Hoechst 33342 h1399

Adenosine 5′-triphosphate sodium hydrate (ATP, A1852), DMEM (D6429), Hanks’ balanced salt solution (HBSS, H4641), and HEPES sodium salt (H7006) were purchased in Sigma-Aldrich, St. Louis, MO, USA; Hoechst 33342 (H1399), and propidium iodide (P1304MP) was purchased from Molecular Probes, Eugene, OR, USA; reverse transcriptase MMLV (SK022S), SYBR Green I PCR Master Mix (PK147L) was purchased from Evrogen, Moscow, Russia; Fura-2AM (cat. no. F1221), and fetal bovine serum (10099141) was purchased from Thermo Fisher Scientific, Waltham, MA, USA; Selenium and SeSo nanoparticles (courtesy of Dr. SV Gudkov, Prokhorov General Physics Institute, Russian Academy of Sciences, Moscow, Russia).

MCs were inoculated and cultured on a 35 mm glass base dish (IWAKI 3910-035, Tokyo, Japan) in DMEM with 10% FBS and normal glucose. After serum starvation in DMEM with 0.5% FBS for 24 h, cells were incubated in DMEM with 0.5% FBS and normal glucose or high glucose for 20 h. Then, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized in PBS with 0.25% Triton-X for 15 min. After blocking with 10% albumin for 20 min, cells were incubated with rabbit anti-USF1 antibody (ab180717, Abcam, Cambridge, UK) diluted to 1:100 for 1 h. After washing with PBS, cells were incubated with Alexa-594 Goat anti-Rabbit IgG (A11072, Invitrogen, Waltham, MA, USA) as the second antibody for 30 min. Nuclei in the cells were stained with Hoechst 33342 (H1399, Invitrogen) diluted 1:1000.

We obtained lovastatin (PHR1285), mevalonate (41288), 25-hydroxycholesterol (H1015), MG-132 (M8699), Filipin (F9765), geranylgeraniol (G3278), protease inhibitor cocktail (P8340), N-Ethylmaleimide (E3876), and paraformaldehyde (PFA) (P6148) from Sigma; [¹⁴C]-acetic acid sodium salt (NEC084H001) from Perkin Elmer; and FuGENE HD transfection reagent (E2312) from Promega; G418 (345810), digitonin (300410), henylmethylsulfonyl fluoride (PMSF) (52332), leupeptin (108975), pepstatin A (516481) and ALLN (208719) from Merck; Hoechst 33342 (H1399) from Invitrogen. Lipoprotein-deficient serum (LPDS) [41 (link)] and delipidated-fetal calf serum (FCS) [42 (link)] was prepared from FCS (S1580, Biowest) by ultracentrifugation in our laboratory [25 (link)].

H1299 cells (2 × 10⁴ cells) were seeded on 200 μL of genipin-mixed collagen gels in 16 mm glass dishes or collagen-coated 16 mm glass dishes and cultured for 24 h. Then, the cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and washed three times with PBS. Next, the cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature and washed three times with PBS. The cells were blocked with 0.1 or 0.5% bovine serum albumin in PBS for 30–60 min at room temperature. Primary antibody solution (1:250 anti-YAP antibody; 14074S, Cell Signaling Technology Inc., Danvers, MA, USA) in PBS was added and incubated at 4 °C overnight. After three washes with PBS, the secondary antibody solution (1:250 Alexa Fluor 488 goat anti-rabbit; A27034, Invitrogen, Waltham, MA, USA and 1:10,000 Hoechst 33342; H1399, Invitrogen) in PBS was added and incubated at room temperature for 1 h. After three washes with PBS, fluorescent images were captured using an A1 confocal imaging system (Nikon Instech, Tokyo, Japan) with a 60× objective. To quantify the localization of YAP, the fluorescence intensity was calculated as the nuclear/cytosol ratio using ImageJ software.

The cells at T0 were digested with 0.05% Trypsin-EDTA at 37°C for 5 min. For T3, T6, or T9, we digested the cells with 0.05% Trypsin-EDTA at 37°C for 5–10 min after the digestion of the cells with 0.02% collagenase at 37°C for 3–5 min. We obtained single cells by pipetting the digested cell sheets. Dissociated cells were resuspended in washing buffer consisting of 2.5% FBS in PBS, counted, and stained with Hoechst 33342 (H-1399; Invitrogen) and SytoxRed dead-cell stain (S34859; Thermo Fisher Scientific). Hoechst positive/Sytox negative single cells were sorted into 1 µl of cell lysis buffer consisting of 0.3% NP40 (28324; Thermo Fisher Scientific), 0.12 dNTPs (N0477; New England BioLab, Ipswich, MA, United States), 1U RNase Inhibitor (2313A; TaKaRa Bio), and 0.11 µM 384 well-unique reverse transcription primer in a 348-well PCR plate (0030129547; Eppendorf, Hamburg, Germany).