9-7 Hongo 2-Chome, Bunkyo-Ku
Tokyo 113-0033, Japan

PCEI-HU <Photoswitchable CENP-E Inhibitor>
Catalog NO. FDV-0037
Research use only, not for human or animal therapeutic or diagnostic use.

Product Background
Mitosis is a fundamental process of cell duplication for the growth and maintenance of tissues in multicellularorganisms. During mitosis, many kinds of proteins play crucial roles in accurate distribution of the replicated genome
into two daughter cells. The replicated genomes are condensed into chromosomes in the prophase and subsequently,
chromosomes move to the center of the mitotic cells, frequently called metaphase plate, along spindle microtubules
in prometaphase. Chromosomes bind to spindle microtubules via kinetochore which is a unique protein complex incentromere. Only when all chromosomes are aligned to the metaphase plate in metaphase, separation of sister
chromosome begins (anaphase). There is an important checkpoint called spindle assembly checkpoint (SAC) from
metaphase to anaphase. SAC monitors accurate alignment of chromosomes in metaphase plate in mitosis to exact
separation of genomic information into daughter cells. Accurate segregation of all chromosomes is the most importantmatter to satisfy the SAC criteria. Centromere protein E (CENP-E) is a key component for chromosome congression
along microtubule spindles to the metaphase plate. CENP-E is a dimeric protein which has three domains, N-terminal
motor domain, C-terminal kinetochore binding domain and coiled-coiled linker region. Recent studies suggest
inhibition of ATPase activity of the CENP-E motor domain suppresses chromosome congression to the metaphase
plate, subsequently SAC detects the misalignment of chromosomes and as a result, anaphase does not start.
Controlling CENP-E activity has a large potential to control mitosis progression. Although some potent CENP-E
inhibitors have been developed so far, these inhibitors are not suitable for reversibly controlling CENP-E activity.

Figure 1

Overview of CENP-E in mitosis

Photoswitchable CENP-E inhibitor (PCEI-HU) is the world’s first photocontrollable CENP-E inhibitor whichcontains an azobenzene-based photo-isomerization unit (Ref.1). PCEI-HU can regulate CENP-E activity ON and
OFF repeatedly by UV and visible light (Vis) irradiation. PCEI-HU in water without light shows potent CENP-E
inhibitory activity, but once UV light irradiation, PCEI-HU lacks its inhibitory activity. Under the condition, CENPE keeps its motor activity for chromosome congression to the metaphase plate. This UV-induced loss of inhibitor
action is recovered by Vis light. After Vis irradiation, PCEI-HU blocks CENP-E activity again and chromatin
congression is clearly suppressed. PCEI-HU is a powerful tool for the fundamental investigation ofprometaphase/metaphase and SAC.

Figure 2. Principle and overview of PCEI-HU

Description
Catalog Number: FDV-0037
Size : 50 g
Formulation : C26H31N6O3Cl
Molecular weight : 511.02 g/mol
Solubility : Soluble in DMSO

Application- Photoswitchable CENP-E inhibitor- Photoswitchable chromosome movement in prometaphase/metaphase during mitosis

Reconstitution and Storage
Reconstitution : Add 100% DMSO into vial to prepare 1-10 mM stock solution.
Storage :Powder : Store at -20 oC with light shedding.
Stock solution :- Make aliquots and store at -20 °C with protection from light.
- Avoid repeated freeze-thaw cycles.
- To avoid photo-induced isomerization of PCEI-HU, one-time use of each aliquot is highly recommended.
<NOTE> PCEI-HU may be isomerized by environmental light such as room light or sunlight. When making
and storing aliquots, please carefully check the light conditions around the experimental space.
[ver. 2024/05]

Download the latest datasheet from www.funakoshi.co.jp(Japanese)www.funakoshi.co.jp/exports (English)

How to use
Cell-based CENP-E inhibition assay
1. Add PCEI-HU (for example, final. 1 M for HeLa cells) in fresh media in the dark condition.
2. Remove the cultured medium and wash cells by PBS several times.
3. Add PCEI-HU containing medium to the cells.
4. Culture cells for 1-2 hours in dark condition
5. UV-irradiate culture dish by any light sources.
6. Vis-irradiate culture dish by any light sources.
*NOTE(1): Photo-irradiation condition should be optimized by users for each experiments. Detail information
is described in the later chapter.
*NOTE(2): All procedures are recommended in the dark condition to avoid non-specific photoisomerization ofPCEI-HU by environmental light source.

Reference data
Absorption spectrum
Left: Absorption spectrum of PCEI-HU. Without light irradiation (w/o light; gray), after 365 nm light irradiation(UV irradiation; red) and after 510 nm light irradiation (Vis irradiation; blue).
Right : Absorbance changes at 338 nm under alternating 365/510 nm illumination.

Inhibition activity for purified CENP-E ATPase of PCEI-HU
ATPase activities of CENP-E with various concentrationsof PCEI-HU were measured. IC50 values indicate,inhibition activity of PCEI-HU after UV irradiation wasabout ten-fold decreased than that after Vis irradiation.

Cytotoxicity of PCEI-HU for long-term culture
HeLa cells were treated with 1.1 M PCEI-HU under three conditions, w/o light, after
UV irradiation and after Vis irradiation for 42 hours. W/o light and Vis irradiation show
potent cytotoxicity. On the other hand, UV irradiation keeps high cell viability.
Treatment of 1 M PCEI-HU, a recommended concentration, for a few hours, has little
effect on cell viability.

Experimental guides
Light source of photoirradiation
For UV irradiation, 350-380 nm light is recommended. In the case of 365 nm light, 33 mW/cm2 for 25 sec is enoughto convert PCEI-HU.
For Vis irradiation, 480-530 nm light is recommended. In the case of 510 nm light, 117 mW/cm2 for 35 sec isenough to convert PCEI-HU.
In this manual, all data were obtained using 365 nm light-emitting diode (LED, C11924-101, Hamamatsu) and 510
nm LED (CS-LED3W_510 nm, Hayasaka).
Photoisomerization ratio of PCEI-HU“CENP-E block form” and “CENP-E non-block form” were in photoisomerization equilibrium. Under the adequate
irradiation of UV (365 nm) light, the ratio of “CENP-E block form”/”CENP-E non-block form” was 7/93%. Under
the adequate irradiation of Vis (510 nm) light converts 86/14%. Please note both UV and Vis light irradiation did
not convert to “CENP-E block form” and “CENP-E non-block form” completely.
Note for live cell imaging
As PCEI-HU is a photo-responsive reagent, excitation light for any fluorophores (fluorescent dyes, proteins, etc.)
should be carefully selected under live-cell imaging. Ideally near-infrared fluorophores excited by over 630 nm
light are compatible with PCEI-HU without any effect on PCEI-HU photoisomerization.
Validated species
PCEI-HU was only validated in human cell lines and human CENP-E protein. There is no data for other species
such as mice, rat, etc.
Controllable capacity
Ref.1 revealed that once chromosomes reached to the metaphase plate, PCEI-HU cannot regulate furtherchromosome movement. Please carefully check the status of chromosome alignment. (See Application data).

[ver. 2024/05]

Download the latest datasheet from www.funakoshi.co.jp(Japanese)www.funakoshi.co.jp/exports (English)

Application data
Cellular localization of chromosomes, CENP-E and tubulin under photo-dependent PCEI-HU
HeLa cells were treated with 1 M PCEI-HU and 20 M MG132*1 under following three conditions. After theseconditions, cells were fixed with PFA and visualized by specific antibodies for CENP-E and -tubulin and DAPI
staining for chromosomes.
1) Add PCEI-HU without light for 2 hours → fixation = “w/o light”
2) Add PCEI-HU without light for 2 hours → after UV irradiation, culture for 30 min = “UV irradiation”
3) Add PCEI-HU without light for 2 hours → after UV irradiation, culture for 5 min→ after Vis irradiation, culture for 30 min = “Vis irradiation”
Under the condition 1), PCEI-HU w/o light works as the “CENP-E block form” and induced misalignment ofchromosomes. CENP-E was highly accumulated in the centrosome. Under the condition 2), UV irradiation
converted PCEI-HU to “CENP-E non-block form” and all chromosomes reached to the metaphase plate. CENP-E
was distributed near metaphase plate. Under condition 3), Vis irradiation recovered CENP-E inhibitory activity ofPCEI-HU and shows a similar result with 1) w/o light.
NOTE *1: MG132 is used as an anaphase inhibitor. Under MG132 treatment, mitosis did not enter anaphase evenwhen all chromosomes were aligned in the metaphase plate.

Live cell imaging of mitotic chromosomes in PCEI-HU treated LLC-PK 1 cells
LLC-PK1 cells were treated with near-infrared DNA staining dye (SiR-DNA, 1 M) and subsequently 1 MPCEI-HU and 20 M MG132 for 2 hours in darkroom. Cells were irradiated with UV light (365 nm) and Vis (510nm) repeatedly and monitored in live-cell (Upper figure). Movement of a specific chromosome (yellow arrow)
was analyzed in kymograph (Lower figure). While after UV irradiation, chromosomes moved to metaphase plate,but after Vis irradiation chromosomes left from metaphase plate. The chromosome was repeatedly regulated byUV/Vis cycles until chromosomes reaching to the metaphase plate.

Elucidation of CENP-E roles after chromosome congression to the metaphase plate
HeLa cells were treated with 1 M PCEI-HU and 20 M MG132 under the following two conditions.
1) Add PCEI-HU without light for 2 hours → after UV irradiation, culture for 3 min→ after Vis irradiation, culture for 30 min (Left figure)2) Add PCEI-HU without light for 2 hours → after UV irradiation, culture for 30 min→ after Vis irradiation, culture for 30 min (Right figure)
When the incubation time after UV irradiation was short such as 3 min, Vis irradiation recovered the suppressionof chromosome movement. However, if the incubation time after UV irradiation was long such as 30 min,chromosomes reached to metaphase plate and could not be controlled by Vis irradiation again. This experimentshowed once chromosomes reached to the metaphase plate, PCEI-HU could not regulate further chromosome
movement.

Functional assay to monitor a movement of Mad2, a SAC-regulatory protein
HeLa cells co-expressing Mad2-GFP and histone H2B-mCherry were treated with PCEI-HU and MG132 under thefollowing two conditions.
1) PCEI-HU w/o light for 2 hours →after UV irradiation, culture for 5 min→after Vis irradiation, culture for 30 min →fixed and observed2) PCEI-HU w/o light for 2 hours →after UV irradiation, culture for 30 min→after Vis irradiation, culture for 30 min →fixed and observed
In both conditions, PCEI-HU w/o light-induced misalignment of chromosomes and aggregation of Mad2 in
misaligned chromosomes. In condition 1), short-term culture after UV irradiation Mad2 remained aggregated in themisaligned chromosome. On the other hand, condition 2) showed long-term culture after UV irradiation induces
correct alignment of chromosomes and Mad2 was clearly diffused in the cytosol. These results indicate Mad2
specifically accumulated in misaligned chromosomes and after diffused in the cell by a complete alignment ofchromosome in the metaphase plate.

[ver. 2024/05]

Download the latest datasheet from www.funakoshi.co.jp(Japanese)www.funakoshi.co.jp/exports (English)Reference
1. Mafy, N. N. et al., J. Am. Chem. Soc., 142, 1763-1767 (2020) Photoswitchable CENP-E Inhibitor Enabling theDynamic Control Chromosome Movement and Mitotic Progression.

Disclaimer/免責事項This product has been commercialized by Funakoshi Co., Ltd.
based on the results of academic research, and the advertisement
text, figures and manuals (hereinafter “Product information”) havebeen prepared based on published research reports on March, 2020.
The academic interpretation at the time of creation of the Product
Information may change in accordance with future developmentsin the relevant research field and expansion of various scientificfindings, and the latest version and certainty of the Product
Information are not guaranteed. The specifications of this productand the Product Information are subject to change without notice.
Please contact us for the latest information.
本商品は学術研究成果を基にフナコシ株式会社が商品化したもので、2020 年 3 月時点における公開研究報告を基に広告文章およびマニュアル（以下、商品資料）を作成しています。今後の当該研究分野の発展および各種学術知見の拡大にともない、商品資料作成時の学術的解釈が変更になる可能性があり、最新性・確実性を保証するものではありません。また、本商品の仕様および商品資料を予告なく変更する場合がございます。最新の情報に関しましては、弊社までご確認いただきますようお願い申し上げます。

Related products:
NucleoSeeingTM <Live Nucleus Green>
NucleoSeeingTM is DNA-responsive green dye for monitoring cell nucleus in live cells. As it shows low
cytotoxicity and phototoxicity, it is very suitable for long-term live imaging of cell nucleus.
Catalog No. FDV-0029
Size
0.1 mgFeatures- Easy and quick procedure- Compatible with 10% FBS- Validated for both adherent cells and floating cells- Little influence on cellular functions- Ex/Em: 488 nm/520 nm (commercial FITC filters are available)

NOTE: NucleoSeeingTM is not compatible with PCEI-HU experiments. For PCEI-HU experiments in live cells,
please use near-infrared (NIR) dyes.

Gatastatin G2 <-Tubulin Inhibitor>
Gatastatin G2 is a second generation of -tubulin specific inhibitor, Gatastatin. Gatastatin G2 has little effects
on /−tubulin polymerization both in vitro and in cells and induced abnormal chromosome alignment andmultipolar formation in mitotic cells.
Catalog No. FDV-0040
Size
0.1 mgFeatures- -tubulin specific inhibitor- blocks GTP-binding of -tubulin- useful for functional analysis of multifunction of -tubulin[ver. 2024/05]

Download the latest datasheet from www.funakoshi.co.jp(Japanese)www.funakoshi.co.jp/exports (English)