibitors on tumor growth are determined after mice are killed. As in vivo imaging is a useful technique for visualizing biological reactions in living animals, a ubiquitin‐luciferase bioluminescence imaging reporter method has been developed for the direct assay of proteasome inhibition in the tumors of living animals.17 The luminescent reporter is a fusion protein containing firefly luciferase and four copies of a mutant ubiquitin (ubiquitin G76V) that resists cleavage by ubiquitin hydrolases. The reporter is rapidly degraded by proteasomes under steady‐state conditions but stabilized in the presence of proteasome inhibitors. Indeed, this ubiquitin‐luciferase reporter has been used to develop and validate proteasome inhibitors in mouse models.18 Fluorescent reporters have also been developed to assess proteasome function in living cells.19, 20 Fluorescent reporters (e.g. green fluorescent protein [GFP]) are useful because they are simple to visualize and require no special reagents (e.g. D‐luciferin). In addition, real‐time imaging of proteasome function becomes feasible and reagent costs can be reduced. However, in vivo imaging of proteasome function using fluorescent reporters in living animals has not been reported to date. Here, we describe an assay for fluorescent in vivo imaging of proteasome function in the tumors of living mice.

Materials and Methods

Materials

Bortezomib was synthesized as described by Adams et al.11 MG‐132 and succinyl‐leucyl‐leucyl‐valyl‐tyrosine‐4‐methylcoumaryl‐7‐amide (Suc‐LLVY‐MCA) were obtained from the Peptide Institute (Osaka, Japan). G418 was purchased from Promega (Madison, WI, USA).

Cell lines and culture conditions

The HEK293 ZsGreen Proteasome Sensor Cell Line, which is a stably transfected human embryo kidney cell line that expresses the ZsProSensor‐1 fusion protein, was obtained from Takara Bio (Tokyo, Japan). The human prostate cancer PC‐3 cell line was obtained from American Type Culture Collection (Rockville, MD, USA). The cells were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (Tissue Culture Biologicals, Tulare, CA, USA), 100 000 U/L penicillin G and 100 mg/L streptomycin. The G418 antibiotic reagent was added to the culture medium at a concentration of 0.2 mg/mL to select for stably transfected cells.

Transfection

PC‐3 cells were transfected with the Proteasome Sensor Vector (Takara Bio) using FuGENE HD (Promega). Stably transfected cell lines were generated by selection with 400 μg/mL G418.

Cytotoxicity

Cells (5 × 103) were incubated with proteasome inhibitors in 96‐well plates for 72 h. Cell viability was determined using a MTT assay.21

Sample preparation for intracellular proteasome activity

To measure intracellular proteasome activity, 5 × 105 cells were cultured in 35‐mm dishes in DMEM for 24 h and then incubated with bortezomib for 8 h. The cells were washed twice with PBS and scraped with a rubber policeman. The cells were then collected by centrifugation at 220g for 10 min and lysed by freezing and thawing in a lysis buffer containing 25 mM Tris‐HCl (pH 7.5), 1 mM DTT, 2 mM ATP and 20% glycerol. Cell debris was removed by centrifugation at 90 000g for 30 min and the supernatants were assayed. To measure proteasome activity in tumors, the tumors were frozen and mechanically disrupted in a ShakeMaster Neo (Bio Medical Science Inc., Tokyo, Japan) in lysis buffer. Tumor debris was removed by centrifugation at 90 000g for 30 min and the supernatants were assayed.

Proteasome activity

To measure proteasome activity, 10 μL of cell or tumor lysate was added to a 96‐well plate along with 90 μL of 50 mM Tris‐HCl buffer (pH 8.0) containing 1 mM DTT, 0.04% SDS and 100 μM Suc‐LLVY‐MCA. The reaction mixture was incubated for 30 min at 37°C. Proteasome activity was measured by monitoring for the increase in fluorescence (excitation, 360 nm; emission, 460 nm) that accompanies the cleavage of 7‐amino‐4‐methylcoumarin from Suc‐LLVY‐MCA using a fluorescence microplate reader (Powerscan HT, DS Pharma Biomedical, Osaka, Japan).22

Fluorescence microscopy and determination of fluorescence intensity

Cells (1 × 105) were incubated in 35‐mm dishes in 2 mL DMEM for 24 h and then incubated for 12 h with 0.1 μg/mL bortezomib or 1 μg/mL MG‐132. Fluorescent proteins were monitored with a LEITZ‐BMRM fluorescence microscope (Leica, Heidelberg, Germany) using the FITC filter. To measure fluorescence intensity, the cells were lysed in a 10 mM Tris‐HCl buffer (pH 7.4) containing 150 mM NaCl, 0.9 mM CaCl2 and 1% Triton X‐100. Cell lysates were measured using an EnVision multilabel plate reader (PerkinElmer, Waltham, MA, USA) with an excitation of 480 nm and emission of 535 nm.

Animal experiments

The mouse experiments were conducted in accordance with a code of practice established by the ethical committee of the Microbial Chemistry Research Foundation (Shizuoka, Japan). Six‐week‐old female ICR nude mice were purchased from Charles River Japan (Yokohama, Japan) and maintained in a specific pathogen‐free barrier facility according to the Institute of Microbial Chemistry guidelines. Tumor xenografts were established by injecting 0.8–2.0 × 107 cells with 50% growth factor‐reduced matrigel (BD Biosciences, Franklin Lake, NJ, USA) subcutaneously near the left lateral flank. Tumors were allowed to grow to approximately 1000 mm3 before administration of bortezomib by tail‐vein injection. Tumor volume was estimated using the following formula: tumor volume(mm3) = (length × width2)/2.

In vivo imaging

In vivo fluorescence imaging was carried out with an OV110 in vivo imaging system (Olympus, Tokyo, Japan) using the GFP filter. Signal intensities from the regions of interest were defined manually, and the data were expressed as average green fluorescence intensity (relative fluorescence units [RFU]). Background green fluorescence intensity was defined from a region of interest drawn over the non‐tumor regions and these values were subtracted from the intensities quantified in tumors.

Statistical analysis

Representative examples with similar results from several independent experiments are shown. The data are expressed as the mean ± SD using descriptive statistics.

Results

Proteasome inhibition induced the accumulation of ZsProSensor‐1 in HEK293PS and PC‐3PS cells

To measure proteasome inhibition in living cells, we used stably transfected HEK293 cells and PC‐3 cells (HEK293PS and PC‐3PS), which each express the ZsProSensor‐1 fusion protein, a proteasome‐sensitive fluorescent reporter. The ZsProSensor‐1 protein is a fusion of the green fluorescent protein ZsGreen and mouse ornithine decarboxylase (MODC), which can be degraded by the proteasome without being ubiquitinated.23 The stably transfected HEK293PS cells expressing the ZsProSensor‐1 fusion protein grew at a similar rate to the parental HEK293 cells (Fig. 1A), but the stably transfected PC‐3PS cells grew significantly more slowly than the parental PC‐3 cells. The sensitivity of transfected cells to proteasome inhibitors was similar to that of the parental cells (Fig. 1B). Intracellular proteasome activities in the parental and transfected cells were equivalently inhibited by the proteasome inhibitor bortezomib (Fig. 1C). In HEK293PS and PC‐3PS cells, the fluorescent protein was undetectable by fluorescence microscopy due to rapid degradation by the proteasome under steady‐state conditions. However, inhibition of proteasome activity significantly induced the accumulation of the fluorescent reporter (Fig. 1D). These results collectively demonstrated that proteasome inhibition induced the accumulation of ZsProSensor‐1 in HEK293PS and PC‐3PS cells. In a quantitative analysis of fluorescence intensity, HEK293PS cells exhibited stronger fluorescence than PC‐3PS cells. Therefore, HEK293PS cells were subsequently used to evaluate protesome inhibition in vivo.

Figure 1.

Figure 1

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Proteasome inhibition increased accumulation of the ZsProSensor‐1 fluorescent protein in HEK293PS and PC‐3PS cells. (A) Cell growth was determined based on cell count. (B) Proteasome inhibitors suppressed the cell growth. Cells were incubated with MG‐132 (closed symbols) or bortezomib (open symbols) for 72 h, and cell viability was determined using a MTT assay. (C) The proteasome inhibitor bortezomib inhibited intracellular proteasome activity. Cells were incubated with bortezomib for 8 h and proteasome activity was determined from the degradation of Suc‐LLVY‐MCA. (D) Proteasome inhibitors induced the accumulation of green fluorescent protein in HEK293PS and PC‐3PS cells. HEK293PS and PC‐3PS cells were incubated with bortezomib (0.1 μg/mL) or MG‐132 (1 μg/mL). Green fluorescent protein levels were monitored by fluorescence microscopy at 12 h and the fluorescence intensities of cell lysates were determined using a fluorometer. RFU, relative fluorescence units.

Proteasome inhibition induced the accumulation of the ZsProSensor‐1 fluorescent protein in tumors of mice bearing HEK293PS xenografts

The tumorigenic potential of the stably transfected HEK293PS cells expressing the proteasome‐sensitive fluorescent reporter was investigated using nude mice (Fig. 2A). The growth of HEK293PS xenograft tumors was similar to that of the parental HEK293 tumors. These results suggest that the tumorigenicity of the transfected cells might be related to the cell growth rates in vitro (Fig. 1A).

Figure 2.

Figure 2

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Proteasome inhibition induced the accumulation of ZsProSensor‐1 in tumors of mice bearing HEK293PS xenografts. (A) Growth of tumor xenografts in nude mice. HEK293 (1 × 107) and HEK293PS (1 × 107) cells suspended in 50% matrigel were subcutaneously inoculated into nude mice on day 0. Tumor volume was measured with calipers. Values are the means of five mice bearing HEK293 and 29 mice bearing HEK293PS xenografts. Bars represent SD. (B) Bortezomib increased the accumulation of ZsProSensor‐1 in tumors of mice bearing HEK293PS xenografts. HEK293 and HEK293PS cells (1 × 107) suspended in 50% matrigel were inoculated into nude mice on day 0. Bortezomib (2 mg/kg) was intravenously administered to mice bearing HEK293 tumors on day 49 and HEK293PS tumors on day 34. Mice were imaged 24 h after drug administration. BF, bright‐field; GFP, green fluorescent protein. (C) Bortezomib inhibited proteasome activity in tumors of mice bearing HEK293PS xenografts. HEK293PS cells (1 × 107) suspended in 50% matrigel were subcutaneously inoculated into nude mice on day 0. Bortezomib (2 mg/kg) was intravenously administered to mice bearing size‐matched tumors. Proteasome activity in tumor lysates was determined at 7 h after drug administration.

We used mice bearing size‐matched tumors to evaluate the potential of this reporter system for in vivo imaging of proteasome inhibitory activity. In HEK293PS tumors, fluorescence was either undetectable or barely detectable above background (Figs 2B, Supporting Information S1). Intravenous administration of bortezomib induced robust fluorescence in tumors at 24 h after administration. Indeed, bortezomib inhibited proteasome activity in tumors of mice bearing HEK293PS xenografts (Fig. 2C). However, in HEK293 tumors fluorescence was undetectable after the administration of bortezomib. Taken together, xenografts made from stably transfected cells expressing a proteasome‐sensitive fluorescent reporter permitted monitoring of proteasome inhibition in living mice. Therefore, HEK293PS tumors are useful for in vivo imaging of proteasome inhibition based on their high sensitivity to proteasome inhibitors.

Proteasome inhibition resulted in ZsProSensor‐1 accumulation in HEK293PS xenograft‐derived tumors in a time‐ and dose‐dependent manner

To define the time‐course of proteasome inhibition in tumors, we intravenously administered bortezomib (a single dose at 1 mg/kg) to mice bearing HEK293PS xenografts and monitored them continuously for 20 days (Figs 3A, S2). Fluorescence levels rapidly increased in the tumors after administration of bortezomib and remained at peak levels for 1–3 days. The accumulated fluorescent protein was then gradually degraded, returning to approximately baseline levels within 15 days. To determine the dose response of proteasome inhibition in tumors, we intravenously injected mice bearing HEK293PS xenografts with one of two doses of bortezomib (1 or 4 mg/kg) (Fig. 3B, Supporting Information Table S1). The administration of 4 mg/kg bortezomib significantly increased the accumulation of the fluorescent reporter in tumors, whereas administration of 1 mg/kg bortezomib resulted in only a slight increase. The fluorescence intensities in tumors of mice administered 2 or 4 mg/kg bortezomib were quantified by the OV110 software (Fig. 3C, Table S2). Administration of 2 mg/kg bortezomib resulted in an 11‐fold increase in the fluorescence intensity compared with the intensity before administration. Furthermore, administration of 4 mg/kg bortezomib showed an 18‐fold increase. These results show that proteasome inhibition induced the accumulation of ZsProsensor‐1 in the tumors of mice bearing HEK293PS xenografts in a time‐ and dose‐dependent manner.

Figure 3.

Figure 3

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Bortezomib increased the accumulation of ZsProSensor‐1 in a time‐ and dose‐dependent manner in the tumors of mice bearing HEK293PS xenografts. (A) Bortezomib caused a time‐dependent increase in tumor fluorescence levels. HEK293PS cells (2 × 107) were subcutaneously inoculated into nude mice on day 0 and bortezomib (2 mg/kg) was intravenously administered on day 56. Mice were imaged continuously for 20 days after drug administration. (B) Bortezomib also caused a dose‐dependent increase in tumor fluorescence levels. HEK293PS cells (1 × 107) were subcutaneously inoculated into nude mice on day 0 and bortezomib (1 or 4 mg/kg) was intravenously administered on day 50. Mice were imaged 24 h after drug administration. (C) Quantitative analysis of green fluorescence intensity in tumors. HEK293PS cells (2 × 107) were subcutaneously inoculated into nude mice on day 0 and bortezomib (2 or 4 mg/kg) was intravenously administered on day 56.

Intravenous administration of bortezomib induced antitumor effects and accumulation of ZsProSensor‐1 in HEK293PS tumors

To elucidate the relationship between antitumor activity and accumulation of the proteasome‐sensitive reporter in tumors, we injected bortezomib intravenously or orally to mice bearing HEK293PS tumors. Intravenous administration of bortezomib significantly suppressed tumor growth, while oral administration did not exert antitumor effects (Fig. 4A). In vivo imaging showed that intravenous, but not oral, administration of bortezomib significantly increased levels of the fluorescent protein in tumors, as described above (Figs 4B, S3). The quantification of green fluorescence intensities in these tumors revealed that intravenous administration of bortezomib increased the average green fluorescence intensity from 11.0 RFU (0 h) to 40.2 RFU (24 h), while oral administration induced only a small increase, from 7.8 RFU (0 h) to 8.8 RFU (24 h) (Fig. 4C, Table S3). These results clearly show that accumulation of the ZsProSensor‐1 fluorescent protein in tumors by proteasome inhibition closely correlated with antitumor activity. This reporter system can therefore be used as an indicator of antitumor activity and the stably transfected HEK293PS cells will serve as powerful tools for the development of new proteasome inhibitors.

Figure 4.

Figure 4

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Intravenous administration of bortezomib had antitumor activity and promoted the accumulation of ZsProSensor‐1 in HEK293PS tumors. (A) Antitumor activity of intravenously administered bortezomib in a HEK293PS xenograft mouse model. HEK293PS cells (1 × 107) suspended in 50% matrigel were subcutaneously inoculated into nude mice on day 0. Bortezomib was administered intravenously (i.v.) or orally (p.o.) once a week beginning on day 42. (B) In vivo imaging 24 h after the administration of bortezomib. Bortezomib was administered intravenously or orally on day 42 and mice were imaged 24 h later. Values are the means of six mice. Bars represent SD. (C) Quantitative analysis of green fluorescence intensity in tumors.

Discussion

In vivo imaging of proteasome function using luciferase has already been reported.18 For bioluminescence imaging using luciferase, administration of d‐luciferin as a substrate for luciferase is required. Therefore, in vivo bioluminescence imaging is expensive and needs great care. In addition, d‐luciferin reached peak plasma concentrations at 15 min post‐intraperitoneal administration and then rapidly decreased. Bioluminescence signal has a short half‐life. Fluorescent reporters (e.g. GFP) do not require special reagents to visualize fluorescence signals. Therefore, operation is simple and low cost, and fluorescence signals are ready to be determined. For these reasons, we used the ZsProSensor‐1 protein as a proteasome‐sensitive fluorescent reporter. This construct is a fusion of a green fluorescent protein, ZsGreen, with a MODC degradation domain, which is targeted for rapid degradation by the proteasome. Inhibition of the proteasome leads to accumulation of the ZsProSensor‐1 fluorescent protein. Quantification of ZsProSensor‐1 fluorescence levels therefore represents an efficient image‐based screening method. Indeed, stably transfected HEK293PS cells expressing the ZsProSensor‐1 protein have already been used for high‐throughput screening assays to identify proteasome inhibitors.20 However, in vivo imaging of proteasome inhibition using a fluorescent reporter in living animals has not been previously reported. The present study represents the first report of in vivo imaging of proteasome function using a fluorescent reporter in the tumors of living mice.

HEK293 cells have often been used as host cells to produce exogenous proteins. However, HEK293 xenografts require 40–50 days to develop into 1000 mm3 tumors in nude mice. Therefore, to obtain reporter cell lines that rapidly form tumors, we attempted to transfect the ZsProSensor‐1 vector into several cancer cell lines, including human prostate cancer PC‐3 cells. However, only PC‐3 cells successfully maintained the ZsProSensor‐1 vector. The stably transfected PC‐3PS cells exhibited low rates of cell and tumor growth (Figs 1A,S4). In addition, PC‐3PS cells accumulated low levels of fluorescent protein in vitro and in vivo (Figs 1D, S4). ZsProSensor‐1 protein expression seemed to be stressful for the host cells. In contrast, the stably transfected HEK293PS cells showed high levels of fluorescent protein accumulation in vitro and in vivo (Figs 1D, 2B). Therefore, HEK293 cells might be more accommodating for the production of exogenous proteins such as ZsProSensor‐1. We therefore used HEK293PS xenografts to monitor proteasome inhibition in vivo.

We successfully detected increased fluorescence levels in HEK293PS tumors in living mice on proteasome inhibition. When bortezomib was intravenously administered to mice bearing HEK293PS xenografts, robust fluorescence was detected in the tumors at 24 h after administration and was maintained for 1–3 days (Figs 3A, S2). The fluorescence signal returned to near‐baseline levels within 15 days. In studies using the ubiquitin‐luciferase reporter, the luciferase photon flux ratio significantly increased 30 min after injection of bortezomib and returned to baseline levels within 46 h when proteasome function returned to normal.17 These data are consistent with the established kinetics of 20S proteasome inhibition by conventional analysis of blood samples from patients treated with bortezomib.24 In contrast, the fluorescence signal derived from the ZsProSensor‐1 reporter was sustained for a significantly longer time than the luciferase reporter. The accumulated ZsProSensor‐1 protein might be resistant to proteolysis by the proteasome. These results suggest that use of the ZsProSensor‐1 reporter could facilitate the monitoring of proteasome inhibition in living mice due to the increased duration of the fluorescence signal. Furthermore, use of other fluorescent proteins instead of ZsGreen might have the potential to increase fluorescence signals.

ZsProSensor‐1 accumulated in HEK293PS xenograft‐derived tumors in response to proteasome inhibition in a dose‐dependent manner (Fig. 3B,C). This accumulation correlated with antitumor activity (Fig. 4). Bortezomib has a beneficial antitumor effect during intravenous administration but not oral administration. This difference was validated using in vivo imaging of the reporter (Fig. 4). Recent attempts to develop second‐generation proteasome inhibitors have focused on orally active compounds such as marizomib (NPI‐0052; salinosporamide A), CEP‐18770 and MLN9708.25, 26, 27 Therefore, in vivo imaging using proteasome‐sensitive fluorescent proteins might contribute to the identification of orally active proteasome inhibitors and facilitate the development of novel antitumor agents.

Disclosure Statement

The authors have no conflict of interest.

Supporting information

Fig. S1. Bortezomib increased the accumulation of ZsProSensor‐1 in the tumors of mice bearing HEK293PS xenografts.

Click here for additional data file. (11MB, pdf)

Fig. S2. Time‐course of proteasome inhibition in tumors.

Click here for additional data file. (10.4MB, pdf)

Fig. S3. In vivo imaging 24 h after bortezomib administration.

Click here for additional data file. (20MB, pdf)

Fig. S4. Bortezomib slightly increased ZsProSensor‐1 fluorescence levels in the tumors of mice bearing PC‐3PS xenografts.

Click here for additional data file. (13.8MB, pdf)

Table S1. Effect of 1 and 4 mg/kg bortezomib on the green fluorescence intensity in tumors of mice bearing HEK293PS xenografts.

Click here for additional data file. (26.7KB, pdf)

Table S2. Effect of 2 and 4 mg/kg bortezomib on the green fluorescence intensity in tumors of mice bearing HEK293PS xenografts.

Click here for additional data file. (26.7KB, pdf)

Table S3. Effect of administration route of bortezomib on the green fluorescence intensity in tumors of mice bearing HEK293PS xenografts.

Click here for additional data file. (28.5KB, pdf)

Acknowledgments

This work was supported by a Grant‐in‐Aid for Scientific Research (C, 23510270) from The Ministry of Education, Culture, Sports, Science and Technology in Japan. The authors thank Ms S. Kakuda and Dr M. Kawada for technical assistance and helpful advice.

(Cancer Sci, 2012; 103: 1730–1736)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Bortezomib increased the accumulation of ZsProSensor‐1 in the tumors of mice bearing HEK293PS xenografts.

Click here for additional data file. (11MB, pdf)

Fig. S2. Time‐course of proteasome inhibition in tumors.

Click here for additional data file. (10.4MB, pdf)

Fig. S3. In vivo imaging 24 h after bortezomib administration.

Click here for additional data file. (20MB, pdf)

Fig. S4. Bortezomib slightly increased ZsProSensor‐1 fluorescence levels in the tumors of mice bearing PC‐3PS xenografts.

Click here for additional data file. (13.8MB, pdf)

Table S1. Effect of 1 and 4 mg/kg bortezomib on the green fluorescence intensity in tumors of mice bearing HEK293PS xenografts.

Click here for additional data file. (26.7KB, pdf)

Table S2. Effect of 2 and 4 mg/kg bortezomib on the green fluorescence intensity in tumors of mice bearing HEK293PS xenografts.

Click here for additional data file. (26.7KB, pdf)

Table S3. Effect of administration route of bortezomib on the green fluorescence intensity in tumors of mice bearing HEK293PS xenografts.

Click here for additional data file. (28.5KB, pdf)

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