Peg300

Scn8a^+/+ and Scn8a^+/mut mice were treated with the HDAC inhibitor TSA or vehicle on a previously published dosing schedule shown to prevent activity-dependent myelination in mice^{11 (link)}. Male and female littermates were randomly assigned to either vehicle or TSA treatment. TSA (Selleck Chemicals, S1045) stock solution (50 mg ml⁻¹ in sterile-filtered DMSO) was kept at −20 °C until the day of dosing, when it was diluted to a concentration of 10 mg ml⁻¹ in 30% PEG300 (Selleck Chemicals, S6704) and 2% Tween 80 (Sigma-Aldrich, P1754) in sterile-filtered DMSO (Tocris, 3176) just before each administration. Then, 10 mg kg⁻¹ of TSA or vehicle was administered each day by intraperitoneal injection^{4 (link)} between P28 (starting after the first EEG recording on the same date) and P45. Mice were weighed every 1–2 days, and their health was monitored throughout the study. We did not observe any deleterious effects of TSA treatment.

Reagents were freshly constituted for experiments. Forskolin (20 μM) and amiloride (10 μM) were purchased from Selleckchem and CFTRinh-172 (20 μM) from MilliporeSigma. Ivacaftor was purchased from Selleckchem and reconstituted on delivery with DMSO (Thermo Fisher Scientific) before aliquoting and storage at –80°C. We prepared single doses of Ivacaftor or vehicle within 1 hour of intraperitoneal (i.p.) administration. Doses were 40 mg/kg Ivacaftor in 5% DMSO, 5% Tween-80, 40% PEG300 (all from Selleckchem), and 50% of 0.9% saline (Grifols) solution. Vehicle was the identical weight-based solution volume without Ivacaftor.

To investigate the effect of SR1078 on cSCC growth in vivo, we conducted an animal experiment. Firstly, 10 eight-week-old SKH-1 mice were randomly divided into two groups (n = 5 per group). The injected dose of SR1078 was 20 mg/kg, and the weight of each mouse was estimated to an average of 25 g. SR1078 was dissolved in the solution including 10% dimethyl sulfoxide (DMSO, Selleck), 40% PEG300 (Selleck), 5% Tween80 (Selleck) and 45% ddH₂O. The agonist group and control group were injected intraperitoneally with 150 µl SR1078 solution and DMSO solution, respectively. In specific, three times injection in a week was performed before cSCC cell inoculation. Then, XL50 cSCC cells from China Center for Type Culture Collection (Wuhan, China, 5 × 10⁶) and NIH/3T3 cells (mouse embryonic fibroblast, 1 × 10⁶) were injected subcutaneously into the backs of mice to establish a cSCC mouse model. After incubation, mice were injected intraperitoneally with SR1078 or DMSO solution daily. Tumor volume was measured every 2 days and calculated as length × width² × 0.5 (mm³). Eighteen days post-injection, the tumor was harvested for cell proliferation evaluation by IHC of Ki67 expression (MA5-14520, Thermo Fisher Scientific) following the guideline.

BALB/c male nude mice (4–5 weeks old) were purchased from BEIJING HFK Biot SCIENCE CO., Ltd. (Beijing, China) and raised in individually ventilated cages in a specific pathogen-free environment in the Laboratory Animal Center of Central South University. To build the xenografted tumor model, HepG2 cells (8 × 10⁶ cells per mouse) were inoculated subcutaneously into the left armpit of nude mice after being sterilized with 75% alcohol. When the tumor volume reached ~50 mm³, nude mice were randomly divided into three groups (n = 5/group): vehicle control (experimental solvent with compound-free) (group 1); 5-FU, 30.0 mg/kg every 2 days (group 2); compound SIOC-XJC-SF02, 20.0 mg/kg every 2 days (group 3). The compound was dissolved in PEG300 (Selleck, Texas, USA) and Tween-80 (Bio Froxx, Einhausen, Germany). Nude mice in all three groups received intraperitoneal (i.p.) injections. The tumor volume of nude mice was estimated every two days via the eq. V = (L × W²)/2, where L is the length, and W is the width of the tumor, and the length and width were measured with vernier calipers. After treatment for 14 days, nude mice of all three groups were sacrificed, and the tumors were removed, weighed, photographed, and fixed in 10% formaldehyde for immunohistochemistry (IHC) and hematoxylin-eosin (H&E) staining.

The following antibodies from Cell Signaling Technology (CST) were used: Phospho-NF-kappa-B p65 (Ser536) Antibody (3031S), NF-kappa-B p65 (C22B4) Rabbit mAb (4764S), Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (4668), SAPK/JNK Antibody (9252), p38 MAPK (D13E1) XP® Rabbit mAb (8690), Phospho-p38 MAPK (Thr180/Tyr182) (12F8) Rabbit mAb (4631), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (9101S), and p44/42 MAPK (Erk1/2) Antibody (4695). Nrf2 antibody was purchased from Abcam (ab137550), RNA Polymerase II antibody was purchased from Sigma (05-623), and β-action was purchased from Proteintech (60008-1-Ig). RSL3 (S8155) was from Selleck, Ferrostatin-1 (SML0583) was from Sigma, and C11-BODIPY 581/591 lipid peroxidation sensor (D3861) was from Life Technologies. LPS (LPS Ultrapure, Escherichia coli 0111: B4) was from Sigma. PEG300 and Tween 80 were from Selleck.

Chemicals used in this study were from the following sources: nilotinib (Selleckchem, S1033), imatinib (Selleckchem, S2475), torin1 (Selleckchem, S2827), Gefitinib (MedChemExpress, HY-50895), GSK621 (Sigma, SML2003), EX229 (MedChemExpress, HY-112769), BC1618 (MedChemExpress, HY-134656), bafilomycin A1 (Selleckchem, S1413), MK2206 (Selleckchem, S1078), SRT1720 (Apexbio, A4180–10), FR180204 (Selleckchem, S7524), PLX-4720 (Selleckchem, S1152). PEG300 (Selleckchem, S6704), DMSO (Sigma, D2650).