Phalloidin atto 594

SMAD2 (L16D3; #3103) and Snail (C15D3; #3879) antibodies were from Cell Signaling Technology. Acetylated α-tubulin (clone 6-11B-1; #T6793) antibody and Phalloidin-Atto 594 (#51927) were from Sigma-Aldrich. Antibody against α-tubulin (DM1A; #CP06) was from Calbiochem, Merck Millipore. Antibodies against p-SMAD2 and p-SMAD3 have been described previously [28 (link), 29 (link)]. Goat anti-Rabbit IgG (H + L) Alexa Fluor 488 conjugate (#A-11008), Goat anti-Mouse IgG (H + L) Alexa Fluor 488 conjugate (#A-11029), and Goat anti-Mouse IgG (H + L) Alexa Fluor 594 conjugate (A-11032) were from Life Technologies. Goat-anti-Rabbit IRDye 800CW (#926-32211) and Goat-anti-Mouse IRDye 680 (#926-32220) were from LI-COR Biosciences.

Before fixation with 4% PFA (43368, Alfa Aesar) for 30 min at r.t., samples were washed with 1X DPBS (14287-050, Gibco). After fixation, samples were washed three more times with 1X DPBS on a rocker for 15 min at 150 r.p.m. Next, 1X DPBS was removed and samples were stained with DAPI (1:2,000 dilution; 62248, Thermo Scientific), Phalloidin-Atto 594 (1:3,000 dilution; 51927, Sigma) and Caspase-3/7 (1:1,000 dilution; C10423">C10423, Invitrogen). After 90 min incubation at r.t., samples were washed three times with 1X DPBS, again using a rocker. Images were acquired with the Operetta CLS (Perkin Elmer) using a ×20 water objective. Optical mode, focus and binning were set up as described above. The first plane imaged was at −15 µm, followed by 50 z-stacks with a distance of 4 µm. 3D reconstruction was performed in Harmony (Perkin Elmer).

CuBr₂ (99.999% trace metal basis), CuBr (99.99%), 2,2’-bipyridyl (99%), oligo(ethylene glycol) methyl ether methacrylate (M_n = 300 g/mol, MeOEGMA), glycidyl methacrylate (97%, GMA), ethylenediamine (99.5%, EtDA), α-bromoisobutyryl bromide (98%, BiBB), copper sulphate (CuSO₄), sodium L-ascorbate (98%), NaN₃ (99.5%) were purchased from Sigma-Aldrich (Czech Republic). Deionized (DI) water was obtained from a Milli-Q purification system (Milli-Q gradient A10, Merck-Millipore). Tetrahydrofuran (THF, Lach-Ner, Czech Republic) was distilled under argon over sodium immediately prior use. All other organic solvents (Lach-Ner, Czech Republic) of analytical grade were used as received. ATRP initiators 11-(2-bromo-2-methyl)propionyloxy)undecyltrichlorosilane and ω-mercaptoundecyl bromoisobutyrate were synthesized according to procedures reported earlier [49 (link),50 (link)]. Human blood plasma, Endothelial Cell Growth Medium 2 Kit, and HUVEC cells were obtained from Promocell (Germany). Penicillin-streptomycin (10 U/mL, 10 μg/mL), trypsin-EDTA (0.05%–0.02%) and Phalloidin–Atto 594 were from Sigma-Aldrich (Czech Republic). LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells and Hoechst 33342 were purchased from Thermo Fisher Scientific (USA).

Zirconium(IV) propoxide (70 wt% in 1-propanol), acetylacetone (Hacac), 1-propanol, absolute ethanol, 3-aminopropyltriethoxysilane (APTES), N-(3-Dimethylaminopropyl)-N′-ethyl carbodiimide (EDC), Phalloidin–Atto 594, N-hydroxysuccinimide (NHS), heat-inactivated fetal bovine serum (FBS), streptomycin, and penicillin were purchased from Merck, DE. Sodium hyaluronate (55 KDa) (HA) was provided by Altergon SRL (Avellino, Italy). 2,7-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Cabru, IT. All the reagents were used without further purification. Hank Balanced Salt Solutions (HBSS) were purchased from Gibco (Milan, IT). Alamar Blue assay was purchased from Serotec, (Milan, IT).

PLGA 75:25 (Resomer^® RG 752 H, Mw = 4–15 kDa) (analytical grade), SC, PVA (Mw = 31–50 kDa, 98–99% hydrolyzed), PTM isethionate (PTM-I), dimethyl sulfoxide (DMSO), phosphoric acid, sodium hydroxide and sodium 1-heptanesulfonate were purchased from Merck (Milan, Italy). All the solvents used were of analytical grade or HPLC grade and were purchased from Carlo Erba Reagenti (Milan, Italy). Ultrapure water used for the buffers was obtained from a Milli-Q^® Plus Purification System (Merck Millipore, Vimodrone Milan, Italy). Solvent evaporation was carried out using a rotating evaporator (Heidolph Laborota 400, Heidolph Instruments, Schwabach, Germany) equipped with a vacuum pump (Diaphragm Vacuum Pump DC-4). Lyophilization was performed with a LyoQuest-85^® freeze drier (Azbil Telstar Technologies, Barcelona, Spain). C2C12 myoblasts, an immortalized murine cell line, were purchased from ECACC 91031101. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), glutamine, amphotericin B and penicillin-streptomycin were purchased from Gibco, Thermo Fisher Scientific (Waltham, MA, USA). Thiazolyl Blue Tetrazolium Bromide (MTT solution), Phalloidin-Atto 488, Phalloidin-Atto 594 and Alexafluor 488 were obtained from Merck.

Cells were seeded at 40,000 per well on coverslips in 24-well plates and transfected with 1 μl jetPEI per well and the appropriate transfection vectors (S10 Table), in triplicate. Forty-eight hours after transfection the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature. The cells were washed twice and permeabilized with 0.2% Triton X-100 in PBS for 20 minutes at room temperature. The cells were washed three times with PBS and blocked with 2% bovine serum albumin and 5% chick serum in PBS for one hour at room temperature. The cells were stained with Alexa Fluor 488 conjugated DYKDDDDK (FLAG) Tag antibody (Cell Signaling Technology, 5407) diluted 1:200 in blocking buffer overnight at 4°C protected from light. The cells were washed three times with 0.1% TWEEN 20 in PBS (PBS-T) and incubated with Atto 594 Phalloidin (Sigma-Aldrich, 51927) diluted 1:100 in PBS for 20 minutes at room temperature protected from light. The cells were washed with PBS-T and stained with DAPI in PBS (1:5000) for 15 minutes at room temperature protected from light. The coverslips were inversely mounted on slides with hard-dry DPX mountant (Leica Biosystems). A Leica TCS SP5 II confocal microscope (Leica Microsystems) was used to image the cells.