Electronic Supplementary Material (ESI) for RSC Advances.
This journal is © The Royal Society of Chemistry 2022

Supplementary information

Direct analysis of stress-fiber formation effect in photodynamic therapy

Atsushi Taninaka1,2, Shunta Ugajin1, Hiromi Kurokawa3, Yu Nagoshi1, Mayuka Kamiyanagi1,Osamu Takeuchi1, Hirofumi Matsui3, and Hidemi Shigekawa1*

1Faculty of Pure and Applied Sciences, University of Tsukuba, 305-8573 Ibaraki, Japan
2TAKANO Co., LTD., Miyano-mura, Kamiina-gun, Nagano, 399-4301 Japan
3Faculty of medicine, University of Tsukuba, 305-8575 Ibaraki, JapanFig. S1 Model for the mechanism of stress fiber formation by PDT. (a) When a photosensitizer(TS) taken up into cells is irradiated with light, singlet oxygen is generated and converted to active
oxygen (ROS)1–3. ROS is known to activate RhoA2,4–7. (b) RhoA activation is regulated byguanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP)4,8,9. (c) RhoA*
activated by GEF acts on Mammalian diaphanous-related formin (mDia) and promotes actin
polymerization10–13. (d), (e) On the other hand, activated RhoA* binds to Rho-associated protein
kinase (ROCK) and promotes phosphorylation of the myosin light chain4,6,7,9,12,14–16. (f) S-fiber isformed by the formed A-filament and myosin-light chain. S-fiber binds to desmosomes, and theirtension determines the elastic modulus of the cell.

Fig. S2 Results of quantifying the intensity of the fluorescence images in the text. The CCD
measurement intensity values of one pixel (6.45 × 6.45 μm2) were averaged over each image.

Fig. S3 Phase difference observation images (upper) and fluorescence observation images(lower) when only SPY555-actin fluorescent probe is inserted.

Fig. S4 Western blotting data used to make Fig. 3b.

Figure S5
Phase contrast images ((a)-(d)) and fluorescence images ((e)-(h)) immediately, 4, and 9 minutes
after light irradiation with an intensity of 34 mW/cm2 for 1 min.

Figure S6
Ultraviolet-visible (UV-Vis) range absorption spectrum of aqueous solution of taraporphyrinsodium. A(545) and A(655) indicate the areas for the irradiation with center wavelengths of 545 and655 nm, respectively.

Figure S7
Results obtained by irradiating RGK1 with light with a center wavelength of 545 nm and anintensity of 235 mW/cm2 for 5 minutes ((a) to (e)) and 10 minutes ((f)-(j)), (a), (f) Phase contrast
images before light irradiation and fluorescence images (b), (g) before light irradiation, (c), (h)immediately after light irradiation, (d), (i) 5 minutes after light irradiation, and (e), (j) 10 minutes
after light irradiation.

Figure S8
Phase contrast images (middle row) and their magnifications (top row), and fluorescence images(bottom row) of RGK1 when light with a central wavelength of 545 nm and an intensity of 235mW/cm2 was irradiated for 5 minutes. (a) Immediately, (b) 5 min, (c) 10 min, (d) 15 min, (e) 20min, (f) 25 min, and (g) 30 min after irradiation.

Movie S1
Movie showing the effect of PDT on RGK1 obtained by irradiation with light with a center
wavelength of 655 nm and an intensity of 87 mW/cm2 for 1 minute.

References
1 M. Ethirajan, Y. Chen, P. Joshi and R. K. Pandey, The role of porphyrin chemistry in tumor
imaging and photodynamic therapy, Chem. Soc. Rev., 2010, 40, 340–362.
2 W. M. Sharman, C. M. Allen and J. E. van Lier, in Methods in Enzymology, Academic Press,
2000, vol. 319, pp. 376–400.
3 C. S. Foote, DEFINITION OF TYPE I and TYPE II PHOTOSENSITIZED OXIDATION,Photochem. Photobiol., 1991, 54, 659–659.
4 A. Wirth, Rho kinase and hypertension, Biochim. Biophys. Acta BBA - Mol. Basis Dis., 2010,1802, 1276–1284.
5 Kajimoto Hidemi, Hashimoto Kyoko, Bonnet Sandra N., Haromy Alois, Harry Gwyneth,
Moudgil Rohit, Nakanishi Toshio, Rebeyka Ivan, Thébaud Bernard, Michelakis Evangelos D.,
and Archer Stephen L., Oxygen Activates the Rho/Rho-Kinase Pathway and Induces RhoB
and ROCK-1 Expression in Human and Rabbit Ductus Arteriosus by Increasing
Mitochondria-Derived Reactive Oxygen Species, Circulation, 2007, 115, 1777–1788.
6 T. Suzuki, M. Tanaka, M. Sasaki, H. Ichikawa, H. Nishie and H. Kataoka, Vascular Shutdown
by Photodynamic Therapy Using Talaporfin Sodium, Cancers, 2020, 12, 2369.
7 S. Cavin, T. Riedel, P. Rosskopfova, M. Gonzalez, G. Baldini, M. Zellweger, G. Wagnières, P.
J. Dyson, H.-B. Ris, T. Krueger and J. Y. Perentes, Vascular-targeted low dose photodynamic
therapy stabilizes tumor vessels by modulating pericyte contractility, Lasers Surg. Med., 2019,51, 550–561.
8 S. Etienne-Manneville and A. Hall, Rho GTPases in cell biology, Nature, 2002, 420, 629–635.
9 S. Hartmann, A. J. Ridley and S. Lutz, The Function of Rho-Associated Kinases ROCK1 andROCK2 in the Pathogenesis of Cardiovascular Disease, Front. Pharmacol., ,DOI:10.3389/fphar.2015.00276.
10H. Mizuno, K. Tanaka, S. Yamashiro, A. Narita and N. Watanabe, Helical rotation of

the diaphanous-related formin mDia1 generates actin filaments resistant to cofilin, Proc. Natl.
Acad. Sci., 2018, 115, E5000–E5007.
11K. Zaoui, S. Honoré, D. Isnardon, D. Braguer and A. Badache, Memo–RhoA–mDia1

signaling controls microtubules, the actin network, and adhesion site formation in migratingcells, J. Cell Biol., 2008, 183, 401–408.
12S. Narumiya, M. Tanji and T. Ishizaki, Rho signaling, ROCK and mDia1, intransformation, metastasis and invasion, Cancer Metastasis Rev., 2009, 28, 65–76.
13Mizuno H. and Watanabe N., The Role of Rotational Movement of a MammalianFormin mDia1 in Actin Remodeling, Seibutsu Butsuri, 2011, 51, 218–221.

14K. Burridge, E. Monaghan-Benson and D. M. Graham, Mechanotransduction: fromthe cell surface to the nucleus via RhoA, Philos. Trans. R. Soc. B Biol. Sci., 2019, 374,20180229.
15J. Liu, Y. Wada, M. Katsura, H. Tozawa, N. Erwin, C. M. Kapron, G. Bao and J. Liu,

Rho-Associated Coiled-Coil Kinase (ROCK) in Molecular Regulation of Angiogenesis,Theranostics, 2018, 8, 6053–6069.
16

K. Rottner and T. E. Stradal, Actin dynamics and turnover in cell motility, Curr. Opin.

Cell Biol., 2011, 23, 569–578.